Subregions of the periaqueductal gray topographically innervate the rostral ventral medulla in the rat.

THE JOURNAL OF COMPARATIVE NEUROLOGY 309~305-327 (1991)

Subregions of the Periaqueductal Gray Topographically Innervate the Rostra1 Ventral Medulla in the Rat ELISABETH J. VAN BOCKSTAELE, GARY ASTON-JONES, VINCENT A. PIERIBONE, MATTHEW ENNIS, AND MICHAEL T. SHIPLEY Division of Behavioral Neurobiology, Department of Mental Health Sciences, Hahnemann University, Philadelphia, Pennsylvania 19102-1192 (E.J.V.B., G.A.-J.); New York University, Department of Biology, New York, New York 10003 (E.J.V.B.); Department of Histology and Neurobiology, Karolinska Institutet, S-104 01 Stockholm, Sweden (V.A.P.); and Department of Anatomy and Cell Biology, University of Cincinnati School of Medicine, Cincinnati, Ohio 45267 (M.E., M.T.S.)

ABSTRACT Previous anatomical and physiological studies have revealed a substantial projection from the periaqueductal gray (PAG) to the nucleus paragigantocellularis (PGi). In addition, physiological studies have indicated that the PAG is composed of functionally distinct subregions. However, projections from PAG subregions to PGi have not been comprehensively examined. In the present study, we sought to examine possible topographic specificity for projections from subregions of the PAG to PGi. Pressure or iontophoretic injections of wheat germ agglutinin-conjugated horseradish peroxidase, or of Fluoro-Gold, placed into the PGi of the rat retrogradely labeled a substantial number .of neurons in the PAG from the level of the Edinger-Westphal nucleus to the caudal midbrain. Retrogradely labeled neurons were preferentially aggregated in distinct subregions of the PAG. Rostrally, at the level of the oculomotor nucleus, labeled neurons were i) compactly aggregated in the ventromedial portion of the PAG corresponding closely to the supraoculomotor nucleus of the central gray, ii) in the lateral and ventrolateral PAG, and iii) in medial dorsal PAG. More caudally, retrogradely labeled neurons became less numerous in the dorsomedial PAG but were more widely scattered throughout the lateral and ventrolateral parts of the PAG. Only few retrogradely labeled neurons were found in the ventromedial part of the PAG at caudal levels. Injections of retrograde tracers restricted to subregions of the PGi suggested topography for aferents from the PAG. Injections into the lateral portion of the PGi yielded the greatest number of labeled neurons within the rostral ventromedial PAG. Medially placed injections yielded numerous retrogradely labeled neurons in the lateral and ventrolateral PAG. Injections placed in the rostral pole of the PGi (medial to the facial nucleus) produced the greatest number of retrogradely labeled neurons in the dorsal PAG. To examine the pathways taken by fibers projecting from PAG neurons to the medulla, and to further specify the topography for the terminations of these afferents in the PGi, the anterograde tracer Phaseolus uu2garis-leucoagglutininwas iontophoretically deposited into subregions of the PAG that contained retrogradely labeled neurons in the above experiments. These results revealed distinct fiber pathways to the rostral medulla that arise from the dorsal, lateralhentrolateral, and ventromedial parts of the PAG. These injections also showed that there are differential but overlapping innervation patterns within the PGi. Consistent with the retrograde tracing results, injections into the rostral ventromedial PAG near the supraoculomotor nucleus yielded anterograde labeling immediately ventral to the nucleus ambiguus in the ventrolateral medulla, within the retrofacial portion of the PGi. Injections into the l a t e r d ventrolateral PAG yielded labeled fibers and terminals predominantly in the ventral nucleus gigantocellularis and in the medial retrofacial portion of the PGi. Injections into the dorsal PAG resulted in anterograde labeling predominantly in the nucleus raphe magnus and in the rostral portion of the PGi medial to the facial nucleus. AcceptedMarch 18,1991 O

1991 WILEY-LISS, INC.

E.J. VAN BOCKSTAELE ET AL.

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The PAG is a heterogeneous region with functions including sensory integration, autonomic regulation, and pain modulation. Our findings indicate that subdivisions of the PAG project to areas in the ventral medulla with analogous functional correlates and suggest that anatomically distinct circuits connecting midbrain and bulbar regions could differentially mediate specific functional components of these processes. Key words: wheat germ agglutinin-conjugatedhorseradish peroxidase, Phaseolus vulgaris-leucoagglutinin,retrograde transport, anterograde transport, nucleus

paragigantocellularis

The periaqueductal gray (PAG), an area surrounding the cerebral aqueduct throughout the midbrain, is a cytoarchitectonically complex region that has been difficult to subdivide anatomically in rat, cat, and monkey (but see Hamilton, '73; Beitz, '82; Shipley et al.,'87). Many physiological studies, however, have suggested that the PAG is compartmentalized into functionally distinct units. Stimulation of various subregions of the PAG in the rat have implicated this structure in antinociception (Mayer et al., '71; Mayer and Liebeskind, '74), analgesia (Depaulis et al., '87; Morgan and Liebeskind, '87; Lakos and Basbaum, '881, and cardiovascular control (Gauthier and Reader, '82). Furthermore, for cat, electrical and chemical stimulation of the PAG have also implicated the PAG in cardiovascular control (Hilton and Redfern, '86; Carrive et al., '87; Carrive et al., '89) as well as respiration (Kabat, '36), and defense behavior (Skultety, '63; Hilton, '82; Hilton and Redfern, '86; Gomita et al., '88; Bandler et al., in press). Stimulation of certain regions of the PAG has also been reported to cause vocalization in monkeys (Jurgens and Pratt, '79; Kirzinger and Jurgens, '85; Larson and Kistler, '86) and in cats (Bandler et al., in press).

In addition to its cytoarchitectonic heterogeneity and functional diversity, PAG neurons contain many neurotransmitters, e.g., serotonin (Steinbusch et al., '78; Clements et al., '851, acetylcholine (Palkovits and Jacobowitz, '74; Ruggiero et al., 'go), dopamine (Versteeg, '76), GABA (Barbaresi and Manfrini, '881, glutamate (Clements et al., '87), and neuropeptides such as enkephalin (Hokfelt et al., '77; Beitz, '82), neurotensin (Jennes et al., '82; Beitz, '83a; Shipley et al., '87), and somatostatin (Finley et al., '81). Although a systematic analysis of the efferent projections of the PAG has not been undertaken, a variety of tracttracing experiments indicate that the PAG has extensive efferent connections in the monkey (Jurgens and Pratt, '79; Mantyh, '83a,b), cat (Holstege and Tan, '881, and rat (Andrezik et al., '81a; Beitz et al., '83b; Bandler and Tork, '87; Bandler and Carrive, '88). Anterograde tracing studies have revealed projections to numerous sites in the forebrain in the monkey (Mantyh, '83a) and the rat (Rizvi et al., '91; Shipley et al., in press). In addition, PAG neurons have been reported to innervate the superior colliculus (Mitchell et al., '88), the cuneiform nucleus (Redgrave et al., '88), the nucleus raphe magnus (Beitz, '82; Fardin et al., '84; Lakos

Abbreviations Amb

AP Aq Bar

CIC CnF CP cNTS

cu

DC DR DTg ECu FG fr gWI IC IML icp ING IPC 111

I0 IV LC 1fP

11

LRt

LSO MG ml mlf Me5

nucleus amhiguus area postrema cerebral aqueduct Barrington's nucleus central nucleus, inferior colliculus cuneiform nucleus cerebral peduncle nucleus of the solitary tract, commissural nucleus cuneatus dorsal cochlear nucleus dorsal raphe nucleus dorsal tegmental nucleus external cuneatus nucleus Fluoro-Gold fasciculus retroflexus genu of the VIM1 nerve inferior colliculus intermediolateral cell column inferior cerebellar peduncle intermediate gray layer, superior colliculus interpeduncular nucleus oculomotor nucleus inferior olive fourth ventricle locus coeruleus longitudinal fasciculus pons lateral lemniscus lateral reticular nucleus lateral superior olive medial geniculate medial lemniscus medial longitudinal fasciculus mesencephalic trigeminal nucleus

Mo5 MVe nVI1 NTS PAC PC PGi PHA-L Pn PrH PrVI PY RN

RVL

RVM s5

sc Scp

SN SOM SP5 sp5c Sp5I Sp50 SUG tz VCA VII VIIn VT WGA-HRP XI1 xscp

motor trigeminal nucleus medial vestibular nucleus facial nerve nucleus of the solitary tract periaqueductal gray posterior commissure nucleus paragigantocellularis Phaseolus uulguris-leucoagglutinin pontine nuclei nucleus prepositus hypoglossi principal sensory trigeminal nucleus pyramidal tract red nucleus rostroventrolateral reticular nucleus rostra1 ventral medulla sensory root trigeminal nerve superior colliculus superior cerebellar peduncle substantia nigra supraoculomotornucleus of the central gray spinal trigeminal tract spinal trigeminal nucleus, caudal spinal trigeminal nucleus, interpolaris spinal trigeminal nucleus, oral superficial gray layer, superior colliculus trapezoid body ventral cochlear nucleus anterior facial nucleus facial nerve ventral tegmental nucleus wheat germ agglutininconjugated horseradish peroxidase hypoglossal nucleus decussation superior cerebellar peduncle

PROJECTIONS FROM PAG TO THE RVM and Basbaum, '881, the nucleus paragigantocellularis (PGi; Beitz, '82; Van Bockstaele et al., '89), the nucleus ambiguus (ter Horst et al., '841, the nucleus of the solitary tract (Bandler and Tork, '871, and the nucleus gigantocellularis (Beitz, '82) in the rat. In monkey, the PAG has also been reported to project to the parabrachial nucleus (Mantyh, '83b) and, in cat, has been reported to project to the spinal cord (Blomqvist and Wiberg, '85). However, there is relatively little information about the cells of origin, projection routes, or terminal topography of the descending projections from PAG. The present study focuses on projections to the rostral ventral medulla as these circuits figure prominently in the role of the PAG in antinociception and autonomic regulation. We demonstrate that there is a high degree of anatomical specificity for projections from subregions of PAG to the PGi, the ventral nucleus gigantocellularis and midline brainstem raphe nuclei. Projections to specific subregions of the rostral ventral medulla from select cell groups within PAG may indicate specific neural substrates for different functions associated with PAG activation.

MATERIALS AND METHODS Forty male Sprague-Dawley rats (Taconic Farms) were used in this study. Rats weighing 300 to 350 g were deeply anesthetized with chloral hydrate (400 mg/kg, intraperitoneally) and placed in a Kopf stereotaxic apparatus for surgery. Anesthesia was maintained throughout the surgical procedure by administering supplemental doses of chloral hydrate every 30 minutes. Body temperature was monitored and maintained at 36 to 38°C throughout the surgery with a heating pad. Following the surgery, the animal was kept warm until fully recovered from anesthesia. Abbreviations for illustrations were taken from the atlas of the rat brain by Paxinos and Watson ('85).

PGi and PAG injection placements The nucleus PGi as originally defined for the rat by Andrezik et al. ('81b) covers a large region in the rostral ventral medulla extending rostrally from the lateral reticular nucleus to the superior olive. It is bordered medially by the inferior olive and the nucleus gigantocellularis pars ventralis in its caudal division, laterally by the spinal trigeminal nucleus and tract, and dorsally by the nucleus ambiguus. Caudally, this area has been termed the "retrofacial PGi" (Taber, '61), corresponding to its location immediately caudal to the facial nucleus and ventral to the nucleus ambiguus. Rostrally, the PGi is bordered medially by the nucleus gigantocellularis pars alpha and laterally by the facial nucleus. As the facial nucleus progresses rostrally, it extends more medially in the ventral caudal pons thereby displacing the rostral pole of the PGi. In its rostral extent, this area is designated here as juxtafacial PGi. For injections into retrofacial PGi, glass microelectrodes were positioned 3.5 mm caudal to the interaural line, 1.5 mm lateral to midline, and 0.5 mm above the ventral brain surface. These coordinates were modified with respect to the desired placement of the injection. Accurate localization of the PGi was aided by recording characteristic cellular activity through injection pipettes as previously described (Aston-Jones et al., '86; Van Bockstaele et al., '89). Injections of WGA-HRP into the ventrolateral PAG were made with the electrode carrier angled at 12" relative to the mediolateral plane. Coordinates for such injections were 0.5-1.5 mm rostral to lambda, 1.0-1.6 mm lateral to

307

midline, and 4.2-5.7 mm ventral to the skull surface. The following coordinates were used for PHA-L injections into i) the rostral ventromedial PAG (area of the supraoculomotor nucleus of the central gray, SOMIPAG), 3.2 mm rostral to interaural zero, 0.2 mm lateral to midline, and 6-6.5 mm ventral to the skull surface; ii) the lateral/ventrolateral PAG, 0.7 mm rostral to interaural zero, 0.5 mm lateral to midline, and 5.5-6.0 mm ventral to the skull surface; and iii) the dorsal PAG, 2.0 mm rostral to interaural zero, 0.1 mm lateral to the midline and 4 mm ventral to the skull surface. All injections were made unilaterally by a dorsal approach and all PHA-L injections into PAG were made with bregma and lambda in the horizontal plane.

WGA-HRP methods Iontophoretic injections of wheat germ agglutininconjugated horseradish peroxidase (WGA-HRP; 1%in phosphate-buffered saline, PBS) were made through glass micropipettes (with inner fiber; 4 pm tips) as previously described (Aston-Jones et al., '86; Van Bockstaele et al., '89). In most cases, injections were made with a constant current device (Finntronics or Midgard) with pulsed current (4 seconds on, 4 seconds off, of +1 FA. Pressure injections (20-40 nl) were made in other animals by means of a micropressure device (Picospritzer; General Valve, Inc.). The pipettes were kept at the injection site for 5-10 minutes before and after depositing the tracer. Twenty-four hours following injections of WGA-HRP, the animals were deeply anesthetized with sodium pentobarbital(45-50 mgkg, i.p.) and transcardially perfused according to the method described by Mesulam ('82). Forty-micron-thick frozen sections were collected and treated with tetramethylbenzidine as the chromogen, as described by Mesulam ('82) with modifications by Shipley ('82). Sections were counterstained with neutral red, dehydrated through a graded series of alcohols, and coverslipped with Permount.

Fluoro-Gold methods Iontophoretic injections of Fluoro-Gold (FG; Fluorochrome, Englewood, CO) were made into the ventral medulla by using a 1% solution in 0.1 M sodium acetate buffer (pH 3.3) in glass micropipettes (with inner fiber, 10 pm tips) and pulsed current of + 1 FA (4 seconds on, 4 seconds off) as described by Pieribone and Aston-Jones ('88). Fixation and tissue processing were conducted as previously described (Van Bockstaele et al., '89). Forty-micron-thick sections were collected into 0.1 M PBS immediately after the post-fixation by cutting on a Vibratome or were cut on a freezing microtome following cryoprotection in 20% sucrose for 12-24 hours after perfusion. The sections were mounted on gelatinized slides and were subsequently dehydrated and coverslipped with DPX mountant. Fluoro-Gold was visualized with ultraviolet epi-illumination on a Leitz Aristoplan microscope with Fluotar objectives. No counterstain was used as background tissue autofluorescence allowed adequate visualization of fiber tracts and nuclear boundaries.

Phaseolus vulgaris-leucoagglutininmethods PHA-L (Vector Laboratories, Burlingame, CA; 2.5% in 0.01 M PBS) was iontophoretically deposited with +5 FA of pulsed current (4 seconds on, 4 seconds off) for 30 minutes (Gerfen and Sawchenko, '84). Animals survived for 7 to 14 days and were then deeply anesthetized with sodium pento-


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Fig. 1. Low-power video-computer-aided plots of coronal sections from the same animal illustrating two different levels (several hundred microns apart) of a pressure injection site of WGA-HRP in the ventral medulla. A WGA-HRP injection in the rostral pole of the PGi area. B: WGA-HRP injection caudal to that in panel A. The injection site was

barbital (45-50 mg/kg, intraperitoneally) and perfused transcardially according to a protocol previously described (Van Bockstaele et al., '89; Wouterlood et al., '87). Forty-micron-thick frozen sections were collected into 0.1 M PBS and incubated with the primary antibody for 48 hours (Goat anti-PHA-L; 1:4,000; Vector Labs, Burlingame, CA). The ABC-peroxidase procedure (Vector Laboratories, Burlingame, CA) was used to detect the primary antibody. For some tissue sections, silver intensification of the diaminobenzidine reaction product, described by Gallyas et al. ('82), was used to enhance the visibility of labeled fibers. Black and white photomicrographs were taken with Kodak Plus-X film ( M A 1251, Kodak Panatomic-X film (ASA 32), or Kodak Tmax 100 film. Color prints of WGAHRP material were made with Ektar 25 film.

accurately plotted by using a computer routine designed to define the dense central core of the injection site (see Methods for details). mlf, medial longitudinal fasciculus; py, pyramidal tract; Sp51, spinal trigeminal nucleus (interpolaris); Amb, nucleus ambiguus; 10, inferior olive; NTS. nucleus tractus solitarius.

as defined by Ross et al. ('84), the ventral division of the nucleus ambiguus or external ambiguual formation described by Bieger and Hopkins ('87), and the ventral respiratory group corresponding to the Botzinger complex of the cat (Merrill, '70; Bianchi and Barillot, '82; Feldman, '86; Saether et al., '87; Ezure et al., '88); ii) the medial portion of the retrofacial PGi; or iii) the most rostral pole of the PGi, located medial to the caudal facial nucleus, designated here as juxtafacial PGi. Large pressure injections of WGA-HRP that covered most of the PGi area (Fig. 1)yielded numerous retrogradely labeled neurons throughout the rostrocaudal extent of the PAG, as seen in the series of plots in Figure 2. Retrogradely labeled neurons were found in greatest densities in specific subregions of the PAC, as previously reported (Van Bockstaele et al., '89): i) the rostral ventromedial aspect of the PAG corresponding to the SOMPAG (Fig. 2C); ii) the Data analysis lateral and ventrolateral PAG, corresponding to tissue The locations of retrogradely labeled neurons were plot- laterally adjacent to the aqueduct and immediately ventral ted by means of a NikoniJoyce Loebl Magiscan computer for to this lateral area, respectively (Fig. 2E); and iii) the video-linked image processing and analysis as previously dorsomedial PAG (Fig. 2C,D). Reciprocal labeling was also described (Shipley et al., '90). In brief, microscopic images observed in the dorsal and lateral/ventrolateral PAG as were digitized and optimized for the signal of interest, and a demonstrated by fine punctate WGA-HRP granules (see light pen was used to delineate anatomical landmarks and Fig. 3). Retrograde labeling in SOM/PAG was predomiplot labeled neurons on the optimized image displayed on a nantly contralateral to injection sites, with only few labeled video monitor. Four subfields were drawn for each section neurons located ipsilaterally. By contrast, for lateral/ with a l o x objective. The computer then assembled a ventrolateral and dorsomedial PAG, retrogradely labeled composite image from these four subfields. neurons were found predominantly ipsilateral to the injecThe injection site was analyzed densitometrically and tion site; however, with large injections (such as the one represented with a pseudocoloring routine to define the documented in Figs. 1-3), retrogradely labeled neurons dense central core of the injection site. were found bilaterally. PHA-L fibers were plotted with a camera lucida at These large injections revealed the overall distribution of various coronal levels to illustrate the pathways from PAG PAG afferents to the PGi; however, they could not resolve to PGi. possible topography in these projections. To directly address this issue, restricted iontophoretic injections of WGAHRP or FG were placed in various subregions of the PGi RESULTS and retrogradely labeled neurons were mapped in the PAG. Retrograde tracing studies As described below, the number and location of labeled Injection placements into the rostral ventral medulla. neurons in the PAG varied systematically with injection Iontophoretic (n = 14) or pressure (n = 6) injections of placements in specific subregions of the PGi. Lateral retrofacial PGi. Injections centered in the latretrograde tracers were located within the nucleus PGi in the rostral ventral medulla (see Methods). Large pressure eral retrofacial region of the PGi, such as the one shown in injections of WGA-HRP covered the PGi almost entirely Figure 4A, produced strong retrograde labeling of neurons and in some cases spread into the dorsally adjacent external in the rostral ventromedial PAG. These neurons were division of the nucleus ambiguus. Smaller, more discrete densely clustered in a distinct band above the contralateral iontophoretic injections of either FG or WGA-HRP were oculomotor nucleus, corresponding to the supraoculomotor restricted to i) the lateral retrofacial portions of the PGi nucleus of the central gray (SOM/PAG; Fig. 2, plot C). This including the rostroventrolateral reticular nucleus (RVL) group of labeled neurons did not extend caudally into the

PROJECTIONS FROM PAG TO THE RVM

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Fig. 2. Low-power video-computer-aided plots of individual coronal sections from the same animal as in Figure 1 depicting retrogradely labeled neurons (triangles) throughout the periaqueductal gray matter following a pressure injection of WGA-HRP into the PGi area. Each triangle represents one retrogradely labeled neuron. The plots are ordered rostrally (A) to caudally (F). Cell numbers and locations were

accurately entered into the computer by direct plotting under high magnification ( 2 0 ~objective) (see Methods for details). pc, posterior comrnissure; fr, fasciculus retroflexus; DR, dorsal raphe; mlf, medial longitudinal fasciculus; scp, superior cerebellar peduncle; VT, ventral tegmental nucleus; 111,oculomotor nucleus; LDT, lateral dorsal tegmental nucleus; DTg, dorsal tegmental nucleus.

310


Fig. 3. Polarized, darkfield-illuminated photomicrograph of WGAHRP retrograde labeling in the PAG following a pressure injection into the PGi area, similar to that shown in Figure 1. This section is adjacent to the one plotted in Figure 2C. Note the dense cluster of retrogradely

labeled neurons in the contralateral rostral ventromedial PAG (at arrow). Retrogradely labeled neurons are also found lateral to the cerebral aqueduct and in dorsal PAG. The star is on the midline and indicates t,he cerebral aqueduct. Bar = 300 Fm.

area of the midline dorsal raphe. The number of labeled neurons in this area was typically 15 to 20 per section (Fig. 4B). In cases where injections encroached on the facial nucleus, retrogradely labeled neurons appeared along the midline in the region of the Edinger-Westphal nucleus immediately rostral to SOMIPAG. However, labeling in the Edinger-Westphal nucleus was not present with injections restricted to the retrofacial area of PGi. Medial retrofacial PGi. Injections into the medial aspect of the retrofacial PGi retrogradely labeled numerous neurons preferentially in the ventrolateral and lateral PAG. These neurons were predominantly ipsilateral to the injection site, but with larger pressure injections retrogradely labeled neurons were also present contralaterally (Fig. 2, plots D and E). These were most numerous in caudal PAG (25-30 neurons per section for iontophoretic injections) immediately dorsal to the lateral wings of the dorsal raphe; scattered labeled cells were also found within the dorsal raphe. Retrogradely labeled neurons were also found more rostrally, lateral to the cerebral aqueduct, but were fewer in number. Medial rostral (juxtafacial) PGi. Injections centered in juxtafacial PGi yielded retrogradely labeled neurons in dorsomedial, ventrolateral and lateral PAG. These neurons were most numerous (25-30 neurons per section) in the

rostral PAG at the level of the oculomotor and red nuclei (Fig. 5). Many labeled neurons were found scattered in the dorsal PAG located on the border between the dorsomedial PAG and the deep layers of the superior colliculus.

Anterograde tracing studies WGA-HRP injections into PAG. Multiple iontophoretic injections of WGA-HRP along the rostrocaudal axis of PAG were used to study the overall density and distribution of anterogradely labeled fibers in PGi from PAG. Two dorsoventrally spaced iontophoretic injections of WGA-HRP were made at each of three rostrocaudal levels of the PAG covering most of the ventrolateral, lateral, and ventromedial PAG area (n = 6) as shown in Figure 6. Following these large injections, anterograde labeling was observed throughout the PGi and several adjacent regions. Figure 7A and B shows descending fibers from such an injection. Overall, the intensity of labeling was greater in rostral than in caudal PGi. Fiber density decreased in the caudal medulla overall; however, varicose fibers aggregated in a restricted region of the mid-caudal medulla, corresponding to the nucleus retroambiguus. Anterograde labeling was also much stronger in medial than in lateral portions of the PGi (Fig. 7'2). PHA-L injections into PAG. As the above retrograde tracing experiments indicated that there is topography in


Fig. 4. A: Ultraviolet epi-illuminated photomicrograph of a coronal section containing an iontophoretic Fluoro-Gold injection centrally placed in PGi. Note the nucleus amhiguus (Amb) dorsal to the injection site. The arrow indicates the ventral surface of the brain and medial is to the left. Bar = 250 pm. B: Ultraviolet epi-illuminated photomicrograph of Fluoro-Gold retrogradely labeled neurons in the rostra1

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ventromedial PAG and the supraoculomotor nucleus of the central gray. The closed arrow indicates a retrogradely labeled Fluoro-Gold neuron. The oculomotor nucleus (111) is located immediately ventral to the retrogradely labeled neurons. The open arrow indicates the midline. Bar = 125 pm.

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Fig. 5. Darkfield-illuminated photomicrograph of retrogradely labeled neurons in the dorsal PAG following an iontophoretic injection of WGA-HRP into the rostral, juxtafacial PGi (medial to the facial nucleus). Note the reciprocal innervation of this same region. Some retrogradely labeled neurons are also present lateral to the cerebral aqueduct but they are most numerous in the dorsal part of the PAG. No

retrogradely- labeled neurons were found in the rostral ventromedial PAG (supraoculomotor nucleus region) following such injections. Star indicates midline and the cerebral aqueduct. SC, superior colliculus. Arrows indicate border between PAG and superior colliculus. Bar = 125 pm.

the projections of PAG to PGi, the anterograde tracer PHA-L was used to address this issue in greater detail. Iontophoretic deposits of PHA-L were made into either (i) the SOM/PAG (n = 6), (ii) the ventrolateral and lateral aspects of the PAG (n = 8) or (iii) the dorsal aspect of the PAG (n = 6). PHA-L fibers from rostral ventromedial PAG. Overall, descending fibers from the rostral ventromedial PAG followed a more medial route than those from other subregions of PAG (described below). Fibers exited the rostral

ventromedial PAG ventrally, skirting the lateral border of the medial longitudinal fasciculus in the midbrain but maintaining an overall medial location (Fig. 8). Scattered innervation occurred in the central gray. In the rostral pons, fibers passed just lateral to the trigeminal motor nucleus and became compressed bilaterally between the superior olive and the VIIth nerve. In the pons, PHA-L fibers remained close to the midline. Just dorsal to the genu of the VIIth nerve, a prominent terminal field with numerous boutons was visible in the area of the

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Fig. 6. Low-power video-computer-aidedplots of three different levels of a WGA-HRP injection site into the ventrolateral periaqueductal gray region. Plot A is the most rostral and plot C the most caudal. SC, superior colliculus; MG, medial geniculate; scp, superior cerebellar peduncle; IC, inferior colliculus; IPC, interpeduncular nucleus; Pn, pontine nuclei.

supragenual nucleus (not shown). Innervation (as indicated Although the fibers were very thin, numerous varicosities by boutons) was also present in the abducens nucleus and could be seen clearly (Fig. 13). In the caudal medulla, fiber density decreased overall, becoming more widely scattered in the facial nucleus. In the rostral medulla, PHA-L fibers from the rostral throughout the tegmentum and resembling fibers of pasventromedial PAG were concentrated medial to and within sage. PHA-L fibers from dorsal PAG. Descending fibers from the contralateral VIIth nucleus. Somewhat more caudally, labeled fibers were prominent in the contralateral retrofa- injections into the dorsal PAG at the level of the oculomotor cial PGi, in a restricted area beneath the nucleus ambiguus nucleus exited the injection site laterally and ventrally to and not in the ventral parts of PGi (Fig. 9A). Scattered ramify in the perilemniscal area of the lateral midbrain fibers were also found in the homotopic area of the ipsilat- tegmentum (Fig. 14) immediately dorsal to and within the eral retrofacial PGi. Bilaterally, fibers in this area were lateral aspect of the substantia nigra. highly varicose and thin in diameter, suggesting a terminal In the rostral pons, fibers innervated the dorsal tegmenfield (Fig. 9B). In the dorsal medulla, anterograde labeling tal nucleus of Gudden and the subjacent medial pontine was present in the prepositus hypoglossi and in the vestibu- tegmentum, and ramified in the ventral gigantocellular lar nuclei. The number of fibers decreased markedly in the nucleus. In the rostral medulla, labeled fibers were primarily caudal medulla. PHA-L fibers from ventrolateral PAG. A PHA-L injec- restricted to the midline nucleus raphe magnus and the tion site in the ventrolateral PAG is shown in Figure 10. laterally adjacent ventral gigantocellular nucleus; many Labeled fibers from the ventrolateral PAG took a distinctly varicose fibers were also present in the rostral pole of the different path compared to the ventromedial-projecting PGi (Fig. 15A) medial to the facial nucleus (Fig. 15B).Some neurons described above, being more laterally located over- of these fibers appeared to arborize in the coronal plane as all. As illustrated in Figure 11,fibers exited the ventrolat- illustrated in Figure 16. Innervation was also observed in eral PAG in two directions: dorsally towards dorsal PAG the lateral prepositus hypoglossi area. At the level of and the inferior colliculus, and ventrolaterally to enter the retrofacial PGi and the caudal medulla, the number of labeled fibers decreased and remained concentrated venregion of the lateral lemniscus. At pontine levels, labeled fibers innervated the dorsolat- trally and medially near the inferior olive. Only scattered eral pontine gray near the locus coeruleus and Barrington's fibers were present within retrofacial PGi. nucleus as well as the subjacent subcoeruleus and supraolivary pontine tegmentum (Ennis et al., in press). There DISCUSSION were scattered fibers within the locus coeruleus, especially in its ventral and medial component, and in the lateral Using retrograde and anterograde tract-tracing with division of the parabrachial nucleus. Two types of PHA-L WGA-HRP, FG, and PHA-L, we have found marked topofibers were intermixed throughout these areas: short smooth graphic specificity for projections from the PAG to the PGi in the rostral ventral medulla. These findings extend our fibers and thin beaded fibers. In the rostral medulla at the level of the facial nucleus, recent analysis of afferents to the PGi (Van Bockstaele et labeled fibers appeared dorsal to the pyramidal tracts and in al., '89) by disclosing distinct projections from separate the rostral pole of PGi medial to the facial nucleus. Thin subregions of the PAG. The subregions include i) the beaded fibers were more prominent in the area of the raphe rostral ventromedial PAG and the SOMPAG; ii) the lateral/ magnus, dorsal to the pyramidal tract, whereas in rostral, ventrolateral PAG, at the level of the dorsal raphe nucleus; juxtafacial PGi fibers were short, thick, and smooth, resem- and iii) the dorsomedial PAG, immediately dorsal to the bling fibers of passage. At the level of retrofacial PGi, cerebral aqueduct throughout the midbrain. Parcellation of the PAG has been controversial due to its labeled fibers appeared ventrally in the tegmentum, innervating the ventral gigantocellular nucleus, nucleus raphe cytoarchitectonic and neurochemical heterogeneity. For magnus, and medial PGi; scattered fibers were also present example, Mantyh ('82) in the rat, cat and monkey was in the retrofacial PGi (Fig. 12A). Many highly varicose unable to distinguish subdivisions within the PAG. This fibers could be seen (Fig. 12B)within the medial PGi area. was later supported by Gioia ('84) in the cat. However,

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Fig. 7. Darkfield-illuminated photomicrographs of anterograde labeling from a WGA-HRP injection into the ventrolateral PAG seen in the saggital plane (A and B) and the frontal plane (C). For A and B, rostra1 is to the left and for C medial is to the right. Arrows in A, B, and C indicate the ventral brain surface. Dorsal is at top. nVII, facial nerve; LSO, lateral superior olive; VII, facial nucleus; NTS, nucleus tractus solitarius; LRt, lateral reticular nucleus; Amb, nucleus ambiguus.


Fig. 8. Camera lucida plots illustrating labeled fibers from an injection of Phaseolus vulgaris-leucoagglutinin into the supraoculomotor nucleus of the central gray. These plots are representative of all cases examined with PHA-L injections into the rostral ventromedial PAG. Plot A represents the most rostral section and plot G the caudal-most. Note the dense innervation in the lateral PGi in plot F

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corresponding to the rostroventrolateral reticular nucleus (Ross et al., '84). See abbreviation list for anatomical landmarks. The fibers illustrated in the plots indicate the efferent trajectory of neurons in the rostral ventromedial PAG to the caudal medulla. The presumptive terminal fields of these projections in the PGi can be seen a t higher magnification in Figure 9B.

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EJ. VAN BOCKSTAELE ET AL.

Fig. 9. A: A photomontage of a darkfield-illuminated coronal section containing a dense terminal field of PHA-L-labeled fibers within rostral lateral PGi (immediately ventral to the nucleus ambiguus: Amb) after an injection into the rostral ventromedial PAG and the supraoculomotor nucleus of the central gray. Open arrow indicates ventral brain surface. Closed arrow indicates the PHA-L-labeled fiber seen at higher magnification in B. Dorsal is at top, lateral is to the right. Bar = 245

pm. 10, inferior olive. B High-power brightfield photomicrograph showing PHA-L-labeled fibers immediately ventral to the nucleus ambiguus (Amb) following an injection in the rostral ventromedial PAG and the supraoculomotor nucleus of the central gray. This field is the same as that shown in A but at higher power and using brightfield illumination. Closed arrow points to the PHA-L labeled fiber indicated in A. Note the numerous thin fibers and varicosities. Bar = 75 pm.

others (Hamilton, '73; Liu and Hamilton, '80; Altman and Bayer, '81; Shipley et al., '87; Conti et al., '88) have presented evidence for systematic cytoarchitectonic variations in the PAG supporting the existence of subdivisions. In the present study, we observed that specific subdivisions of the PAG contained retrogradely labeled neurons following tracer injections into discrete regions of the PGi. Thus, our findings provide evidence for parcellation of the PAG

into discrete subdivisions on the basis of its descending efferent projections to the PGi. In fact, the subregions defined by retrograde transport from the PGi correspond well to those identified cytoarchitectonically by Hamilton ('73) for the cat and Beitz ('85) for the rat. As discussed below, the topographically specific innervation of the PGi by PAG may indicate distinct but functionally related properties for these PAG subdivisions.


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Fig. 10. Brightfield photomicrograph depicting two closely spaced Phaseolus vulgaris-leucoagglutinin iontophoretic injections into the ventrolateral part of the periaqueductal gray. Note the neurons within the injection site that are filled with the tracer. The cerebral aqueduct is at star and dorsal is at the top. Bar = 150 pm.

The innervation patterns described for both WGA-HRP and PHA-L anterogade labeling within PGi must be interpreted with care. The neurons in the PGi are located in a dense matrix of myelinated fibers. As a result, what appears to be moderate anterogade labeling might actually be very dense innervation at the level of individual neurons. Electron microscopy is required to establish the density of synaptic contacts onto neurons in these regions.

Rostra1 ventromedial PAG and SOM projections to the ventrolateral tegmentum PGi-projectingneurons in the rostral ventromedial PAG lie dorsal to the oculomotor nucleus and partially overlap with the supraoculomotor nucleus (SOM). The SOM is a narrow, longitudinal cell column located immediately dorsal to the oculomotor nucleus throughout its length; thus, SOM resides at rostral levels of the PAG. In the cat, cytoarchitectonic studies of the SOM suggest that its constituent neurons are not part of the subjacent oculomotor cell group (Giolli et al., '84, '85, '88; Leichnetz et al., '87). It is also distinguished from the more caudally located dorsal raphe, in that none of its cells contain serotonin (May et al., '87). In the cat, the caudal portion of the SOM was proposed to be preoculomotor in function, based on its projections to the abducens nucleus (Maciewicz et al., '75; May et al., '87) and possibly to the oculomotor nucleus

(Maciewicz et al., '75). This region receives input from the frontal eye field (Leichnetz et al., '871, the superior colliculus (Henkel and Edwards, '78), and medial fastigial nucleus (Beitz, '82; Gonzalo-Ruiz and Leichnetz, '87; May et al., 'go), consistent with a possible role in oculomotor function. PHA-L injections into the rostral ventromedial PAG and the SOM yielded anterograde labeling both in the lateral retrofacial PGi and in the abducens nucleus. It is not yet known whether individual neurons innervate both targets or whether the tracer injection involved two populations of neurons with distinct projections. Our preliminary studies using double retrograde transport from the lateral retrofacial PGi and the abducens nucleus indicate that PGiprojecting and abducens-projecting neurons are interdigitated at certain levels of the SOMPAG, but only few neurons are doubly labeled (Van Bockstaele and AstonJones, unpublished observations). While additional experiments are necessary, these results indicate that the PGiprojecting neurons in the SOMPAG region may not be pre-oculomotorin function. Projections from the SOM/PAG innervate a specific region of the dorsolateral retrofacial PGi immediately ventral to the nucleus ambiguus. Neurons in this target area subserve distinct but related functions in cardiac control and respiration. Neurons immediately ventral to the rostral nucleus ambiguus are part of a longitudinal cell


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D

Fig. 11. Camera lucida plots illustrating the pathway from an injection of Phaseolus uulgaris-leucoagglutinin into the lateral/ ventrolateral quadrant of the periaqueductal gray. These plots are representative of all cases examined with PHA-L injections into the lateraliventrolateral PAG. Plot A represents the most rostral section

and plot G the caudal-most. See abbreviation list for anatomical landmarks. The fibers illustrated in the plots indicate the efferent trajectory of neurons in the lateraliventrolateral PAC to the caudal medulla. The presumptive terminal fields of these projections in the PGi are plotted at higher resolution in Figure 13.


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Fig. 12. A: A photomontage of a darkfield-illuminated coronal section showing both nonvaricose (thick) and varicose (thin) PHA-L fibers in the ventral aspect of the nucleus gigantocellularis and medial PGi following a PHA-L injection into the lateral/ventrolateral PAG. Open arrow indicates ventral brain surface. Closed arrow indicates a PGi region which can be seen at higher magnification in B. Dorsal is at

top and lateral is to the right. Bar = 245 pm. 10,inferior olive, Amh, nucleus ambiguus. B: High-power brightfield photomicrograph of PHA-L fibers in medial PGi from the same field shown in A following an injection into lateral/ventrolateral PAG. Note the numerous varicosities on the fibers in this region. Bar = 75 km.

column of neurons whose discharge is linked wit,h respiration (Feldman, '86; Ellenberger and Feldman, '90). These correspond to the rostral component of the ventral respiratory group, analogous to the Botzinger complex defined in cat (Fedorko and Merrill, '84). In addition, neurons interdigitated with rostral cells of the ventral respiratory group have been implicated in parasympathetic function, primarily as a source of cardioinhibitory vagal preganglionic nerve fibers (Nosaka et al., '79; Sugimoto et al., '79; Hopkins and Armour, '82). These neurons correspond to

cells of the rostral external formation of the nucleus ambiguus as defined by Bieger and Hopkins ('87; see also Ellenberger and Feldman, '90). It is noteworthy that projections from the SOM/PAG do not appear to extend ventrally to the area of C1 adrenergic somata but remain dorsal to selectively innervate the subambiguual portion of the lateral PGi. This anatomical specificity connotes functional specificity as well, as the more ventral PGi area, corresponding to the RVL defined by Ross et al. ('84), is associated with sympathoexcitatory


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Amb

Fig. 13. Camera lucida drawing of the same section as illustrated in Figure 12 showing the different types of fibers present in medial PGi following an injection into the lateraliventrolateral PAG. Fibers outside of the PGi are omitted. Note that the intensity of innervation and

number of varicosities are much greater in the medial aspect of this region. Open arrow indicates ventral brain surface. 10, inferior olive; Amb, nucleus ambiguus.

functions: i) stimulation of this region yields increases in sympathetic nerve discharge and arterial blood pressure (Dampney et al., '82; McAllen et al., '82; Keeler et al., '84; Ross et al., '84; Lovick and Hilton, '85; Guyenet and Brown, '86), ii) lesions or cooling of the C1 area produces marked hypotension (Dampney and Moon, '80; Guertzenstein and Silver, '74), and iii) many neurons in the ventral PGi region project to sympatheticpreganglionicneurons in the intermediolateral cell column (IML) of the spinal cord (Amendt et al., '79; Caverson et al., '83; Milner et al., '88; Ross et al., '81; Tucker and Saper, '85). The ventral Cl/RVL area also differs from the more dorsal subambiguual region in receiving dense innervation from the nucleus tractus solitarius (NTS). In fact, there is marked complimentarity in the innervation of dorsal and ventral retrofacial PGi, from SOM/PAG and NTS, respectively. These specific and complimentary connections are consistent with the role of NTS and Cl/RVL in cardiovascular control (Ross et al., '851, on the one hand, and suggests a role for the SOMPAG in more parasympathetic functions such as cardiac control and respiration. However, few studies have examined the SOM/PAGregion in the rat, and the functions of these neurons are unknown. Our preliminary studies using stimulation of the SOM/PAG in rat have revealed that these neurons may affect respiration (Van

Bockstaele,Akaoka, and Aston-Jones, unpublished observations). Further studies are required to elucidate the role of the SOM/PAG in such processes.

Ventrolateral PAG projections to medial PGi Injections of retrograde tracers into the medial retrofacial PGi yielded robust retrograde labeling in the lateral/ ventrolateral PAG (Van Bockstaele et al., '891, consistent with previous findings (Beitz, '84; Lakos and Basbaum, '88). This was confirmed in the present study using anterograde transport techniques. Injections of PHA-L into the ventrolateral PAG yielded anterograde labeling in medial PGi, nucleus gigantocellularis,and the midline raphe magnus. The present results add to these previous observations in that the projection from the ventrolateral PAG to the PGi is topographically specific. It is well established that stimulation of the ventrolateral PAG elicits analgesia (Gebhart and Toleikis '78; Basbaum and Fields, '84). It has been suggested that this effect may be relayed through medullary nuclei (Behbehani and Fields, '79) as lesions of the nucleus raphe magnus disrupt analgesia produced by stimulation of the ventrolateral PAG (Prieto et al., '83). This effect is distinct from analgesia

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D

F

Fig. 14. Camera lucida plots illustrating the pathway from an injection of Phaseolus uulgaris-leucoagglutinininto the dorsal part of the periaqueductal gray. These plots are representative of all cases examined with PHA-L injections into the dorsal PAG. PlotA represents the most rostral section and plot G the caudal-most. See abbreviation

list for anatomical landmarks. The fibers illustrated in the plots indicate the efferent trajectory of neurons in the dorsal PAG to the caudal medulla. The presumptive terminal fields of these projections in the PGi can be seen at higher magnification in Figure 16.

produced by stimulation of the dorsal PAG (Cannon et al., ’82) which may be conveyed by a separate pathway. In addition, stimulation of the lateraliventrolateral PAG produces blood pressure changes (Carrive et al., ’87, ’89; Bandler et al., in press). Recently, based upon retrograde transport of fluorescent latex microspheres, Carrive et al. (’89) concluded that neurons within functionally distinct regions of the cat midbrain PAG project specifically to the pressor region of C1 neurons in the rostral ventrolateral

medulla, and that these projections may be “involved in the expression of different patterns of emotionally coupled cardiovascular responses” (Carrive et al., ’89).Many of the neurons thought to mediate these effects are restricted to the caudal two thirds of the PAG (Bandler et al., in press). Our results indicate that the rat may resemble the cat in this respect, as ventrolateral PAG neurons retrogradely labeled from PGi were more numerous at caudal than rostral PAG levels.

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Fig. 15. A: A photomontage of a darkfield-illuminated coronal section showing both nonvaricose (thick) and varicose (thin) PHA-L fibers in rostral, juxtafacial PGi (medial to the facial nucleus; VII)from a Phaseolus uulgaris-leucoagglutinin injection into the dorsal PAG. Open arrow indicates ventral brain surface. Closed arrow indicates a PHA-L-labeled fiber which can be seen at higher magnification in B.

Dorsal is at top and lateral is to the right. Bar = 245 km. py, pyramidal tract. B: Brightfield Nomarski photomicrograph illustrating a thin, varicose fiber in rostral PGi from a PHA-L injection into the dorsal PAG. This same fiber can also be observed, at open arrow, in darkfield illumination in A. Bar = 75 km.

However, we find that the projection from ventrolateral PAG to the ventral medulla in the rat predominantly targets the medial aspect of the PGi, while fewer fibers appear to terminate in the lateral PGi pressor region of C1 neurons. The medial PGi contains numerous serotonergic neurons, and there is evidence that these neurons contribute to blood pressure regulation: i) 5-HT neurons in this region project to the IML (Loewy and McKellar, '81; Bowker and Abbott, '90). ii) The increased blood pressure following stimulation of medullary 5-HT neurons (Coote

and MacLeod, '74; Adair et al., '77) can be blocked by serotonergic antagonists (Wing and Chalmers, '74; Howe et al., '83; Mills et al., '88; Lovick, '89). However, cardiovascular effects need not be elicited from a direct projection from 5-HT neurons to sympathetic preganglionic neurons. Other studies have revealed projections from medullary 5-HT neurons to the neighboring nucleus RVL (Hancock, '891, an area which clearly elicits sympathoexcitation in the rat. Hancock showed that 5-HT processes were situated nearby PNMT neurons. Also, Ma-


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VII

Fig. 16. Camera lucida drawing of the same section as in Figure 15 showing the different types of PHA-L-labeled fibers present in the rostral, juxtafacial PGi region of the ventral medulla following an injection into the dorsal PAG. The fibers containing varicosities are located predominantly in the rostral PGi region, medial to the facial

nucleus. Note the long, highly varicose fiber within this region, at arrow, corresponding to the fiber indicated in Figure 15. Small, thicker fibers are present more medially, above the pyramidal tract and resemble fibers of passage cut in cross section. Open arrow indicates ventral brain surface. VII, facial nucleus; py, pyramidal tract.

son and Fields ('89) have shown that medullary midline neurons send collaterals to the lateral medulla. Therefore, whether by a direct or indirect route to IML, neurons in the medial PGi area may be involved in cardiovascular as well as analgesic effects of lateral/ventrolateral PAG stimulation in the rat.

As found for the ventrolateral PAG, stimulation of the dorsal part of the PAG induces analgesia (Fardin et al., '84; Gebhart et al., '84). However, a more prominent effect of dorsal PAG stimulation is strong aversive behavior (Mosset al., '82; Fardin et al., '84) or behavior characterizing the defense reaction (Bandler et al., in press). Many of these behaviors are accompaniedby autonomic changes including increases in blood pressure and heart rate. Lovick recently has suggested that although the dorsal and the ventrolateral parts of the PAG appear to subserve different functions, they may not act independently. In her studies, neurons in the ventrolateral PAG appear to modulate the cardiovascular responses elicited from dorsal PAG stimulation via an interaction in the ventrolateral medulla (Lovick, in press). This is consistent with our anterograde tracing experiments which reveal overlap in the innervation of the PGi from the dorsal and the ventrolateral PAG. We have also found that the juxtafacial PGi projects to the

Dorsal PAG projections to rostral PGi Injections of retrograde tracers centered in rostral, juxtafacial PGi were most effective at labeling neurons in the dorsal PAG (Van Bockstaele et al., '89). PHA-L injections into the dorsal PAG confirmed these findings, yielding anterograde labeling predominantly in the rostral juxtafacia1 PGi and in nucleus raphe magnus. Although dorsal PAG injections of PHA-L and WGA-HRP appear to preferentially innervate midline nuclei, there is overlap in the innervation patterns from dorsal and ventrolateral PAG injections.


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ACKNOWLEDGMENTS retrofacial PGi (Van Bockstaele and Aston-Jones, in press). Therefore, the link between the dorsal and ventrolateral The authors wish to acknowledge the excellent technical PAG may occur via an interneuron in the rostral ventral assistance of Yan Zhu. This work was supported by PHS medulla. grants HL08097, NS20463, NS24698, and DA06214. Dorsal PAG projections to the juxtafacial PGi may influence other processes as well. We (Van Bockstaele and Aston-Jones, in press) and others (Kamiya et al., ’88)have LITERATURE CITED found that a restricted region in the juxtafacial PGi receives prominent innervation from auditory and somatosensory Adair, J.R., B.L. Hamilton, K.A. Scappaticci, C. Helke, and R.A. Gillis (1977) Cardiovascular responses to electrical stimulation of the medullary brainstem nuclei (dorsal column nuclei, the inferior collicuraphe area ofthe cat. Brain Res. 123t141-145. lus and the periolivary pontine reticular formation) and Altman, J., and S.A. Bayer (1981) Development of the brainstem in the rat: projects to corresponding thalamic sensory nuclei (medial V. Thymidine-radiography study of the time of origin of neurons in the geniculate and ventral posterior thalamic nucleus). This midbrain tegmentum. J. Comp. Neurol. 198r677-716. suggests a possible intersection whereby the dorsal PAG Amendt, K., J. Czachurski, K. Dembowsky,and H. Seller (1979) Bulbospinal projections to the intermediolateral cell column: A neuroanatomical may influence and integrate somatosensory and auditory study. J. Autonom. Nerv. Syst. 1t103-117. processes. Functional implications The PAG appears to be a crossroads for circuitry involved in autonomic function and in integrating an animal’s response to threatening stimuli. It is noteworthy that functions represented in the PAG such as cardiovascular regulation and pain modulation are also represented in the ventral medulla, In addition, there is a close relationship between nociception and blood pressure (e.g., nociceptive thresholds are greater in hypertensive patients; Zamir and Shuber, ’80). These effects may be modulated in the rostral ventral medulla, as electrolytic lesions of the rostral medulla that increased the tail flick latencies also blocked pressor responses evoked by PAG stimulation (Lovick, ’85). These results imply that populations of neurons within the rostral medulla may subserve both functions or that separate populations of neurons are closely interdigitated (Siddall and Dampney, ’89). In addition to the above-mentioned functions of the ventrolateral medulla, it should also be noted that this area targeted by rostral ventromedial PAG, ventrolateral PAG, and dorsomedial PAG neurons sends strong projections t o the pontine noradrenergic nucleus locus coeruleus (AstonJones et al., ’86, in press). The LC has been strongly implicated in mechanisms underlying arousal, attention, and vigilance (Aston-Jones et al., ’84; Aston-Jones, ’85; Aston-Jones et al., in press). The PAG then may be indirectly related to arousal mechanisms in addition to its other roles in autonomic, respiratory, and analgesic processes. While the above analysis indicates possible functional relationshipsfrom PAG and PGi, it should be noted that both of these regions are heterogeneous and integrative in nature so that functional interpretations are uncertain at this time. For example, it is difficult to hypothesize the functional significanceof the projection from one subregion of PAG to a group of neurons in the rostral medulla because of the co-mingling of functionally distinct neurons in both of these regions. As discussed by Ellenberger and Feldman (’89), “electrical and chemical stimulation and anatomical tracing studies with target sites in the ventrolateral medulla cannot without stringent and difficult controls provide clearly interpretable results because of the close proximity and incidental inclusion of functionally diverse neurons.” Additional studies of the precise circuits composing and linking these regions will greatly expand our understanding of their functions and relationships.

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Distinct populations of neurons in the ventromedial periaqueductal gray project to the rostral ventral medulla and abducens nucleus.

Glutamate in spinally projecting neurons of the rostral ventral medulla.

Disrupted functional connectivity of periaqueductal gray subregions in episodic migraine.

Depression-like behavior in rat: Involvement of galanin receptor subtype 1 in the ventral periaqueductal gray.

Collateralized projections from neurons in the rostral medulla to the nucleus locus coeruleus, the nucleus of the solitary tract and the periaqueductal gray.

Midbrain periaqueductal gray projections to the dorsomedial medulla in the rabbit.

GABAergic circuitry in the rostral ventral medulla of the rat and its relationship to descending antinociceptive controls.

Inhibition of noradrenergic locus coeruleus neurons by C1 adrenergic cells in the rostral ventral medulla.

Evidence for clonidine presynaptically modulating amino acid release in the rostral ventral medulla: role in hypertension.

Repetitive Electroacupuncture Attenuates Cold-Induced Hypertension through Enkephalin in the Rostral Ventral Lateral Medulla.

Role of the rostral ventrolateral medulla in the generation of synchronized sympathetic rhythmicities in the rat.

The effect of foot-shock on the noxious-evoked activity of neurons in the rostral ventral medulla.

Analysis of morphine-induced changes in the activity of periaqueductal gray neurons in the intact rat.

Longitudinal neuronal organization of defensive reactions in the midbrain periaqueductal gray region of the rat.

Mechanism Underlying the Analgesic Effect Exerted by Endomorphin-1 in the rat Ventrolateral Periaqueductal Gray.

- rat model of Parkinson disease.

Ultrastructure of substance P immunoreactive elements in the periaqueductal gray matter of the rat.

Excitatory amino acid binding sites in the periaqueductal gray of the rat.

Organization of medullary adrenergic and noradrenergic projections to the periaqueductal gray matter in the rat.

Cortically projecting cells in the periaqueductal gray matter of the rat. A retrograde fluorescent tracer study.

The effect of GABA and its antagonists on midbrain periaqueductal gray neurons in the rat.

Projections of anterior hypothalamic neurones to the dorsal and ventral periaqueductal grey in the rat.

Projections from the rostral ventrolateral medulla to brainstem monoamine neurons in the rat.

Responses of single units in the midline medulla to stimulation of the rostral ventrolateral medulla.