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The organization of afferent projections to the midbrain periaqueductal gray of the rat
Neur**
D306-4522~82/010133-27503.00!0 Pergamon Press Ltd 0 1982 IBRO
I. pp. t33 to 159. 1982
THE ORGANIZATION OF AFFERENT PROJECTIONS TO THE MIDBRAIN PERIAQUE~UCTAL GRAY OF THE RAT A. J. BEITZ Department of Anatomy, University of South Carolina, School of Medicine, Columbia, South Carolina 29028, U.S.A. Abstract-The retrograde transport technique was utilized in the present study to investigate the afferent projections to the ~riaqueductai gray of the rat. Iontophoretic injections of horseradish peroxidase were made into the periaqueductai gray of 22 experimental animals and into regions adjacent to the periaqueductai gray in 6 control animals. Utilization of the retrograde transport method permitted a quantitative analysis of the afferent projections not only to the entire periaqueductai gray, but also to each of its four intrinsic subdivisions. The largest cortical input to this midbrain region arises from areas 24 and 32 in the medial prefrontal cortex. The basal forebrain provides a significant input to the periaqueductal gray and this arises predominantly from the ipsilateral lateral and medial preoptic areas and from the horizontal limb of the diagonal band of Broca. The hypothalamus was found to provide the largest descending input to the central gray. Numerous labeled ceils occurred in the ventromedial hypothalamic nucleus, the lateral hypothalamic area, the posterior hypothalamic area, the anterior hypothalamic area, the perifornicai nucleus and the area of the tuber cinereum. The largest mesencephaiic input to the periaqueductai gray arises from the nucleus cuneiformis and the substantia nigra. The periaqueductal gray was found to have numerous intrinsic connections and contained a significant number of labeled cells both above and below the injection site in each case. Other structures containing significant label in the midbrain and isthmus region included the nucleus subcuneiformis~ the ventral tegmentai area, the locus coeruieus and the parabrachiai nuclei. The meduliary and pontine reticular formation provide the largest input to the periaqueductai gray from the lower brain stem. The midline raphe magnus and superior central nucleus also supply a significant fiber projection to the central gray. Both the trigeminal complex and the spinal cord provide a minor input to this region of the midbrain. The sources of afferent projections to the periaqueductai gray are extensive and allow this midbrain region to be influenced by motor, sensory and limbic structures. In addition, evidence is provided which indicates that the four subdivisions of the central gray receive differential projections from the brain stem as well as from higher brain structures.
The mesencephaiic periaqueductal gray (PAG) or central gray is formed by a mantle of cells which surround the cerebral aqueduct. The rodent periaqueductal gray has recently been shown to consist of four anatomical subdivisions3,49 (1) a medial subdivision which is composed of small, primarily bipolar neurons which closely surround the cerebral aqueduct; (2) a ventrolateral division which contains predominantly large fusiform neurons, large multipolar neurons and small bipolar neurons, the majority of these cells are oriented at a lW150” angle with respect to the aqueduct; (3) a dorsoiateral division comprised predominantly of triangular-shaped cells, small multipolar neurons and small bipolar neurons, the majority of which are oriented at a 30-70” angle with respect to the cerebral aqueduct; and (4) a dorsal subdivision containing primarily small bipolar and multipolar neurons which show no orientational preference. This division lies immediately dorsal to the cerebral aqueduct. The PAG has received a great deal of attention within the past decade because of its unique role in Abbreviations:
mesencephaiic benzidine.
both stimulation and chemically-induced analgesia. Since the initial observation that stimulation of this in rats produced profound analgesia5’ region numerous laboratories have demonstrated that the PAG is an important site for stimulation-produced analgesia33.36,54 as well as a central site for morphine’s antinociceptive action.26,33+4s Although the central gray has attracted much attention because of its role in analgesia_ it is nonetheless involved in several other functions. It appears to be involved in vocalization,” rage reactions,36 control of reproductive behavior” and pressor responses.59 The role played by the PAG in the above functions is dependent on the interconnections of this midbrain area with other regions of the central nervous system (CNS). This has recently been clearly demonstrated by Sakuma & Pfaff5’ with regard to the PAG’s involvement in reproductive function. Although the efferent projections of the PAG have been studied in some detail’O~ss the afferent connections of this region have been largely neglected. Grofova, Ottersen & Rinvik” studied mesencephalic and diencephalic projections to dorsal portions of the PAG, but apart from this investigation very little work has been done on the afferent projection system to the central gray. The present investigation was designed to construct a
HRP, horseradish peroxidase; PAG, periaqueductai gray; TMB, tetramethyi133
134 detailed
A. J. Beitz map
of the CNS
regions
which
supply
input
to the PAG
utilizing the retrograde transport technique. In addition to mapping the sources of input to the central gray as a whole, the origin of the specific projections to each of the four PAG subdivisions described above were analyzed.
EXPERIMENTAL
PROCEDURES
Sources of afferent projections to the midbrain periaqueductal gray were studied in 22 male Sprague-Dawley rats by means of the retrograde transport of horseradish peroxidase (HRP). Each rat was anesthetized with chloral hydrate (0.35 g/kg ip.) and a small deposit of HRP (Sigma type VI) was placed sterotaxically into the PAC. The HRP was delivered by microelectrophoresis via a glass micropipette filled with a 13:< solution of HRP in 0.05 M tris buffer (pH 7.6). The size of the micropipette tip was in the 10-25 pm diameter range to minimize the amount of retrograde labeling following the uptake of HRP by fibers of passage. *’ The driving force for the microelectrophoretic injections was supplied by a 1.8 pa positive current delivered by a constant current source (Midgard Electronics, Newton, Massachusetts) at a pulse rate of 7 s on, 7 s OR. for a duration of 615 min. The animals were killed 2436 hours postoperatively by intracardiac perfusion. under anesthesia, with 500 ml of a O.SO:, paraformaldehyde-2”;, glutaraldehyde fixative made up in 0.12 M phosphate buffer followed by 500 ml of 0.12 M phosphate buffer containing 506 sucrose and 5% polyethylene glycol. The brains were removed, placed in the same 5”” sucroese~-5p, polyethylene glycol solution for an additional 30 min, and subsequently cut in the coronal plane on a freezing microtome at 40 ilrn. Sections through the entire brain and spinal cord were collected and reacted with tetramethylbenzidine (TMB) and hydrogen peroxide according to the procedure described by Mesulam3’ with the following modifications. (I) The 20min incubation time in both the pre-reaction soak solution (a mixture of Mesulam’s solution A and B) and the enzymatic reaction solution (a mixture of solution A and B to which H202 was added) was reduced to 12 min and 10 min. respectively. This reduction in incubation time was found to reduce significantly the number of filamentous or crystalline artifacts present on tissue sections without significantly affecting neuronal labeling. (2) The improved stabilization procedure recently developed by Adams’ was employed rather than stabilization in a chilled ethanolic solution of sodium nitroferricyanide as Mesulam”s originally recommended. This procedure utilizes methyl salicylate to both clear the tissue and stabilize the TMB reaction product. (3) The tissue sections were stained with a 1”” solution of pyronin-Y (Eastman Kodak) in acetate buffer (pH 3.3). The use of this particular stain has several advantages. It allows the staining process to be performed at the same pH and in the same buffer as the initial TMB reaction, which increases the stability of the reaction product. In addition, it enhances tissue contrast without decreasing the visibility of the TMB reaction product and is thus superior to neutral red in this regard. Every second section through the cerebrum. diencephaIon and brain stem was examined in each experimental animal for the presence of retrogradely-labeled neurons containing dark blue granules throughout their cytoplasm. In addition. every fifth section through the spinal cord of
five experimental animals was scanned for HRP-positive neurons. In order to quantitate the afferent contribution from the cortex and basal forebrain and from diencephalic, brain stem and spinal cord nuclei to the PAG, counts of the total number of labeled perikarya were made on each of the sections examined from these regions for each case. Counts were made under bright-field illumination at a magnification of 100 x In cases where it was uncertain if a neuron contained HRP-reaction product at the initial magnification of 100 x . the cell was reexamined at a magnification of 400x to ascertain if it contained dark blue HRPpositive granules throughout its cytoplasm. The above quantitation procedure allowed us to determine the relative contribution of afferent input from nuclei within the diencephalon and brain stem to the entire PAG as well as to its intrinsic subdivisions. In addition to a quantitative analysis of the number of labeled cells in each case. the location of labeled neurons were carefully plotted onto drawings of selected sections through each region. This allowed an accurate determination of the location of HRPpositive neurons within diencephalic. basal forebrain and brain stem nuclear groups. One of the major problems encountered in analyzing retrograde transport studies utilizing HRP as the retrograde marker is determining the size of the injection site.3’ In an attempt to determine quantitatively the size of the injection site, sections through the injection site were analyzed in several of our experimental cases on a computerized image processing system. This system consists of a spatial data systems image digitizer. a colorado video memory, and a comtal vision one interactive color display and real-time processor. A dedicated PDP-I I./O3 performs real-time image analysis functions in a multiprocessing mode with a DEC PDP-8e. This system can convert photometric data from the stained brain sections mto digital form, providing discrimination of up to 256 density levels. A hard copy character representation of optical density bins throughout the section is produced on a line printer (see Fig. 1) and the precise extent of the HRP injection size is subsequently analyzed on each print out. In addition to the 22 animals in which HRP injections were made into the PAG, an additional 6 animals were used as controls. The brains from these 6 cases were processed in an identical manner to that described above for experimental animals. In 2 of these cases (R-179 and R-180), iontophoretic HRP injections were made into nucleus cuneiformis on one side. Control HRP injections were also made into the dorsal raphe nucleus (R-58). the superior colliculus (R-27 & R-66) and the cerebral aqueduct. The nucleus cuneiformis parallels the ventrolateral aspect of the PAG throughout most of its rostra-caudal extent and the dorsal raphe nucleus is embedded in the ventrocaudal portion of the central gray. Because of their close proximity to the PAG they were often involved in the outer fringe of our PAG Injection sites. Injections of HRP were thus made into these two nuclei as a means of comparing the resultant labeling with the results of PAG injections. Likewise, the results of superior colliculus injections were compared to dorsally-placed PAG injections. Finally. an injection was made into the cerebral aqueduct to determine if any neuronal labeling occurred in the CNS as a result of leakage into the ventricular system.
Two possible sources of artifacts should be considered evaluating the present results: (1) cells labeled by spread
in of
Afferent
projections
to the periaqueductal
HRP to regions adjacent to the PAG, and (2) cells labeled as a result of uptake by injured fibers passing through the midbrain PAG. If we first consider the possibility of labeling occurring due to uptake and subsequent retrograde transport of HRP from neighboring PAG areas, this source of artifact can probably be ruled out in 6 of our cases (R-36, R-37, R-45, R-49, R-57 and R-53) since the visible HRP reaction product was confined to the midbrain central gray in each case. In an additional 5 cases (R-33, R-38, R-43, R-51 and R-59) the core of the HRP reaction was confined to the PAG. Although there was some spread of HRP into surrounding areas in these cases, recent evidence has shown that the area of effective uptake of HRP is restricted to tissue very close to the pipette tip.48.65 Since both the micropipette tip and the dense core of the HRP reaction was localized to the PAG in these 5 animals, the above finding would argue against uptake and subsequent transport of the enzyme by synaptic regions outside the PAG in these cases. The technique of iontophoresis of HRP into CNS sites will not only label neuron cell bodies by the physiological process of uptake and subsequent retrograde transport of the enzyme from axon terminals, but will also label cells whose axons pass through the site of injection and are damaged by the injection procedure.22S48 Thus, although there are advantages in using the iontophoretic technique, in that extremely small deposits of HRP can be made, there is still a risk of labeling neurons ciu uptake or by injection into damaged fibers of passage, as had been earlier noticed in cases where pressure injections of HRP were made into the CNS.Z’,34.66.67 Differentiation between neurons labeled by terminal uptake and those from damaged axons of passage is not possible utilizing the retrograde transport technique at the light-microscopic level. Only anterograde tracing techniques in conjunction with electron microscopy or electrophysiological techniques can definitely establish the presence of axon terminations in regions such as the PAG where fibers are passing through to other CNS areas. Although the PAG is an area traversed by many fiber tracts of different origin,12 the use of extremely small pipette tips and small amounts of current in the present study would argue against a major amount of uptake of HRP by injured fibers of passage.
RESULTS
Description of horseradish peroxidase injection sites The HRP deposits in 6 of the 22 rats which received injections into the midbrain PAG were restricted to the central gray. The limited spread of HRP in these 6 animals was initially determined under microscopic examination at a magnification of 40 x and subsequently confirmed utilizing the computer-assisted image processing system. In an additional 5 animals the core of the HRP injection site was confined to the PAG, but faint reaction product was evident in adjacent structures, notably the superior colliculus, the dorsal raphe nucleus or the nucleus cuneiformis. In the remaining 11 animals the core HRP reaction product spread beyond the PAG dorsally into the overlying inferior or superior colliculus, laterally into the nucleus cuneiformis or ventrally into the tegmentum.
gray
135
Because of the HRP injection site was confined to the PAG in cases R-36, R-37, R-45, R-49, R-53 and R-57 these cases will be utilized throughout the rest of this paper to demonstrate the afferent input to the PAG. Moreover, in cases R-33, R-38, R-43, R-51 and R-59 the dense core of the HRP reaction product was confined to the PAG. Since there was limited spread of HRP in these cases they will also be used in the discussion of afferents to the midbrain central gray. In addition to the above cases, one other case (R-50) will be included to demonstrate the afferents to the caudal part of the ventrolateral subdivision of the PAG. As indicated in Table 1, a portion of the injection site from this animal involved the dorsal raphe nucleus which is almost unavoidable at this ventral and cauda1 level of the central gray. Finally, in one of our cases (R-38) the dark core of the HRP injection site covered much of the dorso-ventral extent of the PAG and nearly encompassed its entire rostrocaudal length (Fig. 3). Because of the large size and good confinement of the iontophoretic deposit in this case (only faint reaction product was evident in the overlying deep layer of the superior colliculus), it will be utilized to illustrate the broad spectrum of afferent input to the PAG. The location and size of the HRP injection site for the experimental cases discussed above and for the 6 control animals are listed in Table 1 and representative examples are shown in Figs 2 & 3.
Sources of qfierent input to the periaqueductal gray As indicated above, case R-38 will be used to summarize the nuclear groups within the forebrain, diencephalon, brain stem and spinal cord which project to the midbrain central gray. Over 60 different structures were found to project from these regions to the PAG. The areas containing label are listed in Tables 2, 3 and 4 (Case R-38) together with their laterality and the total number of labeled cells in each. The location of most of these structures is illustrated in Figs 4-9. Each projecting structure was identified in at least two experiments. The following commentary supplies supplementary information on the distribution of labeled cells in selected areas. Forebrain. The largest number of labeled cells in the basal forebrain region were in the preoptic area and in the horizontal limb of the diagonal band of Broca (Figs 4 and 10). Numerous cells were also found in cortical areas 24 and 32 as defined by Krieg.30” An analysis of the results of smaller HRP placements (R-51 and R-59) indicate that the projection from this region of the cerebral cortex is limited to ventral and ventrolateral portions of the PAG (Fig. 5). In several of our experimental cases with large injections in which the HRP reaction product spread outside the PAG (R-26, R-28, R-35 and R-46) labeling occurred in the nucleus accumbens, area 4. area 14 and area 29. The only cases with restricted injection sites in which labeling could be demonstrated in these regions were R-50 and R-51.
A. J. Beitz
136
Fig. 1. A. Low power photomicrograph of a coronal section through the rostra1 midbrain from case R-59. The HRP injection site (arrow) involves the ventral and ventrolateral portions of the periaqueductal gray (PAG). 40 pm, pyronin-Y, mag. = IS x B. A hardcopy computer image of the same section shown in 1A. The numbers and letters on the print out are numerical representations of density bins throughout the section. The greatest density is represented by the numbers l--9, while decreasing units of density are indicated by the letters A-W. The central core of the HRP reaction product (represented by the numbers 1-9) is encircled with a solid black line (arrow). The outer fringe of the injection site (containing faint reaction product in 1A) is represented by the letters A-G and is enclosed with a dotted line. The remainder of the section outside the HRP deposit is represented by the letters H-W except for areas that have stained intensely with pyronin-Y. Thus the superior colliculus (SC) and the interpeduncular nucleus (ip) contain letters in the D--G range but are clearly not part of the injection site. C. The section shown in 1A was projected onto the computer print out depicted in 1B and a tracing of the iniection site was subseauentlv made onto the projected image. The resulting tracing is illustrated in IC depicting the core of the injection site (b&k) and the surrounding spread of HRP (stipple). _I
Abbreviations a,
ag, aha. apl. apm, c, CA, CC, ces, Cl. cm, CP. cu, dpb. dpm, DSCP. F. FR. g, ge, gP, hldb. HRP. IC. ice. icp. io. ip, iVv. ip. 1, lgd. lgr. Iha, LM, lP, Iv, md, mg. MLF. MP, MT. mv. nb. ni,
used on figures
arcuate nucleus cerebral aqueduct anterior hypothalamic area lateral preoptic area medial preoptic area nucleus cuneatus anterior commissure corpus callosum nucleus centralis superioris nucleus cuneatus lateralis centromedial nucleus posterior commissure nucleus cuneiformis nucleus dorsalis parabrachialis dorsal premamillary nucleus decussation of superior cerebellar peduncle fornix fasciculus retroflexus nucleus supra geniculatus nucleus gelatinosus globus pallidus horizontal limb of the diagonal band horseradish peroxidase internal capsule nucleus centralis colliculus inferioris nucleus pericentralis colliculus inferioris inferior olive nucleus interpeduncularis fourth ventricle nucleus lateralis posterior thalami nucleus lateralis thalami nucleus dorsalis corporis genicalati lateralis nucleus ventralis corporis geniculati lateralis lateral hypothalamic area medial lemniscus nucleus lateralis posterior thalami nucleus vestibularis lateralis nucleus dorsalis medialis thalami medial geniculate body medial longitudinal fasciculus mamillary peduncle mamillothalamic tract nucleus vestibularis medialis nucleus basalis (magnocellular preoptic nucleus) nucleus interpositus
Pt, Pv, rd,
nucleus of the lateral olfactory tract nucleus medialis nucleus lateralis optic chiasm optic tract periaqueductal gray posterior hypothalamus parafascicular nucleus substantia nigra pars lateralis pyramidal tract nucleus paratenialis nucleus parvocellularis hypothalamic subnucleus reticularis dorsalis medulla
re. rgc, rgca, rh, rm. rn, rpc, rpca, rpo. rpt. rv,
gata nucleus reuniens nucleus reticularis gigantocellularis nucleus reticularis gigantocellularis pars a nucleus rhomboideus nucleus raphe magnus red nucleus nucleus reticularis pontis caudalis nucleus reticularis pontis caudalis pars a nucleus reticularis pontis oralis nucleus reticularis tegmenti pontis subnucleus reticularis ventralis medulla oblon-
nlot, nm. nl, oc. OT, PAG, ph. Pf. PP. PT,
SC. scu, sg. sl, SM, snc, snr, snl. so, sol, st, tag, tc, tp. v. vmh, vpm vta, zi.
gata superior colliculus nucleus subcuneiformis nucleus sagulum nucleus septi lateralis stria medullaris substantia nigra pars compacta substantia nigra pars reticulata substantia nigra pars lateralis nucleus supraopticus nucleus solitarius nucleus of the stria terminalis nucleus triangularis area of the tuber cinerium nucleus trapezoideus ventral thalamic complex nucleus ventromediahs nucleus ventralis premamillaris ventral tegmental area zona incerta
oblon-
137
A
R-36
R-38 138
Atrerent
projections
to the periaqueductal
139
gray
Table 1. The size and location of the horseradish peroxidase injection site and the animal survival time for the twelve experimental cases and six control cases discussed in the text
Case Number (1) R-33 (2) R-36 (3) R-37 (4) R-38 (5) R-43 (6) R-45 (7) R-49 (8) R-50
(9) R-51
(10) R-53 (11) R-51 (12) R-59 (13) R-27 (14) (15) (16) (17) (18)
R-54 R-58 R-66 R-179 R-180
Location of injection site Medial PAG subdivision Lateral PAG Dorsolateral PAG subdivision (caudal part) Entire PAG, deep layer of Superior colliculus Dorsal PAG subdivision (caudal part) Dorsolateral PAG subdivision (rostra1 part) Dorsolateral PAG subdivision (caudal part) Ventrolateral PAG subdivision (caudal part), lateral portion of dorsal raphe N. Ventrolateral PAG subdivision (rostra1 part), rostra1 most portion of dorsal raphe N. Dorsolateral PAG subdivision (rostra1 part) Rostroventral PAG Ventrolateral PAG subdivision (rostra1 part) Deep layer of superior colliculus Cerebral aqueduct Dorsal raphe nucleus Superior colliculus Nucleus cuneiformis Nucleus cuneiformis
Hypothalamus. The hypothalamus will be treated separately from the remainder of the diencephalon because it provides a substantial input to the PAG. The lateral hypothalamus is heavily labeled following iontophoretic deposits of the tracer enzyme into ventral (R-51) and ventrolateral portions (R-50 & R59) of the PAG, while the ventromedial hypothalamic nucleus (VMN) contains numerous marked cells following dorsolateral PAG injections (R-45 and R-49). A careful analysis of the location of labeled cells within the VMN indicated a topographical organization in this projection. Thus, the majority of neurons containing HRP reaction product occur in dorsomedial portions of the nucleus following HRP deposits into dorsal portions of the PAG (Fig. 6) while more ven-
Diameter of injection site (mm)
Longitudinal extent of injection site (mm)
Animal survival time (h)
0.32 0.36 0.21
0.90 0.70 0.50
31 29 29
0.96
2.40
32
0.40
1.60
28
0.80
1.00
28
0.55
1.90
24
0.91
1.50
28
1.28
2.00
32
0.38
0.90
30
0.81 0.55
1.90 1.20
30 34
0.80
1.70
28
None 0.88 2.2 1.29 1.05
None 1.50 3.10 2.10 2.40
30 28 30 36 36
trally-placed PAG injections result in heavier labeling in ventrolateral portions of the nucleus (Figs 5 and 10). The topography of lateral hypothalamic projections to the PAG was not as clear as VMN projections, however, there was a tendency for more dorsally-placed HRP deposits to label more lateral portions of this hypothalamic area. Both the anterior hypothalamic area and the posterior hypothalamic area contained significant label following iontophoretic injections into the central gray. Marked cells were found to predominate in cauda1 and lateral portions of the anterior hypothalamic area. The lateral portion of this hypothalamic region (the lateral anterior nucleus of Bleier, Cohn & Siggelkow6) was especially heavily labeled in case R-59. The
Fig. 2. Photomicrographs through the center of the horseradish peroxidase injection site from cases R-36, R-37 and R-38. A. Photomicrograph of a coronal section through the midbrain depicting the HRP deposit (arrow) in case R-36. 40 pm, Pyronin-Y, mag. = 12 x B. A tracing of the stained section shown in A illustrating the core (black) and surrounding spread (dots) of the injection site. The periaqueductal gray is indicated by the diagonal lines. C. A photomicrograph of a coronal section through the caudal midbrain depicting the HRP deposit (arrow) in case R-37. 40 pm, Pyronin-Y, mag. = 12 x D. Tracing of the stained section shown in C illustrating the extent of the HRP reaction product in case R-37. E. A photomicrograph of a coronal section through the midbrain depicting the HRP deposit (arrow) in case R-38. 40 pm, Pyronin-Y, mag. = 12 x F. This tracing diagram illustrates the full extent of the injection site in case R-38. Sagittal reconstructions of the injection sites from the three cases illustrated in this figure are shown in Fig. 3.
N\<
7 I
H
140
A. J. Beitz
d
d
c+r V
c+r V
R-36
R-37
rostra1
R-38 d
c+r V
d
R-59 Fig. 3. Sagittal reconstructions of the periaqueductal gray through the center of the horseradish peroxidase injection site for cases R-36, R-37, R-38. R-45 and R-59. The PAG is depicted by the diagonal lines. The core of the injection site in each case is indicated in black, while the outer fringe of the injection site is illustrated with stipple. Each drawing illustrates the rostrocaudal extent of the HRP deposit for the above cases. The PAG was reconstructed at a point 0.76 mm from the midline for case R-36 while the center of the injection site was determined to be 0.62 mm, 0.48 mm, 0.43 mm and 0.33 mm from the midline for cases R-37. R-38, R-45 and R-59, respectively. The rostra1 (r). caudal (c), dorsal (d) and ventral (v) directions are indicated.
cells in the posterior hypothalamic area were also predominantly localized to lateral parts of this region and were often found along the medial aspect of the mamillothalamic tract. The perifornical nucleus contained a relatively large number of marked cells following injections of HRP into the retrogradely-filled
ventrolateral subdivision of the central gray but contained no label following dorsolateral placements of the enzyme. These neurons were localized in a region immediately dorsal and medial to the fibers of the fornix. In addition to the hypothalamic regions listed in
group
1. 2. 3. 4.
Zona incerta Ventral lateral geniculate Lat. habenular n. Ant. pretectal n.
n.
Ant. hypothalamic a. Lat. hypothalamic a. Ventromedial hypothalamic n. Dorsomedial hypothalamic n. Post. hypothalamic a. Infundibular n. Periventricular hypothalamic n. Dorsal premamillary n. A. of the tuber cinerium Perifornical n. Med tuberal a. Lat. tuberal a. Dorsal hypothal. a.
Diencephalon
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Hypothalamus
1. Prefrontal cortex 2. Cingulate gyrus 3. Diagonal band Broca horiz. limb 4. Med. Preoptic A. 5. Lat. preoptic A. 6. Bed n. of stria terminalis 7. Magnocellular preoptic N. 8. Subst. innominata 9. Central amygdala
Forebrain
Nuclear
40 0 0 13
23 31 36 7 7 5 3 13 5 3 4 1 6
20 6 17 28 50 4 17 5 3
Ipsi
2 0 0 4
0 0 2 0 0 0 0 0 0 0 1 0 0
Contra
R-38 Entire right half of PAG
20 3 0 4
0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 2 2 0 0 0 0
Ipsi
0. 0 0 2
0 0 0 0 0 3 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0
Contra
R-43 Dorsal PAG subdivision (Caudal part)
23 3 0 0
2 3 36 0 3 0 0 3 3 0 3 0 0
0 0 0 0 0 0 0 0 0
Ipsi
2 0 0 12
1 1 0 0 2 0 0 0 0 0 0 0 0
Contra
R-45 Dorsolateral subdivision (Rostra1 Part
73 0 0 5
7 0 0 2
5 0 17 3 0 0 0 4 0 0 0 0 0
z”
i?
18 38 104 2 26 0 0 81 6 0 2 0 4
:m
Contra
gLo
Ipsi
R-49 Dorsolateral subdivision (Caudal part)
12 0 48 0
3 75 2 3 17 2 0 1 5 12 11 3 8
92 5 60 7 1
37 16 26 34
Ipsi
1 0 33 2
2 0 0 4 5 3 1
0
3 37 1 1 3
23 0 26 1 0
11 1 14 9
Contra
R-50 Ventrolateral subdivision (Caudal part)
18 5 0 0
27 123 3 1 12 4 0 3 32 16 5 2 19
39 24 2 31 27 10 5 0 1
Ipsi
3 0 3 0
3 31 0 0 0 0 0 0 5 1 1 1 1
8 4 0 3 5 0 3 0 0
Contra
R-59 Ventrolateral subdivision (Rostra1 part)
Table 2. A summary of the distribution of horseradish peroxidase labeled neurons in the forebrain, hypothalamus and diencephalon following iontophoretic injections of horseradish peroxidase which encompassed the entire periaqueductal gray on one side or which were confined to particular periaqueductal gray subdivisions. The case number and the horseradish peroxidase injection site are listed at the top of the table and the nuclear groups within the forebrain, hypothalamus and diencephalon which contained labeled neurons are indicated on the left. The laterality (ipsi = ipsilateral, contra = contralateral) of labeling for each nuclear group is also indicated
group
1. 2. 3. 4. 5. 6. I. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
Sup. colliculus (deep layer) Cuneiform n. Subcuneiform n. Substantia nigra pars compacta Substantia nigra pars lateralis Substantia nigra pars reticulata Parabigeminal n. Periparabigeminal a. Paralemniscal a. N. of the brachium of the inf coil Inf. colliculus external n. Locus coeruleus Raphe dorsalis Lat. parabrachial n. Med. parabrachial n. PAG (rostra1 to injection site) PAG (at injection) PAG (caud. to injection) Pontopeduncular n. Vent. tegmental a.
Midbrain & isrhmus
Nuclear
105 3 0 4 4 9 0 0 0 0 3 0 14 3 0 40 7 5 2
1
Ipsi 2 21 0 0 0 2 0 0 10 0 0 0 2 7 0 0 21 3 0 0
Contra
R-38 Entire right half of PAG
83 0 0 4 0 0 0 10 0 0 0 0 2 0 10 30 30 4 0
0
Ipsi 0 60 0 0 5 0 0 0 8 0 0 0 0 2 0 4 27 10 3 0
Contra
R-43 Dorsal PAG subdivision (Caudal part)
10 36 5 0 0 5 0 0 0 0 0 0 0 2 4 0 13 27 0 0
Ipsi 4 34 19 0 4 2 0 0 2 0 0 4 0 0 3 0 12 8 0 2
Contra
R-45 Dorsolateral subdivision (Rostra1 part)
4 152 3 0 20 2 3 28 2 2 11 3 2 0 0 33 8 32 0 0
Ipsi
IO 0 0 5 0 2 12 2 0 0 0 0 0 0 3 39 12 0 0
0
Contra
R-49 Dorsolateral subdivision (Caudal part)
0 43 2 I 2 4-l 0 0 0 0 0 3 25 18 20 22 20 0 9 10
Ipsi
11 30 0 5 3 38 3 0 11 0 2 4 20 9 9 23 57 0 4 10
Contra
R-SO Ventrolateral subdivision (Caudal part)
0 4 12 8 0 4
0 0
0
0 0
1 0 0 0
0
1 11 12 0 4
14 7 0 0
Contra 11 32 8 0 3 2 0 0 0 1 0 0 4 3 6
Ipsi
R-59 Ventrolateral subdivision (Rostra1 part)
Table 3. A summary of the distribution of horseradish peroxidase labeled neurons, in the midbrain and isthmus region following iontophoretic injections of horseradish peroxidase into the periaqueductal gray and its intrinsic subdivisions. The case number and the location of the horseradish peroxidase injection site are listed at the top of the table and the nuclear groups within the midbrain and isthmus region which contained labeled cells are indicated on the left. The laterality (ipsi = ipsilateral, contra = contralateral) of the labeling for each nuclear group is indicated
K z
? LI
N. pontis oralis N. pontis caudalis N. pontis caudalis pars a Reticulotegmental n. Sup. central n. Raphe magnus Vent. n. of lat lemniscus N. lat. dorsalis tegmenti Spinal n. of V N. suprageniculatus
N. N. N. N. N. N. N. N.
gigantocellularis paragigantocellularis gigantocell pars a prepositus hypoglossi cuneatus ret. medulla oblongata pars ventralis ret. medulla oblongata pars dorsalis solitarius
1. Dorsal horn 2. Ventral horn
Spinal cord
1. N. medialis 2. N. interpositus 3. N. lateralis
Cerebellum
1. 2. 3. 4. 5. 6. 7. 8.
Medulla
1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
Pons
Nuclear group
Table 4. A summation of the distribution
0 0
0 0 2
2 0 2 2 1 7 2 2
7 8 7 0 3 5 0 0 0 0
0 0
0 4 0
0 2 3 7 0 0 0 0
0 2 4 0 0 0 0 0 2 0
0 0 0
0 0 0 0 0 0 0 0
3 2 0 0 0 2 2 0 0 0
0 2 0
0 2 2 0 0 2 0 0
0 0 7 0 0 0 0 0 0 0
Contra
Ipsi
Ipsi
Contra
R-43 Dorsal PAG subdivision (Caudal part)
R-38 Entire right half of PAG
-
0 0 0
0 0 0 0 0 0 0 0
13 2 2 0 2 0 0 0 0 0
Ipsi
0 2 4
0 0 0 0 0 0 0 0
5 5 2 4 0 0 0 0 0 0
Contra
R-45 Dorsolateral subdivision (Rostra1 part)
0 0
0 0 0
0 0 0 0 0 2 0 0
2 3 3 0 0 0 1 0 0 0
Ipsi
0 0
0 4 0
0 0 0 0 0 0 0 0
3 0 0 0 0 0 2 0 4 0
Contra
R-49 Dorsolateral subdivision (Caudal part)
0 0
7 0 0
0 7 0 12 0 3 0 4
35 0 4 3 17 16 0 4 0 2
Ipsi
4 0
0 4 0
0 2 0 5 0 0 0 0
21 5 2 2 50 0 0 16 0 6
Contra
R-SO Ventrolateral subdivision (Caudal part)
injections of
0 0
1 0 0
3 2 2 2 0 5 3 0
8 7 0 0 2 3 0 7 0 2
Ipsi
2 0
B
g 6
0 8 2 0
0 2
5 0
% g B a a %. 2.
5 12
Contra
R-59 Ventrolateral subdivision (Rostra1 part)
of horseradish peroxidase labeled neurons, in the pons, medulla, cerebellum and spinal cord following iontophoretic horseradish peroxidase into the periaqueductal gray and its intrinsic subdivisions
Fig. 4. A charting diagram illustrating the location of labeled cells (black dots) in a series of coronal sections through the forebrain and dicncephalon from case R-38. The most rostra1 section is illustrated in A, while the most caudal section is shown in G. The injection site is illustrated in F and G. Note that the majority of labeled cells occur ipsilateral to the injection site. The abbreviations are indicated on p. 136. 144
Afferent projections to the periaqu~u~tal
gray
Fig. 5. A charting diagram illustrating the location of labeled cells (black dots) in a series of coronal sections through the forebrain and diencephalon following an injection of horseradish peroxidase into the dorsolateral periaqueductal gray (case R-45).
146
A. J. Beitz
Fig. 6. A charting diagram illustrating the location of labeled ceils (black dots) in a series of coronal sections
through
the forebrain
and diencephalon following a horseradish ventral periaqueductal gray (case R-51).
Table 2, there were a few hypothalamic
nuclei
which
in cases R-51 and R-59. The ipsilateral submamillothalamic nucleus contained 13 labeled cells in case R-59 and 8 marked cells in case R-51. This nucleus was not labeled following injections into dorsal, dorsolateral or medial portions of the central gray. The dorsal tuberal nucleus contained 7 retrogradely-marked cells in case R-59 and only 3 cells in R-51. Other nuclear groups labeled in these 2 cases include the parvocellular nucleus, and tubera) magnocellular nucleus,
contained
a small
number
of labeled
cells
peroxidase
injection
into the
the ventral premamillary nucleus and the paraventricular nucleus. Di~~cephtrlon. Next to the hypothalamus, the subthalamus provides the largest diencephalic input to the PAG. This input arises from the zona incerta which contained labeled cells in the majority of our cases (Fig. 4). Marked neurons were very prominent throughout the zona incerta following injections into dorsal and dorsolateral portions of the PAG (see Table 2). HRP injections involving the ventral and ventrolateral parts of the central gray resulted in only
Afferent projections to the periaqueductaf gray
Fig. 7. A charting diagram illustrating the location of horseradish peroxidase-labeled cells (black dots) in a series of representative coronal sections through the brain stem from case R-38. The midbraindiencephalic junction is illustrated in A, while the medullary-spinal cord junction is depicted in H. The core of the HRP injection site is represented in B and C. Note the predominance of labeled neurons ipsilateral to the injection site. The abbreviations are indicated on p. 136. a small amount of label in this subthalamic region. This projection appeared to be topographically organized in that dorsal PAG injections labeled more medial portions of the zona incerta than did ventral PAG injections. The ventral lateral geniculate nucleus and the Iateral habenular nucleus contained significant Iabel following ventrally placed HRP deposits in the PAG, while the anterior pretectal nucleus was most heavily labeled with dorsally-placed HRP deposits. Although not indicated in Table 2 a few labeled cells were ob-
served in the thalamic parafasicular nucleus in two of our cases (R-50 and R-51) and in an area just medial to the parafasicular nucleus in three of our cases (R-49, R-SO and R-51). This region adjacent to the parafasicular nucleus appears to correspond to the A-l 1 catecholamine group of Dahlstrom & Fuxe.r3 ~i~b~u~~ und isthmus region. Of ail the brain regions found to project to the PAG, the midbrain contained the greatest number and diversity of labeled structures. The nucleus cuneiformis contained the highest number of marked cells within the mesen-
148
A. J. Beitz
Fig. 8. A charting diagram illustrating the location of labeled neurons (black dots) in a series of sections through the brain stem from case R-45. The core of the injection site (black) and the outer limit of the HRP reaction product (stipple) are indicated in A and B.
representative
cephalon in all our cases. This tegmental nuclear group was found to provide bilateral input to the PAG with a majority of HRP-labeled cells occurring in the ipsilateral nucleus (Fig. 11). A topography in the nucleus cuneiformis projection to the PAG was noted in that HRP injections into dorsal protions of the PAG resulted in heavier labeling in medial parts of this nucleus. Ventral injections into the central gray produced more labeling in the lateral part of the nucleus cuneiformis. The midbrain area containing the second largest number of labeled cells was the PAG itself. Labeled neurons were found bilaterally throughout the PAG both rostra1 and caudal to the injection sites. In some cases (R-49, R-50 and R-51) the majority of labeled cells occurred in the contralateral PAG. Injections into the ventral PAG resulted
in numerous labeled cells primarily in the dorsal parts of the central gray (Fig. 6). Following injections into the dorsal and dorsolateral subdivisions of the PAG, however, HRP-positive cells occurred almost equally in both dorsal and ventral portions of the central gray. Injections of HRP into the medial subdivision of the central gray (R-33) is of special interest in that it produced heavier labeling in the nucleus cuneiformis and in the PAG than in any other CNS region. All three divisions of the substantia nigra contained marked cells in one or more of the experimental cases. The pars reticulata of the substantia nigra contained the greatest number of HRP-positive neurons following injections into ventral portions of the PAG (R-50, R-51) and the label was most prominent with ventrocaudal HRP deposits (R-50). Label neurons were
Fig. 9. A charting diagram illustrating the d~str~butjon of Labeled cells (black dots) on representative brain stem sections following an injection of horseradish peroxidase into the ventral periaqueductai gray (case R-51). The core of the injection site (black) and the outer limit of the HRP reaction product (stipple) are indicated in A, 9, and G. found predominantly in ventromedial parts of the pars reticulata in all cases. The greatest amount of labeling in the pars lateralis occurred following dorsal HRP placements in the central gray (Table 3) while the pars compacta was only labeled with ventrocaudai injections (R-50, R-35). The medial and lateral parabrachial nuclei contained significant label following injections of the tracer enzyme into the ventrolateral subdivision of the PAG while the periparabigeminal area was significantly labeled with caudal dorsolateral injections. The ipsilateral and contralateral deep layer of the superior colliculus contained marked cells in many of
the experimental cases. The heaviest labeling occurred in case R-59 following an injection into the ventrolateral subdivision of the central gray. The subcuneiform nucleus was labeled following injections of HRP into the medial, dorsolateral and ventrolateral subdivisions. Label occurred primarily in an area lateral and dorsolateral to the red nucleus. Smaller numbers of labeied cells were observed in the locus coeruleus, the ventral tegmental area and the paralemniscal area (Table 4). I%ns end cereMftlm. The ipsilaterat pontine reticular formation contained the heaviest labeling at this brain stem level. Retrogradely-labeled cells were most
150
A. J. Beitz
dense in the nucleus reticularis pontis oralis, especially following HRP injections into ventral and ventrolateral portions of the PAG (R-50, R-51 and R-59). The nucleus reticularis pontis caudalis and the nucleus reticularis pontis caudalis pars x contained significant label in case R-38 as well as in most of our experimental cases (see Table 4). The majority of labeled cells in the nucleus pontis oralis were localized in the ventrolateral part of this nucleus. while labeling in the nucleus pontis caudalis occurred along its medial border. Marked cells were observed in the ventral portion of the superior central nucleus following HRP injections into the ventrolateral subdivision of the PAC. The deep cerebellar nuclei contained a few labeled cells in almost all of our cases. The contralateral nucleus lateralis and nucleus interpositus posterior were found to contain labeled neurons primarily along their ventral aspect. The nucleus medialis was only labeled following injections into ventral portions of the PAG. HRP-filled cells were confined to the caudal subdivision of this cerebellar nucleus. Mrtlulltr. The heaviest labeling at this level of the brain stem occurred in the reticular formation. The nucleus reticularis medulla oblongata pars ventralis and the nucleus reticularis gigantocellularis pars a contained the largest number of labeled neurons. HRP-positive cells were found throughout the former nucleus but were localized to lateral portions of the nucleus gigantocellularis pars a. The nucleus reticularis gigantocellularis and the nucleus paragigantocellularis lateralis also contained marked cells localized to the caudal portion of both nuclei. Spinrrl cord. As indicated under Experimental Procedures, the spinal cord was examined in only five experimental cases and in two control animals (R-66 and R-180). A small amount of labeling was observed in three experimental animals (R-50, R51 and R-59) and the majority of cells in these cases were confined to lamina IV and V. The remaining two experimental cases contained no labeled cells in the spinal cord. Injections into either the superior colliculus (R-66) or into the nucleus cuneiformis (R-180) also resulted in a small amount of labeling within the dorsal horn of the spinal cord, predominantly in lamina IV. However, since the injection site in case R-50 and R-51 did not spread into the nucleus cuneiformis or the overlying superior colliculus. this could not account for the spinal cord labeling resulting from this PAG injection. These results would suggest that the ventral portion of the PAG is the only part of the central gray which receives direct spinal cord input.
HRP-labeled cells were not detected in any brain structures in case R-54 in which HRP was injected directly into the cerebral aqueduct. This negative result ruled out, at least in the present study: (1) a possible uptake of HRP by the supraependymal plexus adjacent to the aqueduct and subsequent retro-
grade transport to the somata of these axons;‘,9 (2) a possible uptake of HRP by brain parenchyma adjacent to the third, fourth and lateral ventricles following HRP transport within the cerebrospinal fluid. A comparison of the results of control injections into the superior colliculus, nucleus cuneiformis and dorsal raphe nucleus with the results of HRP injections into the PAG indicate some similarities between all 4 regions. More importantly, however, a comparison of these results indicate major differences both qualitative and quantitative between the control cases and the PAG injections. These findings suggest that the majority of labeling listed in Table 2 is the result of direct uptake of HRP from the PAG and that it is not due to uptake an subsequent retrograde transport of the enzyme from adjacent CNS structures. The heaviest labeling following PAG injections, for instance, occurred in the ventromedial hypothalamic nucleus, the lateral hypothalamic nucleus, the lateral preoptic area and the zona incerta (Table 2). By contrast, injections into the nucleus cuneiformis resulted in heavy labeling in the central nucleus of the amygdala, the lateral tuberal nucleus and the substantia nigra pars reticulata. The greatest number of labeled cells occurred in the zona incerta, the nucleus of the posterior commissure and the pretectal area subsequent to injections into the superior colliculus, while dorsal raphe injections resulted in significant labeling within the lateral habenular nucleus, the dorsal hypothalamic area. and the prefrontal cortex. DISCUSSION The midbrain periaqueductal gray has been shown in the present investigation to receive a broad spectrum of afferent input from brain stem, hypothalamic. basal forebrain and cortical regions. These connections would allow the PAG to be influenced by the limbic system, the sensory system and to some extent the autonomic and motor systems. These systems, moreover, appear to be related to specific portions of the central gray. The sensory system, for instance appears to have indirect projections to all divisions of the PAG. but the dorsolateral subdivision receives the largest input. The limbic and motor systems, on the other hand, project to ventral and ventrolateral portions of the PAG especially to its caudal one half. The major projections from each of these systems to the PAG will be considered below. Limhic
and autonomic
input
to the periaqueductal
gray
Because the limbic system and central portion of the autonomic system are closely related in many respects. especially, at the level of the hypothalamus. they will be discussed together. Aferents ,fiom corficcd reyion.s. The present results showing labeling of the medial prefrontal cortex after HRP injections into the PAG are compatible with an earlier degeneration study in which restricted lesions were placed in the medial frontal cortex of the squir-
Afferent projections to the ~riaqueductal rel monkey. 32 Leichnetz and Astruc, however, indicate that degenerating fiber terminal debris was found in the dorsolateral margin of the PAG. In the present study, the majority of labeling in the prefrontal cortex occurred following HRP injections into the ventrolateral subdivision of the PAG rather than the dorsolateral subdivision. The difference in the projection to the PAG in the present study of the rodent PAG, as compared with the study by Leichnetz & Astruc3’ in the monkey, may reflect a species variability in this pathway. However, negative results obtained with silver techniques used by these authors do not exclude a projection to the ventrolateral PAG. In support of our findings Sotnichencko6’ identified degenerating terminals within the greater part of the ipsilateral PAG following medial prefrontal cortex lesions in the cat. This investigator also indicated that the heaviest degeneration occurred at the most rostra1 levels of the central gray, which agrees well with the results of the present investigation. The heaviest labeling found in the prefrontal cortex occurred in our case R-51 with an injection into the rostroventral PAC. It is of interest that studies of the efferent projections of the PAG have demonstrated an input to the dorsomedial nucleus of the thalamus’O~ss which in turn provides a major input to the prefrontal cortex.h4 This loop which exists between the PAG and the prefrontal cortex may be one of several routes by which the PAG is interrelated with the limbic system. Other hmbic cortical areas were labeled in some of our cases with large HRP injections which were not confined to the PAG. Labeled neurons were observed in area 29 and in the subiculum in these cases. These 2 cortical areas, however, were also labeled following injections into the nucleus cuneiformis and thus we could not conclude with certainty that this labeling was the result of uptake of HRP from the central gray. In addition to containing labeled cells following large HRP injections into the PAG, area 29 contained marked cells in 2 cases with small injections of HRP which were limited to the PAG (R-50 and R-51). This finding would indicate that this cortical region does project to the central gray. Data which lend support to a possible projection from area 29 and also from the subiculum to the PAG are found in a recent report by Jurgens & Prattz8 and in an earlier study by Nauta.42 The former investigators found evidence of a projection from the cingulate gyrus to the PAG in the monkey, utilizing an autoradiographic tracing technique, while the latter investigator employed degeneration techniques to demonstrate a hippocampal projection to this region in the cat. A,flerenfs from fhe amyydala and basuf forebrain. A small number of labeled cells were observed in the central nucleus of the amygdala following injections of HRP into the ventrolateral PAG. This finding is consistent with the autoradiographic findings of Hopkins & Holstege ” in the cat. These investigators also describe a proiection to the rostra1 dorsolateral . ”
gray
151
central gray from the central nucleus of the amygdala. In the present study, however, no labeled cells were observed in the amygdala following HRP injections into the rostra1 portion of the dorsolateral PAG subdivision. Fibers from the bed nucleus of the stria terminalis have been described with anterograde methods by Conrad & Pfaff” to project to the PAG. The present experiments confirm these results with a retrograde method and show that labeling occurs in the bed nucleus after injections of the tracer enzyme into the ventrolateral subdivision of the PAG. Moreover. the labeled cells are localized to ventral portions of the bed nucleus. In the present study, labeled cells were also found in the horizontal limb of the diagonal band, predominantly along its dorsal aspect. Conrad & Pfaff,” on the other hand, found no evidence of terminations of diagonal band axons in the central gray. These authors, however, indicated that the projections they describe are primarily those of the vertical nucleus of the diagonal band and only a few medial and anterior cells of the horizontal nucleus were included in their injection sites. We found no labeled cells in the vertical limb of the diagonal band in our cases and thus the negative findings of Conrad & Pfaff are probably related to the exclusion of the dorsal part of the horizontal limb in their injection sites. Large neurons which were labeled in the horizontal limb were observed to merge laterally and posteriorly with large labeled cells of the substantia innominata and the magnocellular preoptic nucleus. These results are in agreement with autoradiographic data.63 The distribution of large labeled neurons in the horizontal limb of the diagonal band, magnocellular preoptic nucleus and substantia innominata agrees well with the distribution of magno~ellular basal forebrain neurons described by Divac.14 Neurons were also labeled in the nucleus accumbens in case R-50 where the HRP injection was centered in the caudal part of the ventrolateral PAG subdivision. This finding agrees well with the results of Nauta, Smith, Fauli & Domesick who described projections from the nucleus accumbens to the caudoventral central gray substance in the rat. The nucleus accumbens has been postulated to serve as a functional link between the limbic system and basal ganglia4’. The projection from the nucleus accumbens to the PAG is of interest in light of the present results which suggest that the PAG may also serve as an area of interface between the limbic and motor systems. Afirents from the diencephulon. Limbic efferents to the PAG from the diencephalon arose prodominantly from the hypothalamus and the lateral habenular nucleus. In addition, the hypothalamus probably supplies information related to autonomic function to the central gray. Quantitatively the lateral hypothalamus provides the largest hypothalamic input to the PAG. This input, moreover, is directed predominantly to the ventrolateral subdivision of this region. This finding is in agreement with the results of fiber degener-
152
A. J. Beitz
ation42.71 and autoradiographic data43.4“ in the rat which also show fiber projections to the ventral portion of the PAG. The ventromedial hypothalamic nucleus provided the largest hypothalamic input to both the medial and dorsolateral subdivisions of the PAC. This finding is supported by the autoradiographic study of Krieger, Conrad & Pfaff3’ and the retrograde transport study of Grofova, Ottersen & Rinvik.” Krieger and coworkers3’ identified significant fiber projections to dorsal and medial aspects of the rodent central gray. Likewise. the work of Grofova tit trl.,‘” indicates that the ventromedial nucleus appears to be the most prominent hypothalamic source of afferents to the dorsal feline PAC. The dorsal premamillary nucleus was heavily labeled in case R-49 where the injection was localized to the dorsolateral subdivision of the PAG. Evidence in support of this projection can be found in the work of Krieger et L(/.31 These investigations found a dense projection from this nuclear region to the lateral aspect of the central gray along its rostrocaudal extent. A significant number of marked cells were found in the perifornical area following injections of HRP into the ventral central gray as well as injections into the dorsal raphe nucleus. These findings are in good agreement with the results of Sakai and coworkers.56 Finally, the present results, as well as those of Herkenham & Nauta,23 demonstrate a lateral habenular projection to the PAG. specifically to its ventral portion. .4,fl&~71t.sfiokn rhr hmin SIU~ The central gray receives input from certain brain stem nuclei which have been implicated in autonomic function. These include projections from the nucleus solitarus. the nucleus reticularis gigantocellularis, the nucleus paragigantocellularis and the parabrachial nuclei. These connections are related predominantly to the ventrolateral subdivision of the PAG. In addition, the PAG
receives input from the midbrain ventral area, a component of the limbic system.43 Motor
input
to the periuyueductul
tegmental
grq
The motor system also appears to be connected primarily with ventral portions of the PAG. Thus, the motor cortex. cerebellum and substantia nigra project predominantly to ventral and ventrolateral parts of the central gray. It has been demonstrated that vocalization can be elicited from the rodent PAG by electrical stimulation.68 and this region may serve as a relay station for motor impulses related to vocalization. Of interest in this regard is the report by Jiirgens & Prattz8 which indicates that the PAG plays an important role in the vocal expression of emotion. This finding may partially explain the overlap in the projections from the limbic system and motor system to the central gray.
The PAG receives input from several sensory regions of the central nervous system. A small input to the ventral portion of the central gray arises from lamina IV and V of the spinal cord. This is consistent with earlier degeneration studies3”,3’ and with more recent physiological studies’“. Lamina IV and V contain neurons which respond to noxious stimuli’” and also contains a large majority of the cells of origin of that portion of the spinothalamic tract which projects to the lateral thalamus.‘” Input from this part of the dorsal horn would thus provide a direct route by which nociceptive information could reach the PAG. The dorsal portion of the PAG receives input from the trigeminal nuclei. especially subnucleus oralis and interpolaris. In addition, the PAG receives input from the superior colliculus and pretectal area. PAG afferents from the superior colliculus were previously reported by Grofova and coworkers’” in the cat. These findings were confirmed and extended in the
Fig. 10. Representative photomicrographs of horseradish peroxidase-labeled cells III the forebrain and diencephalon. A. Low power photomicrograph (mag. = 60 x ) of the medial prefrontal cortex from case R-51. The arrow points to a labeled cell shown at higher magnification (600 x ) in B. C. LOW, magnilication photomicrograph (60 x ) illustrating the medial preoptic area and optic chiasm (oc) from case R-51. The arrow depicts the location of the labeled neuron illustrated in 7d at high magnitication (600 x ). t. Photomicrograph illustrating several labeled cells in the dorsal premamillary nucleus from cast R-49 (mag. = 230 x ). The third ventricle (III) is indicated and the HRP-positive cell at the arrow tip is shown at higher magnifcatlon (600 x ) in F. G. Photomicrograph of the posterior hypothalamic area from case R-49 (msg. = 230 x )_The arrow depicts the location of the labeled neuron shown at high magnification (600 x ) in 7H. Fig. 1 I. Representative photomicrographs of horseradish peroxidase-labeled cells observed In brain stem sections. A. Photomicrograph of the cerebral aqueduct (ag) and adjacent periaqueductal gray from case R-49 (msg. = 230x ). The arrow depicts a small labeled neuron in the medial aubdivision of the PAG which is shown at higher magnification (600x) in B. C. Photomicrograph illustrating a labeled neuron (arrow) in the nucleus cuneiformis from case R-59 (mag. = 230 x ). The periaqueductal gray (PAG) is indicated to the right of the nucleus cuneiformis. The labeled cell is shown at higher magnilication (600 x ) in 5D. E. Photomicrograph of a labeled cell (arrow) in the midbrain interpeduncular nucleus from case R-50 (msg. = 230 x ). The mamillary peduncle (mp) is shown on the right side of the photograph. The labeled neuron is shown at higher magnification (600 x ) in 5F. Low powcf- photomicrograph of the ventrolateral midbrain from case R-50 illustrating the location of a labeled cell (arrow) in the substantia nigra (mag = 60 x ). The cell is shown at higher magnilication in 5H (msg. = 600 x ).
Fig. 10. 153
Fig. 11 154
Afferent projections to the periaqueductal gray present investigation. Both the ispilateral and contralateral deep layer of the superior colliculus were labeled in our material and were especially prominent following dorsolateral and ventrolateral injections. Golgi studies have shown that cells in the deep layer of the superior colliculus extend their dendrites into the dorsal PAG49”. The evidence provided by Ram6n y Cajal would suggest the possibility that some of the marked cells in the ipsilateral superior colliculus were not labeled by retrograde axonal transport of HRP but rather by dendritic transport. Although it is not possible to differentiate which type of transport occurred in our experimental cases, support for an ipsilateral collicular projection to the PAG can be found in the autoradiographic study of Graham.18 This investigator depicts terminal label within the dorsolateral and ventrolateral portion of the ipsilateral PAG following amino acid injections into the stratum griseum profundum of the superior colliculus. The dorsolateral portion of the PAG also receives a large projection from the zona incerta which appears to have a role in the integration of impulses from many sensory regions. l9 This finding is supported by the recent autoradiographic studies of Ricardo53 which demonstrate zona incerta fiber terminations in the lateral central gray. It is noteworthy that the PAG is the recipient of efferent projections from the above sensory structures, since all of these brain regions have been found in physiological studies to be involved in pain mechanisms.16~29~52~6’ Functional
implications
The experimental results presented above suggest that in the rat the PAG receives a wide variety of ascending and descending neural input. Perhaps the most remarkable single point to emerge from our data is that the central gray is affected by a very great diversity of neural mechanisms. The limbic, autonomic and motor systems for instance, were found to provide input to the ventral PAG. It is possible that this region may represent an area of interface between these three systems. Coincidentally, the ventrocaudal portion of the PAG has been implicated in both stimulation and chemically produced analgesia16~72 and has been shown to receive nociceptive input from the periphery.’ 5s8 This region thus appears to be instrumental in pain control.58 Although pain is in some respects similar to other sensory modalities, it is more akin to motivational systems in some of its characteristics.35 Thus, unlike most other sensory modalities, it is almost invariably accompanied by an affective state. This effective dimension can be viewed as analogous to a ‘drive’ state in that it can command the attention of the organism and result in behaviors aimed at restoring homeostasis. The connections of the PAG demonstrated in the present investigation would also allow this region to serve as an interface between the limbic system and the sensory system. Thus, the limbic projections to the PAG may provide a route through which the limbic system can exert
155
some control over incoming nonciceptive input via descending pathways arising in the PAG.” The ventral portion of the PAG not only receives limbic input but reciprocates this input by projecting either directly or indirectly to limbic structures. Projections from the ventral PAG, have been described to the lateral hypothalamus, the dorsal medial nucleus of the thalamus and the ventral tegmental area.55 This anatomical substrate for interaction between the limbit system and the PAG may subserve the affectivemotivation components of pain. The present results also demonstrate a substantial hypothalamic input to the PAG. The hypothalamus is also the recipient of a dense fiber projection from the midbrain central gray.10~55 These findings in conjunction with the present results indicate that a reciprocal relationship exists between these two structures. This reciprocal interconnectivity, moreover, appears to be topographically organized. Thus, the present investigation has demonstrated a strong projection from the lateral hypothalamus to the ventrolateral subdivision of the PAG. Conversely, the most prominent PAG input to the lateral hypothalamus arises from its ventral portion.55 The dorsolateral PAG is interconnected with the posterior and dorsal hypothalamus in a similar fashion. This interrelationship between the hypothalamus and PAG probably reflects the coordinated involvement of these two structures in a number of brain functions. For instance, the hypothalamic-PAG system may play a major role in controlling feminine reproductive behavior as suggested by Krieger et al. 31 In support of this hypothesis, stimulation of the central gray has been shown to elict lordosis behavior in female rats,47 while lesions in this region depress lordosis.” Moreover, estrogen concentrating cells have been localized to the lateral aspect of the central gray. 46 In addition to a role in reproductive behavior this system may be involved in the central pressor effects of angiotensin II. The centrally mediated pressor activity of angiotensin is dependent upon activation of angiotensin receptors located in the preoptic hypothalamic region’ and in the dorsal PAG.59 Tissue within the rodent dorsal PAG and superior colliculus has also been shown to be essential in the maintenance of central pressor response to angiotensin II.59 Coincidentally, angiotensin II has been localized to the ventral PAG and the preoptic hypothalamic region.8 The possible involvement of the hypothalamic-PAG system in reproduction and in central pressor responses are but two examples of a number of functions that this system may subserve. As alluded to above, our anatomical data suggest that the PAG may serve as a functional interface between the limbic system and motor system. There is considerable evidence that limbic forebrain structures are important in ‘drives’ and ‘motivational’ processes40*41 contributing to the initiation of actions, but little is known about the neural mechanisms by which limbic processes gain access to the motor system. The subject of the interface between motivation
156
A. J. Beitz
and action-between limbic and motor systems--can be traced back to the classical experiments of Hess.24 Hess demonstrated that attack, feeding and other complex, biologically-significant behaviors could be elicited by electrical stimulation of the hypothalam~ts in unanesthetized cats. The neural processes resulting from electrical stimulation of the hypothalamus or limbic forebrain must eventually influence the motor system to produce attack, feeding or other behaviors observed. However. little progress has been made in elucidating the neural mechanisms by which the motor system is activated in such elicited behaviors. A major reason for the neglect of this important problem has been the absence of relevant anatomical evidence. Recently, anatomical evidence has been obtained which suggest that the nucleus accumbens, the lateral habenular nucleus and the ventral tegmental area may be three key structures linking the limbic system with the motor system. 41v43The present investigation demonstrates that the PAG not only receives direct connections from nucleus accumbens, ventral tegmental area and lateral habenular nucleus. but also receives direct input from important motor areas (i.e. motor cortex, cerebellum, substantia nigra) and limbic structures. The PAG is thus in a position to integrate both motor and limbic information and convey impulses to the spinal apparatus ~iu several alternative direct and indirect descending pathways. Studies of the efferent projections of the PAG’“*5s indicate that the central gray can also influence the limbic system t)icl connections to the lateral hypothalamus, ventral tegmental area and dorsal medial nucleus of the thalamas. In addition, the PAG may indirectly influence the motor system uitr projections to the zona incerta, the centromedian nucleus of the thalamas. the parafascicular nucleus and the reticular formation. These anatomical data suggest that the PAG may serve as an integrator of information derived from the limbic and motor systems. Its possible role in limbic-motor integration. however, requires further study.
One of the goals of this study was to determine if differences exist between the afferent input to the four subdivisions of the rodent PAG. The results of the present study clearly indicate a differential input to these four subdivisions from all levels of the CNS. In general, HRP injections involving the ventrolateral subdivision resulted in significant labeling in the basal forebrain. the lateral hypothalamus, the substantia nigra, the contralateral superior colliculus, and the brain stem reticular formation. A deposit of HRP into the dorsolateral subdivision, on the other hand, produced heavy labeling in the medial hypothalamus. This occurred primarily in the ventromedial hypothalamic nucleus, the dorsal premamillary nucleus and the posterior hypothalamic nucleus. In addition,
dorsolateral PAG injections resulted in significant labeling in the nucleus cuneiformis, the anterior pretectal nucleus, the substantia nigra pars lateralis and the zona incerta. Input to the medial subdivision of the PAG was examined in only one case (R-33) in which the core HRP reaction product was localized to this subdivision. Although not listed in Tables 3 and 4, the input to the medial subdivision was quantitatively much less than that to other PAG subdivisions. The largest numbers of labeled neurons in the brain resulting from the placement of HRP in this division occurred in the nucleus cuneiformis (I 1 cells), the rostra1 PAG (7 cells ipsilaterally, 2 cells contralaterally), the raphe dorsalis (8 cells) and the zona incerta (7 cells). Input to the caudal portion of the dorsal subdivision was examined in case R-43. The dorsal subdivision is unique, in contrast with the other divisions. in that it is the only PAG subdivision which does not receive hypothalamic input. Injections of HRP into this PAG subdivision result in a large number of marked cells in the nucleus cuneiformis (both ipsilaterally and contralaterally), in the PAG itself (both rostra1 and cauda1 to the injection site), in the paralemniscal area and the zona incerta. Finally, differences were also noted in the afferent projections to rostra1 and caudal portions of the PAG (see Tables 2-4). Although most of these differences were purely quantitative. a number of qualitative variations also occurred. The caudal portion of the ventrolateral subdivision. for instance, receives input from the locus coeruleus. the substantia nigra pars compacta, the reticulotegmental nucleus and the dorsal premamillary nucleus, while the rostra1 portion of this division does not. In conclusion. the present study has demonstrated that the rodent periaqLleductal gray receives a broad spectrum of input from a diversity of neural systems, including the limbic system. motor system, sensory system and autonomic system. A comparison of these results with studies of the efferent projections of the PAG’“-5” suggest that many brain regions arc reciprocally connected with the central gray. Many brain stem and diencephalic nuclei which have been implicated as components of the endogcnous analgesia system,4,‘“,3s for instance, not only receive PAG input but send projections back to this midbrain region. These reciprocal projections may be part of an important feedback rnech~~nisnl which allows moditication of PAG output. Needless to say, much physiological experimentation is required before the importance of these reciprocal connections is fully understood. Ackno~~t~c!yi~munrsThe A. McDonald aration
for their
author thanks Drs Jim Buggy and helpful
of this manuscript.
Research Grant Foundation.
BNS
comments
The
7906486
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on thr
prep-
was supported
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by
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Richardson tion in periaqueductal and periventricular sites. J. Neurosurg. 47, 178-183. study of the efferent projections of the midbrain central gray in the cat. (Ph.D. 55. Ruda M. T. (1976) Autoradiographic thesis). Philadelphia PA, Univ. of Pennsylvania. of the nucleus raphe dorsalis in the cat as 56. Sakai K., Salvert D., Touret M. & Jouvet M. (1977) Afferent connections visualised by the horseradish peroxidase technique. Bruin Res. 137, 1 l-35. mechanisms for integration of female reproductive behavior in the rat. 57. Sakuma Y. & Pfaff D. W. (1979) Mesencephalic Am. J. Physiol. 237, R285-R290. H. 0. (1980) Differential effects of noxious and nonnoxious 58. Sanders K.. Klein C., Mayer T., Heym C. & Handwerker input on neurons according to location in ventral periaqueductal gray or dorsal raphe nucleus. Bruin Res. 186, 83-97. S. N. & Hubbard J. I. (1979) Angiotensin binding and pressor activity in the rat ventricular 59. Sirett N. E., Thornton system and midbrain. 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gray
159
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D306-4522~82/010133-27503.00!0 Pergamon Press Ltd 0 1982 IBRO
I. pp. t33 to 159. 1982
THE ORGANIZATION OF AFFERENT PROJECTIONS TO THE MIDBRAIN PERIAQUE~UCTAL GRAY OF THE RAT A. J. BEITZ Department of Anatomy, University of South Carolina, School of Medicine, Columbia, South Carolina 29028, U.S.A. Abstract-The retrograde transport technique was utilized in the present study to investigate the afferent projections to the ~riaqueductai gray of the rat. Iontophoretic injections of horseradish peroxidase were made into the periaqueductai gray of 22 experimental animals and into regions adjacent to the periaqueductai gray in 6 control animals. Utilization of the retrograde transport method permitted a quantitative analysis of the afferent projections not only to the entire periaqueductai gray, but also to each of its four intrinsic subdivisions. The largest cortical input to this midbrain region arises from areas 24 and 32 in the medial prefrontal cortex. The basal forebrain provides a significant input to the periaqueductal gray and this arises predominantly from the ipsilateral lateral and medial preoptic areas and from the horizontal limb of the diagonal band of Broca. The hypothalamus was found to provide the largest descending input to the central gray. Numerous labeled ceils occurred in the ventromedial hypothalamic nucleus, the lateral hypothalamic area, the posterior hypothalamic area, the anterior hypothalamic area, the perifornicai nucleus and the area of the tuber cinereum. The largest mesencephaiic input to the periaqueductai gray arises from the nucleus cuneiformis and the substantia nigra. The periaqueductal gray was found to have numerous intrinsic connections and contained a significant number of labeled cells both above and below the injection site in each case. Other structures containing significant label in the midbrain and isthmus region included the nucleus subcuneiformis~ the ventral tegmentai area, the locus coeruieus and the parabrachiai nuclei. The meduliary and pontine reticular formation provide the largest input to the periaqueductai gray from the lower brain stem. The midline raphe magnus and superior central nucleus also supply a significant fiber projection to the central gray. Both the trigeminal complex and the spinal cord provide a minor input to this region of the midbrain. The sources of afferent projections to the periaqueductai gray are extensive and allow this midbrain region to be influenced by motor, sensory and limbic structures. In addition, evidence is provided which indicates that the four subdivisions of the central gray receive differential projections from the brain stem as well as from higher brain structures.
The mesencephaiic periaqueductal gray (PAG) or central gray is formed by a mantle of cells which surround the cerebral aqueduct. The rodent periaqueductal gray has recently been shown to consist of four anatomical subdivisions3,49 (1) a medial subdivision which is composed of small, primarily bipolar neurons which closely surround the cerebral aqueduct; (2) a ventrolateral division which contains predominantly large fusiform neurons, large multipolar neurons and small bipolar neurons, the majority of these cells are oriented at a lW150” angle with respect to the aqueduct; (3) a dorsoiateral division comprised predominantly of triangular-shaped cells, small multipolar neurons and small bipolar neurons, the majority of which are oriented at a 30-70” angle with respect to the cerebral aqueduct; and (4) a dorsal subdivision containing primarily small bipolar and multipolar neurons which show no orientational preference. This division lies immediately dorsal to the cerebral aqueduct. The PAG has received a great deal of attention within the past decade because of its unique role in Abbreviations:
mesencephaiic benzidine.
both stimulation and chemically-induced analgesia. Since the initial observation that stimulation of this in rats produced profound analgesia5’ region numerous laboratories have demonstrated that the PAG is an important site for stimulation-produced analgesia33.36,54 as well as a central site for morphine’s antinociceptive action.26,33+4s Although the central gray has attracted much attention because of its role in analgesia_ it is nonetheless involved in several other functions. It appears to be involved in vocalization,” rage reactions,36 control of reproductive behavior” and pressor responses.59 The role played by the PAG in the above functions is dependent on the interconnections of this midbrain area with other regions of the central nervous system (CNS). This has recently been clearly demonstrated by Sakuma & Pfaff5’ with regard to the PAG’s involvement in reproductive function. Although the efferent projections of the PAG have been studied in some detail’O~ss the afferent connections of this region have been largely neglected. Grofova, Ottersen & Rinvik” studied mesencephalic and diencephalic projections to dorsal portions of the PAG, but apart from this investigation very little work has been done on the afferent projection system to the central gray. The present investigation was designed to construct a
HRP, horseradish peroxidase; PAG, periaqueductai gray; TMB, tetramethyi133
134 detailed
A. J. Beitz map
of the CNS
regions
which
supply
input
to the PAG
utilizing the retrograde transport technique. In addition to mapping the sources of input to the central gray as a whole, the origin of the specific projections to each of the four PAG subdivisions described above were analyzed.
EXPERIMENTAL
PROCEDURES
Sources of afferent projections to the midbrain periaqueductal gray were studied in 22 male Sprague-Dawley rats by means of the retrograde transport of horseradish peroxidase (HRP). Each rat was anesthetized with chloral hydrate (0.35 g/kg ip.) and a small deposit of HRP (Sigma type VI) was placed sterotaxically into the PAC. The HRP was delivered by microelectrophoresis via a glass micropipette filled with a 13:< solution of HRP in 0.05 M tris buffer (pH 7.6). The size of the micropipette tip was in the 10-25 pm diameter range to minimize the amount of retrograde labeling following the uptake of HRP by fibers of passage. *’ The driving force for the microelectrophoretic injections was supplied by a 1.8 pa positive current delivered by a constant current source (Midgard Electronics, Newton, Massachusetts) at a pulse rate of 7 s on, 7 s OR. for a duration of 615 min. The animals were killed 2436 hours postoperatively by intracardiac perfusion. under anesthesia, with 500 ml of a O.SO:, paraformaldehyde-2”;, glutaraldehyde fixative made up in 0.12 M phosphate buffer followed by 500 ml of 0.12 M phosphate buffer containing 506 sucrose and 5% polyethylene glycol. The brains were removed, placed in the same 5”” sucroese~-5p, polyethylene glycol solution for an additional 30 min, and subsequently cut in the coronal plane on a freezing microtome at 40 ilrn. Sections through the entire brain and spinal cord were collected and reacted with tetramethylbenzidine (TMB) and hydrogen peroxide according to the procedure described by Mesulam3’ with the following modifications. (I) The 20min incubation time in both the pre-reaction soak solution (a mixture of Mesulam’s solution A and B) and the enzymatic reaction solution (a mixture of solution A and B to which H202 was added) was reduced to 12 min and 10 min. respectively. This reduction in incubation time was found to reduce significantly the number of filamentous or crystalline artifacts present on tissue sections without significantly affecting neuronal labeling. (2) The improved stabilization procedure recently developed by Adams’ was employed rather than stabilization in a chilled ethanolic solution of sodium nitroferricyanide as Mesulam”s originally recommended. This procedure utilizes methyl salicylate to both clear the tissue and stabilize the TMB reaction product. (3) The tissue sections were stained with a 1”” solution of pyronin-Y (Eastman Kodak) in acetate buffer (pH 3.3). The use of this particular stain has several advantages. It allows the staining process to be performed at the same pH and in the same buffer as the initial TMB reaction, which increases the stability of the reaction product. In addition, it enhances tissue contrast without decreasing the visibility of the TMB reaction product and is thus superior to neutral red in this regard. Every second section through the cerebrum. diencephaIon and brain stem was examined in each experimental animal for the presence of retrogradely-labeled neurons containing dark blue granules throughout their cytoplasm. In addition. every fifth section through the spinal cord of
five experimental animals was scanned for HRP-positive neurons. In order to quantitate the afferent contribution from the cortex and basal forebrain and from diencephalic, brain stem and spinal cord nuclei to the PAG, counts of the total number of labeled perikarya were made on each of the sections examined from these regions for each case. Counts were made under bright-field illumination at a magnification of 100 x In cases where it was uncertain if a neuron contained HRP-reaction product at the initial magnification of 100 x . the cell was reexamined at a magnification of 400x to ascertain if it contained dark blue HRPpositive granules throughout its cytoplasm. The above quantitation procedure allowed us to determine the relative contribution of afferent input from nuclei within the diencephalon and brain stem to the entire PAG as well as to its intrinsic subdivisions. In addition to a quantitative analysis of the number of labeled cells in each case. the location of labeled neurons were carefully plotted onto drawings of selected sections through each region. This allowed an accurate determination of the location of HRPpositive neurons within diencephalic. basal forebrain and brain stem nuclear groups. One of the major problems encountered in analyzing retrograde transport studies utilizing HRP as the retrograde marker is determining the size of the injection site.3’ In an attempt to determine quantitatively the size of the injection site, sections through the injection site were analyzed in several of our experimental cases on a computerized image processing system. This system consists of a spatial data systems image digitizer. a colorado video memory, and a comtal vision one interactive color display and real-time processor. A dedicated PDP-I I./O3 performs real-time image analysis functions in a multiprocessing mode with a DEC PDP-8e. This system can convert photometric data from the stained brain sections mto digital form, providing discrimination of up to 256 density levels. A hard copy character representation of optical density bins throughout the section is produced on a line printer (see Fig. 1) and the precise extent of the HRP injection size is subsequently analyzed on each print out. In addition to the 22 animals in which HRP injections were made into the PAG, an additional 6 animals were used as controls. The brains from these 6 cases were processed in an identical manner to that described above for experimental animals. In 2 of these cases (R-179 and R-180), iontophoretic HRP injections were made into nucleus cuneiformis on one side. Control HRP injections were also made into the dorsal raphe nucleus (R-58). the superior colliculus (R-27 & R-66) and the cerebral aqueduct. The nucleus cuneiformis parallels the ventrolateral aspect of the PAG throughout most of its rostra-caudal extent and the dorsal raphe nucleus is embedded in the ventrocaudal portion of the central gray. Because of their close proximity to the PAG they were often involved in the outer fringe of our PAG Injection sites. Injections of HRP were thus made into these two nuclei as a means of comparing the resultant labeling with the results of PAG injections. Likewise, the results of superior colliculus injections were compared to dorsally-placed PAG injections. Finally. an injection was made into the cerebral aqueduct to determine if any neuronal labeling occurred in the CNS as a result of leakage into the ventricular system.
Two possible sources of artifacts should be considered evaluating the present results: (1) cells labeled by spread
in of
Afferent
projections
to the periaqueductal
HRP to regions adjacent to the PAG, and (2) cells labeled as a result of uptake by injured fibers passing through the midbrain PAG. If we first consider the possibility of labeling occurring due to uptake and subsequent retrograde transport of HRP from neighboring PAG areas, this source of artifact can probably be ruled out in 6 of our cases (R-36, R-37, R-45, R-49, R-57 and R-53) since the visible HRP reaction product was confined to the midbrain central gray in each case. In an additional 5 cases (R-33, R-38, R-43, R-51 and R-59) the core of the HRP reaction was confined to the PAG. Although there was some spread of HRP into surrounding areas in these cases, recent evidence has shown that the area of effective uptake of HRP is restricted to tissue very close to the pipette tip.48.65 Since both the micropipette tip and the dense core of the HRP reaction was localized to the PAG in these 5 animals, the above finding would argue against uptake and subsequent transport of the enzyme by synaptic regions outside the PAG in these cases. The technique of iontophoresis of HRP into CNS sites will not only label neuron cell bodies by the physiological process of uptake and subsequent retrograde transport of the enzyme from axon terminals, but will also label cells whose axons pass through the site of injection and are damaged by the injection procedure.22S48 Thus, although there are advantages in using the iontophoretic technique, in that extremely small deposits of HRP can be made, there is still a risk of labeling neurons ciu uptake or by injection into damaged fibers of passage, as had been earlier noticed in cases where pressure injections of HRP were made into the CNS.Z’,34.66.67 Differentiation between neurons labeled by terminal uptake and those from damaged axons of passage is not possible utilizing the retrograde transport technique at the light-microscopic level. Only anterograde tracing techniques in conjunction with electron microscopy or electrophysiological techniques can definitely establish the presence of axon terminations in regions such as the PAG where fibers are passing through to other CNS areas. Although the PAG is an area traversed by many fiber tracts of different origin,12 the use of extremely small pipette tips and small amounts of current in the present study would argue against a major amount of uptake of HRP by injured fibers of passage.
RESULTS
Description of horseradish peroxidase injection sites The HRP deposits in 6 of the 22 rats which received injections into the midbrain PAG were restricted to the central gray. The limited spread of HRP in these 6 animals was initially determined under microscopic examination at a magnification of 40 x and subsequently confirmed utilizing the computer-assisted image processing system. In an additional 5 animals the core of the HRP injection site was confined to the PAG, but faint reaction product was evident in adjacent structures, notably the superior colliculus, the dorsal raphe nucleus or the nucleus cuneiformis. In the remaining 11 animals the core HRP reaction product spread beyond the PAG dorsally into the overlying inferior or superior colliculus, laterally into the nucleus cuneiformis or ventrally into the tegmentum.
gray
135
Because of the HRP injection site was confined to the PAG in cases R-36, R-37, R-45, R-49, R-53 and R-57 these cases will be utilized throughout the rest of this paper to demonstrate the afferent input to the PAG. Moreover, in cases R-33, R-38, R-43, R-51 and R-59 the dense core of the HRP reaction product was confined to the PAG. Since there was limited spread of HRP in these cases they will also be used in the discussion of afferents to the midbrain central gray. In addition to the above cases, one other case (R-50) will be included to demonstrate the afferents to the caudal part of the ventrolateral subdivision of the PAG. As indicated in Table 1, a portion of the injection site from this animal involved the dorsal raphe nucleus which is almost unavoidable at this ventral and cauda1 level of the central gray. Finally, in one of our cases (R-38) the dark core of the HRP injection site covered much of the dorso-ventral extent of the PAG and nearly encompassed its entire rostrocaudal length (Fig. 3). Because of the large size and good confinement of the iontophoretic deposit in this case (only faint reaction product was evident in the overlying deep layer of the superior colliculus), it will be utilized to illustrate the broad spectrum of afferent input to the PAG. The location and size of the HRP injection site for the experimental cases discussed above and for the 6 control animals are listed in Table 1 and representative examples are shown in Figs 2 & 3.
Sources of qfierent input to the periaqueductal gray As indicated above, case R-38 will be used to summarize the nuclear groups within the forebrain, diencephalon, brain stem and spinal cord which project to the midbrain central gray. Over 60 different structures were found to project from these regions to the PAG. The areas containing label are listed in Tables 2, 3 and 4 (Case R-38) together with their laterality and the total number of labeled cells in each. The location of most of these structures is illustrated in Figs 4-9. Each projecting structure was identified in at least two experiments. The following commentary supplies supplementary information on the distribution of labeled cells in selected areas. Forebrain. The largest number of labeled cells in the basal forebrain region were in the preoptic area and in the horizontal limb of the diagonal band of Broca (Figs 4 and 10). Numerous cells were also found in cortical areas 24 and 32 as defined by Krieg.30” An analysis of the results of smaller HRP placements (R-51 and R-59) indicate that the projection from this region of the cerebral cortex is limited to ventral and ventrolateral portions of the PAG (Fig. 5). In several of our experimental cases with large injections in which the HRP reaction product spread outside the PAG (R-26, R-28, R-35 and R-46) labeling occurred in the nucleus accumbens, area 4. area 14 and area 29. The only cases with restricted injection sites in which labeling could be demonstrated in these regions were R-50 and R-51.
A. J. Beitz
136
Fig. 1. A. Low power photomicrograph of a coronal section through the rostra1 midbrain from case R-59. The HRP injection site (arrow) involves the ventral and ventrolateral portions of the periaqueductal gray (PAG). 40 pm, pyronin-Y, mag. = IS x B. A hardcopy computer image of the same section shown in 1A. The numbers and letters on the print out are numerical representations of density bins throughout the section. The greatest density is represented by the numbers l--9, while decreasing units of density are indicated by the letters A-W. The central core of the HRP reaction product (represented by the numbers 1-9) is encircled with a solid black line (arrow). The outer fringe of the injection site (containing faint reaction product in 1A) is represented by the letters A-G and is enclosed with a dotted line. The remainder of the section outside the HRP deposit is represented by the letters H-W except for areas that have stained intensely with pyronin-Y. Thus the superior colliculus (SC) and the interpeduncular nucleus (ip) contain letters in the D--G range but are clearly not part of the injection site. C. The section shown in 1A was projected onto the computer print out depicted in 1B and a tracing of the iniection site was subseauentlv made onto the projected image. The resulting tracing is illustrated in IC depicting the core of the injection site (b&k) and the surrounding spread of HRP (stipple). _I
Abbreviations a,
ag, aha. apl. apm, c, CA, CC, ces, Cl. cm, CP. cu, dpb. dpm, DSCP. F. FR. g, ge, gP, hldb. HRP. IC. ice. icp. io. ip, iVv. ip. 1, lgd. lgr. Iha, LM, lP, Iv, md, mg. MLF. MP, MT. mv. nb. ni,
used on figures
arcuate nucleus cerebral aqueduct anterior hypothalamic area lateral preoptic area medial preoptic area nucleus cuneatus anterior commissure corpus callosum nucleus centralis superioris nucleus cuneatus lateralis centromedial nucleus posterior commissure nucleus cuneiformis nucleus dorsalis parabrachialis dorsal premamillary nucleus decussation of superior cerebellar peduncle fornix fasciculus retroflexus nucleus supra geniculatus nucleus gelatinosus globus pallidus horizontal limb of the diagonal band horseradish peroxidase internal capsule nucleus centralis colliculus inferioris nucleus pericentralis colliculus inferioris inferior olive nucleus interpeduncularis fourth ventricle nucleus lateralis posterior thalami nucleus lateralis thalami nucleus dorsalis corporis genicalati lateralis nucleus ventralis corporis geniculati lateralis lateral hypothalamic area medial lemniscus nucleus lateralis posterior thalami nucleus vestibularis lateralis nucleus dorsalis medialis thalami medial geniculate body medial longitudinal fasciculus mamillary peduncle mamillothalamic tract nucleus vestibularis medialis nucleus basalis (magnocellular preoptic nucleus) nucleus interpositus
Pt, Pv, rd,
nucleus of the lateral olfactory tract nucleus medialis nucleus lateralis optic chiasm optic tract periaqueductal gray posterior hypothalamus parafascicular nucleus substantia nigra pars lateralis pyramidal tract nucleus paratenialis nucleus parvocellularis hypothalamic subnucleus reticularis dorsalis medulla
re. rgc, rgca, rh, rm. rn, rpc, rpca, rpo. rpt. rv,
gata nucleus reuniens nucleus reticularis gigantocellularis nucleus reticularis gigantocellularis pars a nucleus rhomboideus nucleus raphe magnus red nucleus nucleus reticularis pontis caudalis nucleus reticularis pontis caudalis pars a nucleus reticularis pontis oralis nucleus reticularis tegmenti pontis subnucleus reticularis ventralis medulla oblon-
nlot, nm. nl, oc. OT, PAG, ph. Pf. PP. PT,
SC. scu, sg. sl, SM, snc, snr, snl. so, sol, st, tag, tc, tp. v. vmh, vpm vta, zi.
gata superior colliculus nucleus subcuneiformis nucleus sagulum nucleus septi lateralis stria medullaris substantia nigra pars compacta substantia nigra pars reticulata substantia nigra pars lateralis nucleus supraopticus nucleus solitarius nucleus of the stria terminalis nucleus triangularis area of the tuber cinerium nucleus trapezoideus ventral thalamic complex nucleus ventromediahs nucleus ventralis premamillaris ventral tegmental area zona incerta
oblon-
137
A
R-36
R-38 138
Atrerent
projections
to the periaqueductal
139
gray
Table 1. The size and location of the horseradish peroxidase injection site and the animal survival time for the twelve experimental cases and six control cases discussed in the text
Case Number (1) R-33 (2) R-36 (3) R-37 (4) R-38 (5) R-43 (6) R-45 (7) R-49 (8) R-50
(9) R-51
(10) R-53 (11) R-51 (12) R-59 (13) R-27 (14) (15) (16) (17) (18)
R-54 R-58 R-66 R-179 R-180
Location of injection site Medial PAG subdivision Lateral PAG Dorsolateral PAG subdivision (caudal part) Entire PAG, deep layer of Superior colliculus Dorsal PAG subdivision (caudal part) Dorsolateral PAG subdivision (rostra1 part) Dorsolateral PAG subdivision (caudal part) Ventrolateral PAG subdivision (caudal part), lateral portion of dorsal raphe N. Ventrolateral PAG subdivision (rostra1 part), rostra1 most portion of dorsal raphe N. Dorsolateral PAG subdivision (rostra1 part) Rostroventral PAG Ventrolateral PAG subdivision (rostra1 part) Deep layer of superior colliculus Cerebral aqueduct Dorsal raphe nucleus Superior colliculus Nucleus cuneiformis Nucleus cuneiformis
Hypothalamus. The hypothalamus will be treated separately from the remainder of the diencephalon because it provides a substantial input to the PAG. The lateral hypothalamus is heavily labeled following iontophoretic deposits of the tracer enzyme into ventral (R-51) and ventrolateral portions (R-50 & R59) of the PAG, while the ventromedial hypothalamic nucleus (VMN) contains numerous marked cells following dorsolateral PAG injections (R-45 and R-49). A careful analysis of the location of labeled cells within the VMN indicated a topographical organization in this projection. Thus, the majority of neurons containing HRP reaction product occur in dorsomedial portions of the nucleus following HRP deposits into dorsal portions of the PAG (Fig. 6) while more ven-
Diameter of injection site (mm)
Longitudinal extent of injection site (mm)
Animal survival time (h)
0.32 0.36 0.21
0.90 0.70 0.50
31 29 29
0.96
2.40
32
0.40
1.60
28
0.80
1.00
28
0.55
1.90
24
0.91
1.50
28
1.28
2.00
32
0.38
0.90
30
0.81 0.55
1.90 1.20
30 34
0.80
1.70
28
None 0.88 2.2 1.29 1.05
None 1.50 3.10 2.10 2.40
30 28 30 36 36
trally-placed PAG injections result in heavier labeling in ventrolateral portions of the nucleus (Figs 5 and 10). The topography of lateral hypothalamic projections to the PAG was not as clear as VMN projections, however, there was a tendency for more dorsally-placed HRP deposits to label more lateral portions of this hypothalamic area. Both the anterior hypothalamic area and the posterior hypothalamic area contained significant label following iontophoretic injections into the central gray. Marked cells were found to predominate in cauda1 and lateral portions of the anterior hypothalamic area. The lateral portion of this hypothalamic region (the lateral anterior nucleus of Bleier, Cohn & Siggelkow6) was especially heavily labeled in case R-59. The
Fig. 2. Photomicrographs through the center of the horseradish peroxidase injection site from cases R-36, R-37 and R-38. A. Photomicrograph of a coronal section through the midbrain depicting the HRP deposit (arrow) in case R-36. 40 pm, Pyronin-Y, mag. = 12 x B. A tracing of the stained section shown in A illustrating the core (black) and surrounding spread (dots) of the injection site. The periaqueductal gray is indicated by the diagonal lines. C. A photomicrograph of a coronal section through the caudal midbrain depicting the HRP deposit (arrow) in case R-37. 40 pm, Pyronin-Y, mag. = 12 x D. Tracing of the stained section shown in C illustrating the extent of the HRP reaction product in case R-37. E. A photomicrograph of a coronal section through the midbrain depicting the HRP deposit (arrow) in case R-38. 40 pm, Pyronin-Y, mag. = 12 x F. This tracing diagram illustrates the full extent of the injection site in case R-38. Sagittal reconstructions of the injection sites from the three cases illustrated in this figure are shown in Fig. 3.
N\<
7 I
H
140
A. J. Beitz
d
d
c+r V
c+r V
R-36
R-37
rostra1
R-38 d
c+r V
d
R-59 Fig. 3. Sagittal reconstructions of the periaqueductal gray through the center of the horseradish peroxidase injection site for cases R-36, R-37, R-38. R-45 and R-59. The PAG is depicted by the diagonal lines. The core of the injection site in each case is indicated in black, while the outer fringe of the injection site is illustrated with stipple. Each drawing illustrates the rostrocaudal extent of the HRP deposit for the above cases. The PAG was reconstructed at a point 0.76 mm from the midline for case R-36 while the center of the injection site was determined to be 0.62 mm, 0.48 mm, 0.43 mm and 0.33 mm from the midline for cases R-37. R-38, R-45 and R-59, respectively. The rostra1 (r). caudal (c), dorsal (d) and ventral (v) directions are indicated.
cells in the posterior hypothalamic area were also predominantly localized to lateral parts of this region and were often found along the medial aspect of the mamillothalamic tract. The perifornical nucleus contained a relatively large number of marked cells following injections of HRP into the retrogradely-filled
ventrolateral subdivision of the central gray but contained no label following dorsolateral placements of the enzyme. These neurons were localized in a region immediately dorsal and medial to the fibers of the fornix. In addition to the hypothalamic regions listed in
group
1. 2. 3. 4.
Zona incerta Ventral lateral geniculate Lat. habenular n. Ant. pretectal n.
n.
Ant. hypothalamic a. Lat. hypothalamic a. Ventromedial hypothalamic n. Dorsomedial hypothalamic n. Post. hypothalamic a. Infundibular n. Periventricular hypothalamic n. Dorsal premamillary n. A. of the tuber cinerium Perifornical n. Med tuberal a. Lat. tuberal a. Dorsal hypothal. a.
Diencephalon
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Hypothalamus
1. Prefrontal cortex 2. Cingulate gyrus 3. Diagonal band Broca horiz. limb 4. Med. Preoptic A. 5. Lat. preoptic A. 6. Bed n. of stria terminalis 7. Magnocellular preoptic N. 8. Subst. innominata 9. Central amygdala
Forebrain
Nuclear
40 0 0 13
23 31 36 7 7 5 3 13 5 3 4 1 6
20 6 17 28 50 4 17 5 3
Ipsi
2 0 0 4
0 0 2 0 0 0 0 0 0 0 1 0 0
Contra
R-38 Entire right half of PAG
20 3 0 4
0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 2 2 0 0 0 0
Ipsi
0. 0 0 2
0 0 0 0 0 3 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0
Contra
R-43 Dorsal PAG subdivision (Caudal part)
23 3 0 0
2 3 36 0 3 0 0 3 3 0 3 0 0
0 0 0 0 0 0 0 0 0
Ipsi
2 0 0 12
1 1 0 0 2 0 0 0 0 0 0 0 0
Contra
R-45 Dorsolateral subdivision (Rostra1 Part
73 0 0 5
7 0 0 2
5 0 17 3 0 0 0 4 0 0 0 0 0
z”
i?
18 38 104 2 26 0 0 81 6 0 2 0 4
:m
Contra
gLo
Ipsi
R-49 Dorsolateral subdivision (Caudal part)
12 0 48 0
3 75 2 3 17 2 0 1 5 12 11 3 8
92 5 60 7 1
37 16 26 34
Ipsi
1 0 33 2
2 0 0 4 5 3 1
0
3 37 1 1 3
23 0 26 1 0
11 1 14 9
Contra
R-50 Ventrolateral subdivision (Caudal part)
18 5 0 0
27 123 3 1 12 4 0 3 32 16 5 2 19
39 24 2 31 27 10 5 0 1
Ipsi
3 0 3 0
3 31 0 0 0 0 0 0 5 1 1 1 1
8 4 0 3 5 0 3 0 0
Contra
R-59 Ventrolateral subdivision (Rostra1 part)
Table 2. A summary of the distribution of horseradish peroxidase labeled neurons in the forebrain, hypothalamus and diencephalon following iontophoretic injections of horseradish peroxidase which encompassed the entire periaqueductal gray on one side or which were confined to particular periaqueductal gray subdivisions. The case number and the horseradish peroxidase injection site are listed at the top of the table and the nuclear groups within the forebrain, hypothalamus and diencephalon which contained labeled neurons are indicated on the left. The laterality (ipsi = ipsilateral, contra = contralateral) of labeling for each nuclear group is also indicated
group
1. 2. 3. 4. 5. 6. I. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
Sup. colliculus (deep layer) Cuneiform n. Subcuneiform n. Substantia nigra pars compacta Substantia nigra pars lateralis Substantia nigra pars reticulata Parabigeminal n. Periparabigeminal a. Paralemniscal a. N. of the brachium of the inf coil Inf. colliculus external n. Locus coeruleus Raphe dorsalis Lat. parabrachial n. Med. parabrachial n. PAG (rostra1 to injection site) PAG (at injection) PAG (caud. to injection) Pontopeduncular n. Vent. tegmental a.
Midbrain & isrhmus
Nuclear
105 3 0 4 4 9 0 0 0 0 3 0 14 3 0 40 7 5 2
1
Ipsi 2 21 0 0 0 2 0 0 10 0 0 0 2 7 0 0 21 3 0 0
Contra
R-38 Entire right half of PAG
83 0 0 4 0 0 0 10 0 0 0 0 2 0 10 30 30 4 0
0
Ipsi 0 60 0 0 5 0 0 0 8 0 0 0 0 2 0 4 27 10 3 0
Contra
R-43 Dorsal PAG subdivision (Caudal part)
10 36 5 0 0 5 0 0 0 0 0 0 0 2 4 0 13 27 0 0
Ipsi 4 34 19 0 4 2 0 0 2 0 0 4 0 0 3 0 12 8 0 2
Contra
R-45 Dorsolateral subdivision (Rostra1 part)
4 152 3 0 20 2 3 28 2 2 11 3 2 0 0 33 8 32 0 0
Ipsi
IO 0 0 5 0 2 12 2 0 0 0 0 0 0 3 39 12 0 0
0
Contra
R-49 Dorsolateral subdivision (Caudal part)
0 43 2 I 2 4-l 0 0 0 0 0 3 25 18 20 22 20 0 9 10
Ipsi
11 30 0 5 3 38 3 0 11 0 2 4 20 9 9 23 57 0 4 10
Contra
R-SO Ventrolateral subdivision (Caudal part)
0 4 12 8 0 4
0 0
0
0 0
1 0 0 0
0
1 11 12 0 4
14 7 0 0
Contra 11 32 8 0 3 2 0 0 0 1 0 0 4 3 6
Ipsi
R-59 Ventrolateral subdivision (Rostra1 part)
Table 3. A summary of the distribution of horseradish peroxidase labeled neurons, in the midbrain and isthmus region following iontophoretic injections of horseradish peroxidase into the periaqueductal gray and its intrinsic subdivisions. The case number and the location of the horseradish peroxidase injection site are listed at the top of the table and the nuclear groups within the midbrain and isthmus region which contained labeled cells are indicated on the left. The laterality (ipsi = ipsilateral, contra = contralateral) of the labeling for each nuclear group is indicated
K z
? LI
N. pontis oralis N. pontis caudalis N. pontis caudalis pars a Reticulotegmental n. Sup. central n. Raphe magnus Vent. n. of lat lemniscus N. lat. dorsalis tegmenti Spinal n. of V N. suprageniculatus
N. N. N. N. N. N. N. N.
gigantocellularis paragigantocellularis gigantocell pars a prepositus hypoglossi cuneatus ret. medulla oblongata pars ventralis ret. medulla oblongata pars dorsalis solitarius
1. Dorsal horn 2. Ventral horn
Spinal cord
1. N. medialis 2. N. interpositus 3. N. lateralis
Cerebellum
1. 2. 3. 4. 5. 6. 7. 8.
Medulla
1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
Pons
Nuclear group
Table 4. A summation of the distribution
0 0
0 0 2
2 0 2 2 1 7 2 2
7 8 7 0 3 5 0 0 0 0
0 0
0 4 0
0 2 3 7 0 0 0 0
0 2 4 0 0 0 0 0 2 0
0 0 0
0 0 0 0 0 0 0 0
3 2 0 0 0 2 2 0 0 0
0 2 0
0 2 2 0 0 2 0 0
0 0 7 0 0 0 0 0 0 0
Contra
Ipsi
Ipsi
Contra
R-43 Dorsal PAG subdivision (Caudal part)
R-38 Entire right half of PAG
-
0 0 0
0 0 0 0 0 0 0 0
13 2 2 0 2 0 0 0 0 0
Ipsi
0 2 4
0 0 0 0 0 0 0 0
5 5 2 4 0 0 0 0 0 0
Contra
R-45 Dorsolateral subdivision (Rostra1 part)
0 0
0 0 0
0 0 0 0 0 2 0 0
2 3 3 0 0 0 1 0 0 0
Ipsi
0 0
0 4 0
0 0 0 0 0 0 0 0
3 0 0 0 0 0 2 0 4 0
Contra
R-49 Dorsolateral subdivision (Caudal part)
0 0
7 0 0
0 7 0 12 0 3 0 4
35 0 4 3 17 16 0 4 0 2
Ipsi
4 0
0 4 0
0 2 0 5 0 0 0 0
21 5 2 2 50 0 0 16 0 6
Contra
R-SO Ventrolateral subdivision (Caudal part)
injections of
0 0
1 0 0
3 2 2 2 0 5 3 0
8 7 0 0 2 3 0 7 0 2
Ipsi
2 0
B
g 6
0 8 2 0
0 2
5 0
% g B a a %. 2.
5 12
Contra
R-59 Ventrolateral subdivision (Rostra1 part)
of horseradish peroxidase labeled neurons, in the pons, medulla, cerebellum and spinal cord following iontophoretic horseradish peroxidase into the periaqueductal gray and its intrinsic subdivisions
Fig. 4. A charting diagram illustrating the location of labeled cells (black dots) in a series of coronal sections through the forebrain and dicncephalon from case R-38. The most rostra1 section is illustrated in A, while the most caudal section is shown in G. The injection site is illustrated in F and G. Note that the majority of labeled cells occur ipsilateral to the injection site. The abbreviations are indicated on p. 136. 144
Afferent projections to the periaqu~u~tal
gray
Fig. 5. A charting diagram illustrating the location of labeled cells (black dots) in a series of coronal sections through the forebrain and diencephalon following an injection of horseradish peroxidase into the dorsolateral periaqueductal gray (case R-45).
146
A. J. Beitz
Fig. 6. A charting diagram illustrating the location of labeled ceils (black dots) in a series of coronal sections
through
the forebrain
and diencephalon following a horseradish ventral periaqueductal gray (case R-51).
Table 2, there were a few hypothalamic
nuclei
which
in cases R-51 and R-59. The ipsilateral submamillothalamic nucleus contained 13 labeled cells in case R-59 and 8 marked cells in case R-51. This nucleus was not labeled following injections into dorsal, dorsolateral or medial portions of the central gray. The dorsal tuberal nucleus contained 7 retrogradely-marked cells in case R-59 and only 3 cells in R-51. Other nuclear groups labeled in these 2 cases include the parvocellular nucleus, and tubera) magnocellular nucleus,
contained
a small
number
of labeled
cells
peroxidase
injection
into the
the ventral premamillary nucleus and the paraventricular nucleus. Di~~cephtrlon. Next to the hypothalamus, the subthalamus provides the largest diencephalic input to the PAG. This input arises from the zona incerta which contained labeled cells in the majority of our cases (Fig. 4). Marked neurons were very prominent throughout the zona incerta following injections into dorsal and dorsolateral portions of the PAG (see Table 2). HRP injections involving the ventral and ventrolateral parts of the central gray resulted in only
Afferent projections to the periaqueductaf gray
Fig. 7. A charting diagram illustrating the location of horseradish peroxidase-labeled cells (black dots) in a series of representative coronal sections through the brain stem from case R-38. The midbraindiencephalic junction is illustrated in A, while the medullary-spinal cord junction is depicted in H. The core of the HRP injection site is represented in B and C. Note the predominance of labeled neurons ipsilateral to the injection site. The abbreviations are indicated on p. 136. a small amount of label in this subthalamic region. This projection appeared to be topographically organized in that dorsal PAG injections labeled more medial portions of the zona incerta than did ventral PAG injections. The ventral lateral geniculate nucleus and the Iateral habenular nucleus contained significant Iabel following ventrally placed HRP deposits in the PAG, while the anterior pretectal nucleus was most heavily labeled with dorsally-placed HRP deposits. Although not indicated in Table 2 a few labeled cells were ob-
served in the thalamic parafasicular nucleus in two of our cases (R-50 and R-51) and in an area just medial to the parafasicular nucleus in three of our cases (R-49, R-SO and R-51). This region adjacent to the parafasicular nucleus appears to correspond to the A-l 1 catecholamine group of Dahlstrom & Fuxe.r3 ~i~b~u~~ und isthmus region. Of ail the brain regions found to project to the PAG, the midbrain contained the greatest number and diversity of labeled structures. The nucleus cuneiformis contained the highest number of marked cells within the mesen-
148
A. J. Beitz
Fig. 8. A charting diagram illustrating the location of labeled neurons (black dots) in a series of sections through the brain stem from case R-45. The core of the injection site (black) and the outer limit of the HRP reaction product (stipple) are indicated in A and B.
representative
cephalon in all our cases. This tegmental nuclear group was found to provide bilateral input to the PAG with a majority of HRP-labeled cells occurring in the ipsilateral nucleus (Fig. 11). A topography in the nucleus cuneiformis projection to the PAG was noted in that HRP injections into dorsal protions of the PAG resulted in heavier labeling in medial parts of this nucleus. Ventral injections into the central gray produced more labeling in the lateral part of the nucleus cuneiformis. The midbrain area containing the second largest number of labeled cells was the PAG itself. Labeled neurons were found bilaterally throughout the PAG both rostra1 and caudal to the injection sites. In some cases (R-49, R-50 and R-51) the majority of labeled cells occurred in the contralateral PAG. Injections into the ventral PAG resulted
in numerous labeled cells primarily in the dorsal parts of the central gray (Fig. 6). Following injections into the dorsal and dorsolateral subdivisions of the PAG, however, HRP-positive cells occurred almost equally in both dorsal and ventral portions of the central gray. Injections of HRP into the medial subdivision of the central gray (R-33) is of special interest in that it produced heavier labeling in the nucleus cuneiformis and in the PAG than in any other CNS region. All three divisions of the substantia nigra contained marked cells in one or more of the experimental cases. The pars reticulata of the substantia nigra contained the greatest number of HRP-positive neurons following injections into ventral portions of the PAG (R-50, R-51) and the label was most prominent with ventrocaudal HRP deposits (R-50). Label neurons were
Fig. 9. A charting diagram illustrating the d~str~butjon of Labeled cells (black dots) on representative brain stem sections following an injection of horseradish peroxidase into the ventral periaqueductai gray (case R-51). The core of the injection site (black) and the outer limit of the HRP reaction product (stipple) are indicated in A, 9, and G. found predominantly in ventromedial parts of the pars reticulata in all cases. The greatest amount of labeling in the pars lateralis occurred following dorsal HRP placements in the central gray (Table 3) while the pars compacta was only labeled with ventrocaudai injections (R-50, R-35). The medial and lateral parabrachial nuclei contained significant label following injections of the tracer enzyme into the ventrolateral subdivision of the PAG while the periparabigeminal area was significantly labeled with caudal dorsolateral injections. The ipsilateral and contralateral deep layer of the superior colliculus contained marked cells in many of
the experimental cases. The heaviest labeling occurred in case R-59 following an injection into the ventrolateral subdivision of the central gray. The subcuneiform nucleus was labeled following injections of HRP into the medial, dorsolateral and ventrolateral subdivisions. Label occurred primarily in an area lateral and dorsolateral to the red nucleus. Smaller numbers of labeied cells were observed in the locus coeruleus, the ventral tegmental area and the paralemniscal area (Table 4). I%ns end cereMftlm. The ipsilaterat pontine reticular formation contained the heaviest labeling at this brain stem level. Retrogradely-labeled cells were most
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dense in the nucleus reticularis pontis oralis, especially following HRP injections into ventral and ventrolateral portions of the PAG (R-50, R-51 and R-59). The nucleus reticularis pontis caudalis and the nucleus reticularis pontis caudalis pars x contained significant label in case R-38 as well as in most of our experimental cases (see Table 4). The majority of labeled cells in the nucleus pontis oralis were localized in the ventrolateral part of this nucleus. while labeling in the nucleus pontis caudalis occurred along its medial border. Marked cells were observed in the ventral portion of the superior central nucleus following HRP injections into the ventrolateral subdivision of the PAC. The deep cerebellar nuclei contained a few labeled cells in almost all of our cases. The contralateral nucleus lateralis and nucleus interpositus posterior were found to contain labeled neurons primarily along their ventral aspect. The nucleus medialis was only labeled following injections into ventral portions of the PAG. HRP-filled cells were confined to the caudal subdivision of this cerebellar nucleus. Mrtlulltr. The heaviest labeling at this level of the brain stem occurred in the reticular formation. The nucleus reticularis medulla oblongata pars ventralis and the nucleus reticularis gigantocellularis pars a contained the largest number of labeled neurons. HRP-positive cells were found throughout the former nucleus but were localized to lateral portions of the nucleus gigantocellularis pars a. The nucleus reticularis gigantocellularis and the nucleus paragigantocellularis lateralis also contained marked cells localized to the caudal portion of both nuclei. Spinrrl cord. As indicated under Experimental Procedures, the spinal cord was examined in only five experimental cases and in two control animals (R-66 and R-180). A small amount of labeling was observed in three experimental animals (R-50, R51 and R-59) and the majority of cells in these cases were confined to lamina IV and V. The remaining two experimental cases contained no labeled cells in the spinal cord. Injections into either the superior colliculus (R-66) or into the nucleus cuneiformis (R-180) also resulted in a small amount of labeling within the dorsal horn of the spinal cord, predominantly in lamina IV. However, since the injection site in case R-50 and R-51 did not spread into the nucleus cuneiformis or the overlying superior colliculus. this could not account for the spinal cord labeling resulting from this PAG injection. These results would suggest that the ventral portion of the PAG is the only part of the central gray which receives direct spinal cord input.
HRP-labeled cells were not detected in any brain structures in case R-54 in which HRP was injected directly into the cerebral aqueduct. This negative result ruled out, at least in the present study: (1) a possible uptake of HRP by the supraependymal plexus adjacent to the aqueduct and subsequent retro-
grade transport to the somata of these axons;‘,9 (2) a possible uptake of HRP by brain parenchyma adjacent to the third, fourth and lateral ventricles following HRP transport within the cerebrospinal fluid. A comparison of the results of control injections into the superior colliculus, nucleus cuneiformis and dorsal raphe nucleus with the results of HRP injections into the PAG indicate some similarities between all 4 regions. More importantly, however, a comparison of these results indicate major differences both qualitative and quantitative between the control cases and the PAG injections. These findings suggest that the majority of labeling listed in Table 2 is the result of direct uptake of HRP from the PAG and that it is not due to uptake an subsequent retrograde transport of the enzyme from adjacent CNS structures. The heaviest labeling following PAG injections, for instance, occurred in the ventromedial hypothalamic nucleus, the lateral hypothalamic nucleus, the lateral preoptic area and the zona incerta (Table 2). By contrast, injections into the nucleus cuneiformis resulted in heavy labeling in the central nucleus of the amygdala, the lateral tuberal nucleus and the substantia nigra pars reticulata. The greatest number of labeled cells occurred in the zona incerta, the nucleus of the posterior commissure and the pretectal area subsequent to injections into the superior colliculus, while dorsal raphe injections resulted in significant labeling within the lateral habenular nucleus, the dorsal hypothalamic area. and the prefrontal cortex. DISCUSSION The midbrain periaqueductal gray has been shown in the present investigation to receive a broad spectrum of afferent input from brain stem, hypothalamic. basal forebrain and cortical regions. These connections would allow the PAG to be influenced by the limbic system, the sensory system and to some extent the autonomic and motor systems. These systems, moreover, appear to be related to specific portions of the central gray. The sensory system, for instance appears to have indirect projections to all divisions of the PAG. but the dorsolateral subdivision receives the largest input. The limbic and motor systems, on the other hand, project to ventral and ventrolateral portions of the PAG especially to its caudal one half. The major projections from each of these systems to the PAG will be considered below. Limhic
and autonomic
input
to the periaqueductal
gray
Because the limbic system and central portion of the autonomic system are closely related in many respects. especially, at the level of the hypothalamus. they will be discussed together. Aferents ,fiom corficcd reyion.s. The present results showing labeling of the medial prefrontal cortex after HRP injections into the PAG are compatible with an earlier degeneration study in which restricted lesions were placed in the medial frontal cortex of the squir-
Afferent projections to the ~riaqueductal rel monkey. 32 Leichnetz and Astruc, however, indicate that degenerating fiber terminal debris was found in the dorsolateral margin of the PAG. In the present study, the majority of labeling in the prefrontal cortex occurred following HRP injections into the ventrolateral subdivision of the PAG rather than the dorsolateral subdivision. The difference in the projection to the PAG in the present study of the rodent PAG, as compared with the study by Leichnetz & Astruc3’ in the monkey, may reflect a species variability in this pathway. However, negative results obtained with silver techniques used by these authors do not exclude a projection to the ventrolateral PAG. In support of our findings Sotnichencko6’ identified degenerating terminals within the greater part of the ipsilateral PAG following medial prefrontal cortex lesions in the cat. This investigator also indicated that the heaviest degeneration occurred at the most rostra1 levels of the central gray, which agrees well with the results of the present investigation. The heaviest labeling found in the prefrontal cortex occurred in our case R-51 with an injection into the rostroventral PAC. It is of interest that studies of the efferent projections of the PAG have demonstrated an input to the dorsomedial nucleus of the thalamus’O~ss which in turn provides a major input to the prefrontal cortex.h4 This loop which exists between the PAG and the prefrontal cortex may be one of several routes by which the PAG is interrelated with the limbic system. Other hmbic cortical areas were labeled in some of our cases with large HRP injections which were not confined to the PAG. Labeled neurons were observed in area 29 and in the subiculum in these cases. These 2 cortical areas, however, were also labeled following injections into the nucleus cuneiformis and thus we could not conclude with certainty that this labeling was the result of uptake of HRP from the central gray. In addition to containing labeled cells following large HRP injections into the PAG, area 29 contained marked cells in 2 cases with small injections of HRP which were limited to the PAG (R-50 and R-51). This finding would indicate that this cortical region does project to the central gray. Data which lend support to a possible projection from area 29 and also from the subiculum to the PAG are found in a recent report by Jurgens & Prattz8 and in an earlier study by Nauta.42 The former investigators found evidence of a projection from the cingulate gyrus to the PAG in the monkey, utilizing an autoradiographic tracing technique, while the latter investigator employed degeneration techniques to demonstrate a hippocampal projection to this region in the cat. A,flerenfs from fhe amyydala and basuf forebrain. A small number of labeled cells were observed in the central nucleus of the amygdala following injections of HRP into the ventrolateral PAG. This finding is consistent with the autoradiographic findings of Hopkins & Holstege ” in the cat. These investigators also describe a proiection to the rostra1 dorsolateral . ”
gray
151
central gray from the central nucleus of the amygdala. In the present study, however, no labeled cells were observed in the amygdala following HRP injections into the rostra1 portion of the dorsolateral PAG subdivision. Fibers from the bed nucleus of the stria terminalis have been described with anterograde methods by Conrad & Pfaff” to project to the PAG. The present experiments confirm these results with a retrograde method and show that labeling occurs in the bed nucleus after injections of the tracer enzyme into the ventrolateral subdivision of the PAG. Moreover. the labeled cells are localized to ventral portions of the bed nucleus. In the present study, labeled cells were also found in the horizontal limb of the diagonal band, predominantly along its dorsal aspect. Conrad & Pfaff,” on the other hand, found no evidence of terminations of diagonal band axons in the central gray. These authors, however, indicated that the projections they describe are primarily those of the vertical nucleus of the diagonal band and only a few medial and anterior cells of the horizontal nucleus were included in their injection sites. We found no labeled cells in the vertical limb of the diagonal band in our cases and thus the negative findings of Conrad & Pfaff are probably related to the exclusion of the dorsal part of the horizontal limb in their injection sites. Large neurons which were labeled in the horizontal limb were observed to merge laterally and posteriorly with large labeled cells of the substantia innominata and the magnocellular preoptic nucleus. These results are in agreement with autoradiographic data.63 The distribution of large labeled neurons in the horizontal limb of the diagonal band, magnocellular preoptic nucleus and substantia innominata agrees well with the distribution of magno~ellular basal forebrain neurons described by Divac.14 Neurons were also labeled in the nucleus accumbens in case R-50 where the HRP injection was centered in the caudal part of the ventrolateral PAG subdivision. This finding agrees well with the results of Nauta, Smith, Fauli & Domesick who described projections from the nucleus accumbens to the caudoventral central gray substance in the rat. The nucleus accumbens has been postulated to serve as a functional link between the limbic system and basal ganglia4’. The projection from the nucleus accumbens to the PAG is of interest in light of the present results which suggest that the PAG may also serve as an area of interface between the limbic and motor systems. Afirents from the diencephulon. Limbic efferents to the PAG from the diencephalon arose prodominantly from the hypothalamus and the lateral habenular nucleus. In addition, the hypothalamus probably supplies information related to autonomic function to the central gray. Quantitatively the lateral hypothalamus provides the largest hypothalamic input to the PAG. This input, moreover, is directed predominantly to the ventrolateral subdivision of this region. This finding is in agreement with the results of fiber degener-
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A. J. Beitz
ation42.71 and autoradiographic data43.4“ in the rat which also show fiber projections to the ventral portion of the PAG. The ventromedial hypothalamic nucleus provided the largest hypothalamic input to both the medial and dorsolateral subdivisions of the PAC. This finding is supported by the autoradiographic study of Krieger, Conrad & Pfaff3’ and the retrograde transport study of Grofova, Ottersen & Rinvik.” Krieger and coworkers3’ identified significant fiber projections to dorsal and medial aspects of the rodent central gray. Likewise. the work of Grofova tit trl.,‘” indicates that the ventromedial nucleus appears to be the most prominent hypothalamic source of afferents to the dorsal feline PAC. The dorsal premamillary nucleus was heavily labeled in case R-49 where the injection was localized to the dorsolateral subdivision of the PAG. Evidence in support of this projection can be found in the work of Krieger et L(/.31 These investigations found a dense projection from this nuclear region to the lateral aspect of the central gray along its rostrocaudal extent. A significant number of marked cells were found in the perifornical area following injections of HRP into the ventral central gray as well as injections into the dorsal raphe nucleus. These findings are in good agreement with the results of Sakai and coworkers.56 Finally, the present results, as well as those of Herkenham & Nauta,23 demonstrate a lateral habenular projection to the PAG. specifically to its ventral portion. .4,fl&~71t.sfiokn rhr hmin SIU~ The central gray receives input from certain brain stem nuclei which have been implicated in autonomic function. These include projections from the nucleus solitarus. the nucleus reticularis gigantocellularis, the nucleus paragigantocellularis and the parabrachial nuclei. These connections are related predominantly to the ventrolateral subdivision of the PAG. In addition, the PAG
receives input from the midbrain ventral area, a component of the limbic system.43 Motor
input
to the periuyueductul
tegmental
grq
The motor system also appears to be connected primarily with ventral portions of the PAG. Thus, the motor cortex. cerebellum and substantia nigra project predominantly to ventral and ventrolateral parts of the central gray. It has been demonstrated that vocalization can be elicited from the rodent PAG by electrical stimulation.68 and this region may serve as a relay station for motor impulses related to vocalization. Of interest in this regard is the report by Jiirgens & Prattz8 which indicates that the PAG plays an important role in the vocal expression of emotion. This finding may partially explain the overlap in the projections from the limbic system and motor system to the central gray.
The PAG receives input from several sensory regions of the central nervous system. A small input to the ventral portion of the central gray arises from lamina IV and V of the spinal cord. This is consistent with earlier degeneration studies3”,3’ and with more recent physiological studies’“. Lamina IV and V contain neurons which respond to noxious stimuli’” and also contains a large majority of the cells of origin of that portion of the spinothalamic tract which projects to the lateral thalamus.‘” Input from this part of the dorsal horn would thus provide a direct route by which nociceptive information could reach the PAG. The dorsal portion of the PAG receives input from the trigeminal nuclei. especially subnucleus oralis and interpolaris. In addition, the PAG receives input from the superior colliculus and pretectal area. PAG afferents from the superior colliculus were previously reported by Grofova and coworkers’” in the cat. These findings were confirmed and extended in the
Fig. 10. Representative photomicrographs of horseradish peroxidase-labeled cells III the forebrain and diencephalon. A. Low power photomicrograph (mag. = 60 x ) of the medial prefrontal cortex from case R-51. The arrow points to a labeled cell shown at higher magnification (600 x ) in B. C. LOW, magnilication photomicrograph (60 x ) illustrating the medial preoptic area and optic chiasm (oc) from case R-51. The arrow depicts the location of the labeled neuron illustrated in 7d at high magnitication (600 x ). t. Photomicrograph illustrating several labeled cells in the dorsal premamillary nucleus from cast R-49 (mag. = 230 x ). The third ventricle (III) is indicated and the HRP-positive cell at the arrow tip is shown at higher magnifcatlon (600 x ) in F. G. Photomicrograph of the posterior hypothalamic area from case R-49 (msg. = 230 x )_The arrow depicts the location of the labeled neuron shown at high magnification (600 x ) in 7H. Fig. 1 I. Representative photomicrographs of horseradish peroxidase-labeled cells observed In brain stem sections. A. Photomicrograph of the cerebral aqueduct (ag) and adjacent periaqueductal gray from case R-49 (msg. = 230x ). The arrow depicts a small labeled neuron in the medial aubdivision of the PAG which is shown at higher magnification (600x) in B. C. Photomicrograph illustrating a labeled neuron (arrow) in the nucleus cuneiformis from case R-59 (mag. = 230 x ). The periaqueductal gray (PAG) is indicated to the right of the nucleus cuneiformis. The labeled cell is shown at higher magnilication (600 x ) in 5D. E. Photomicrograph of a labeled cell (arrow) in the midbrain interpeduncular nucleus from case R-50 (msg. = 230 x ). The mamillary peduncle (mp) is shown on the right side of the photograph. The labeled neuron is shown at higher magnification (600 x ) in 5F. Low powcf- photomicrograph of the ventrolateral midbrain from case R-50 illustrating the location of a labeled cell (arrow) in the substantia nigra (mag = 60 x ). The cell is shown at higher magnilication in 5H (msg. = 600 x ).
Fig. 10. 153
Fig. 11 154
Afferent projections to the periaqueductal gray present investigation. Both the ispilateral and contralateral deep layer of the superior colliculus were labeled in our material and were especially prominent following dorsolateral and ventrolateral injections. Golgi studies have shown that cells in the deep layer of the superior colliculus extend their dendrites into the dorsal PAG49”. The evidence provided by Ram6n y Cajal would suggest the possibility that some of the marked cells in the ipsilateral superior colliculus were not labeled by retrograde axonal transport of HRP but rather by dendritic transport. Although it is not possible to differentiate which type of transport occurred in our experimental cases, support for an ipsilateral collicular projection to the PAG can be found in the autoradiographic study of Graham.18 This investigator depicts terminal label within the dorsolateral and ventrolateral portion of the ipsilateral PAG following amino acid injections into the stratum griseum profundum of the superior colliculus. The dorsolateral portion of the PAG also receives a large projection from the zona incerta which appears to have a role in the integration of impulses from many sensory regions. l9 This finding is supported by the recent autoradiographic studies of Ricardo53 which demonstrate zona incerta fiber terminations in the lateral central gray. It is noteworthy that the PAG is the recipient of efferent projections from the above sensory structures, since all of these brain regions have been found in physiological studies to be involved in pain mechanisms.16~29~52~6’ Functional
implications
The experimental results presented above suggest that in the rat the PAG receives a wide variety of ascending and descending neural input. Perhaps the most remarkable single point to emerge from our data is that the central gray is affected by a very great diversity of neural mechanisms. The limbic, autonomic and motor systems for instance, were found to provide input to the ventral PAG. It is possible that this region may represent an area of interface between these three systems. Coincidentally, the ventrocaudal portion of the PAG has been implicated in both stimulation and chemically produced analgesia16~72 and has been shown to receive nociceptive input from the periphery.’ 5s8 This region thus appears to be instrumental in pain control.58 Although pain is in some respects similar to other sensory modalities, it is more akin to motivational systems in some of its characteristics.35 Thus, unlike most other sensory modalities, it is almost invariably accompanied by an affective state. This effective dimension can be viewed as analogous to a ‘drive’ state in that it can command the attention of the organism and result in behaviors aimed at restoring homeostasis. The connections of the PAG demonstrated in the present investigation would also allow this region to serve as an interface between the limbic system and the sensory system. Thus, the limbic projections to the PAG may provide a route through which the limbic system can exert
155
some control over incoming nonciceptive input via descending pathways arising in the PAG.” The ventral portion of the PAG not only receives limbic input but reciprocates this input by projecting either directly or indirectly to limbic structures. Projections from the ventral PAG, have been described to the lateral hypothalamus, the dorsal medial nucleus of the thalamus and the ventral tegmental area.55 This anatomical substrate for interaction between the limbit system and the PAG may subserve the affectivemotivation components of pain. The present results also demonstrate a substantial hypothalamic input to the PAG. The hypothalamus is also the recipient of a dense fiber projection from the midbrain central gray.10~55 These findings in conjunction with the present results indicate that a reciprocal relationship exists between these two structures. This reciprocal interconnectivity, moreover, appears to be topographically organized. Thus, the present investigation has demonstrated a strong projection from the lateral hypothalamus to the ventrolateral subdivision of the PAG. Conversely, the most prominent PAG input to the lateral hypothalamus arises from its ventral portion.55 The dorsolateral PAG is interconnected with the posterior and dorsal hypothalamus in a similar fashion. This interrelationship between the hypothalamus and PAG probably reflects the coordinated involvement of these two structures in a number of brain functions. For instance, the hypothalamic-PAG system may play a major role in controlling feminine reproductive behavior as suggested by Krieger et al. 31 In support of this hypothesis, stimulation of the central gray has been shown to elict lordosis behavior in female rats,47 while lesions in this region depress lordosis.” Moreover, estrogen concentrating cells have been localized to the lateral aspect of the central gray. 46 In addition to a role in reproductive behavior this system may be involved in the central pressor effects of angiotensin II. The centrally mediated pressor activity of angiotensin is dependent upon activation of angiotensin receptors located in the preoptic hypothalamic region’ and in the dorsal PAG.59 Tissue within the rodent dorsal PAG and superior colliculus has also been shown to be essential in the maintenance of central pressor response to angiotensin II.59 Coincidentally, angiotensin II has been localized to the ventral PAG and the preoptic hypothalamic region.8 The possible involvement of the hypothalamic-PAG system in reproduction and in central pressor responses are but two examples of a number of functions that this system may subserve. As alluded to above, our anatomical data suggest that the PAG may serve as a functional interface between the limbic system and motor system. There is considerable evidence that limbic forebrain structures are important in ‘drives’ and ‘motivational’ processes40*41 contributing to the initiation of actions, but little is known about the neural mechanisms by which limbic processes gain access to the motor system. The subject of the interface between motivation
156
A. J. Beitz
and action-between limbic and motor systems--can be traced back to the classical experiments of Hess.24 Hess demonstrated that attack, feeding and other complex, biologically-significant behaviors could be elicited by electrical stimulation of the hypothalam~ts in unanesthetized cats. The neural processes resulting from electrical stimulation of the hypothalamus or limbic forebrain must eventually influence the motor system to produce attack, feeding or other behaviors observed. However. little progress has been made in elucidating the neural mechanisms by which the motor system is activated in such elicited behaviors. A major reason for the neglect of this important problem has been the absence of relevant anatomical evidence. Recently, anatomical evidence has been obtained which suggest that the nucleus accumbens, the lateral habenular nucleus and the ventral tegmental area may be three key structures linking the limbic system with the motor system. 41v43The present investigation demonstrates that the PAG not only receives direct connections from nucleus accumbens, ventral tegmental area and lateral habenular nucleus. but also receives direct input from important motor areas (i.e. motor cortex, cerebellum, substantia nigra) and limbic structures. The PAG is thus in a position to integrate both motor and limbic information and convey impulses to the spinal apparatus ~iu several alternative direct and indirect descending pathways. Studies of the efferent projections of the PAG’“*5s indicate that the central gray can also influence the limbic system t)icl connections to the lateral hypothalamus, ventral tegmental area and dorsal medial nucleus of the thalamas. In addition, the PAG may indirectly influence the motor system uitr projections to the zona incerta, the centromedian nucleus of the thalamas. the parafascicular nucleus and the reticular formation. These anatomical data suggest that the PAG may serve as an integrator of information derived from the limbic and motor systems. Its possible role in limbic-motor integration. however, requires further study.
One of the goals of this study was to determine if differences exist between the afferent input to the four subdivisions of the rodent PAG. The results of the present study clearly indicate a differential input to these four subdivisions from all levels of the CNS. In general, HRP injections involving the ventrolateral subdivision resulted in significant labeling in the basal forebrain. the lateral hypothalamus, the substantia nigra, the contralateral superior colliculus, and the brain stem reticular formation. A deposit of HRP into the dorsolateral subdivision, on the other hand, produced heavy labeling in the medial hypothalamus. This occurred primarily in the ventromedial hypothalamic nucleus, the dorsal premamillary nucleus and the posterior hypothalamic nucleus. In addition,
dorsolateral PAG injections resulted in significant labeling in the nucleus cuneiformis, the anterior pretectal nucleus, the substantia nigra pars lateralis and the zona incerta. Input to the medial subdivision of the PAG was examined in only one case (R-33) in which the core HRP reaction product was localized to this subdivision. Although not listed in Tables 3 and 4, the input to the medial subdivision was quantitatively much less than that to other PAG subdivisions. The largest numbers of labeled neurons in the brain resulting from the placement of HRP in this division occurred in the nucleus cuneiformis (I 1 cells), the rostra1 PAG (7 cells ipsilaterally, 2 cells contralaterally), the raphe dorsalis (8 cells) and the zona incerta (7 cells). Input to the caudal portion of the dorsal subdivision was examined in case R-43. The dorsal subdivision is unique, in contrast with the other divisions. in that it is the only PAG subdivision which does not receive hypothalamic input. Injections of HRP into this PAG subdivision result in a large number of marked cells in the nucleus cuneiformis (both ipsilaterally and contralaterally), in the PAG itself (both rostra1 and cauda1 to the injection site), in the paralemniscal area and the zona incerta. Finally, differences were also noted in the afferent projections to rostra1 and caudal portions of the PAG (see Tables 2-4). Although most of these differences were purely quantitative. a number of qualitative variations also occurred. The caudal portion of the ventrolateral subdivision. for instance, receives input from the locus coeruleus. the substantia nigra pars compacta, the reticulotegmental nucleus and the dorsal premamillary nucleus, while the rostra1 portion of this division does not. In conclusion. the present study has demonstrated that the rodent periaqLleductal gray receives a broad spectrum of input from a diversity of neural systems, including the limbic system. motor system, sensory system and autonomic system. A comparison of these results with studies of the efferent projections of the PAG’“-5” suggest that many brain regions arc reciprocally connected with the central gray. Many brain stem and diencephalic nuclei which have been implicated as components of the endogcnous analgesia system,4,‘“,3s for instance, not only receive PAG input but send projections back to this midbrain region. These reciprocal projections may be part of an important feedback rnech~~nisnl which allows moditication of PAG output. Needless to say, much physiological experimentation is required before the importance of these reciprocal connections is fully understood. Ackno~~t~c!yi~munrsThe A. McDonald aration
for their
author thanks Drs Jim Buggy and helpful
of this manuscript.
Research Grant Foundation.
BNS
comments
The
7906486
work
on thr
prep-
was supported
from the National
by
Science
Afferent
projections
to the periaqueductal
gray
157
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