University of Groningen
Spinal efferents and afferents of the periaqueductal grayMouton, Leonora Johanna
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Citation for published version (APA):Mouton, L. J. (1999). Spinal efferents and afferents of the periaqueductal gray: Possible role in pain, sexand micturition. [S.n.].
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69
Five spino-PAG systems
Chapter 6
The Segmental and Laminar Organization of the Spinal Neuronsprojecting to the Periaqueductal Gray (PAG) in the Cat
suggests the Existence of at least Five Separate Spino-PAG Systems
Leonora J. Mouton and Gert Holstege
J. Comp. Neurol. submitted.
AbstractThe present study describes the spinal cord projection to the periaqueductal gray (PAG) in thecat, taking into account different regions of the PAG and all spinal segments The retrogradewheat germ agglutinin-horseradish peroxidase results show that injecting different parts ofthe PAG leads to different laminar and segmental distributions of labeled spinal neurons.Evaluation of these results suggests that the spino-PAG projection is not one general ascend-ing pathway, but consists of at least five anatomically, and probably functionally, separatespino-PAG systems. System I originates in laminae I and V throughout the length of the cord,terminates in all parts of the PAG, and is probably involved in nociception. System II beginsin the ventrolateral part of laminae VI-VII of the C1-C4 spinal cord and terminates in theventrolateral and lateral parts of the rostrocaudal PAG, and in the deep tectum. System IIIoriginates in lamina X of the thoracic and upper lumbar cord, and terminates in the PAG aswell as in the deep tectum. System IV originates in the medial part of laminae VI-VII of thelumbosacral cord and projects predominantly to the lateral and ventrolateral caudal PAG. Itmight play a role in conveying tactile stimuli to the PAG during mating behavior. System Vbegins in the lateral part of lamina I of L6-S2 and in laminae V-VII and X of S1-S3. It termi-nates mainly in the central portion of the lateral and ventrolateral caudal PAG, and probablyrelays information concerning micturition and mating behavior.
IntroductionThe mesencephalic periaqueductal gray(PAG) plays an integrative role in emotionalresponses necessary for basic survival. Stimu-lation in the PAG of the freely moving rat andcat elicits defense behaviors, such as fight,threat display, flight and immobility (Bandlerand Depaulis, 1991; Carrive, 1993). Thesebehaviors include movements, such asrunning, jumping, arching of the back, turningof the head, vocalization, pupil dilatation, earpositioning and piloerection. Stimulation inthe PAG can also elicit cardiovascularchanges (Carrive, 1991, Bandler and Keay,1996, Lovick, 1996), micturition (Blok andHolstege, 1996), mating behavior (Sakumaand Pfaff, 1979) and analgesia (Basbaum andFields, 1984, Besson and Chaouch, 1987,Besson et al. 1991). Other studies (Lonstein
and Stern, 1997; Lonstein et al, 1998; Lon-stein and Stern, 1998) in which different partsof the PAG were lesioned, showed that thePAG is also involved in maternal behavior andmaternal aggression.The question is how the PAG organizes thesecomplex behaviors. Tracing studies revealedseveral of the efferent pathways that the PAGuses to generate its motor activities and anal-gesic effects (see Holstege, 1997 for review).Other studies have demonstrated that manyafferent pathways to the PAG originate fromvarious regions of the limbic system(Holstege, 1991; Shipley et al., 1991), but alsofrom the lower brainstem and spinal cord(Mehler, 1969; Menetrey, 1982; Mantyh,1982; Wiberg and Blomqvist, 1986; Bandlerand Tork, 1987; Yezierski and Mendez, 1991;
70
Chapter 6
Herbert and Saper, 1992).Earlier tracing studies on the projections ofthe spinal cord to the PAG (rat: Keay et al,1997, cat: Wiberg and Blomqvist, 1984; Keayand Bandler, 1992; Wiberg et al., 1987;Yezierski and Mendez, 1991) showed that thespino-PAG neurons are located bilaterally inthe cervical cord and contralaterally in thesacral cord, predominantly in lamina I, lami-nae IV-V, and laminae VI-VIII, and alsocontralaterally in the cervical and lumbar en-largements. In these latter parts many PAGprojecting neurons were seen in lamina I. Thespino-PAG neurons were found to terminatepredominantly in the lateral and ventrolateral,and to a limited extent in the dorsal PAG(Björkeland and Boivie, 1984). Recently,more precise studies on the projections fromthe upper cervical (Keay and Bandler, 1992)and lumbosacral cord (VanderHorst et al.,1996) to the PAG in the cat demonstrated theexistence of specific groups of neurons in thespinal cord projecting to different areas of thePAG. With respect to the C1 to C3 segments,Keay and Bandler (1992) reported that a ma-jority of lamina I neurons projected to the lat-eral PAG and a majority of laminae VII-VIIIneurons to the ventrolateral PAG. With respectto the L3-Coc3 segments a study from ourgroup (VanderHorst et al., 1996) showed theexistence of at least two different groups ofneurons in the lumbosacral cord projectingto the PAG. One group of neurons extendingfrom the lateral part of lamina I of L6-S2 intolaminae V-VII of S2 projects to the centralpart of the lateral caudal PAG, and anothergroup, located in the medial part of laminaeVII and VIII of the L5-S1 segments, projectsto the lateral part of the lateral caudal PAGand adjacent tegmentum. These findings ar-gue against the idea of the existence of onlyone general spino-PAG projection system,with only one function -the relay of nocicep-tive information- and they raise the questionof how the spino-PAG system is reallyorganized and what other functions, besidesthe control of nociception, are involved.
The present paper presents a detailed map ofthe complete spino-PAG projections in the cat,involving all spinal segments. The resultssuggest that the spino-PAG projection is notone general ascending pathway, but consistsof at least five anatomically separate spino-PAG systems, probably conveying differenttypes of information.
Materials and MethodsSurgical proceduresA total of 14 adult cats was used. The surgi-cal procedures, pre- and postoperative care,as well as the handling and housing of theanimals followed protocols approved by theFaculty of Medicine of the University ofGroningen.For surgery the animals were initiallyanesthetized with intramuscular ketamin(Nimatek, 0.1 ml/kg) and xylazine (Sedamun,0.1 ml/kg), after which they were keptanesthetized either by ventilation with a mix-ture of O2, N2O and halothane or by intrave-nous 6% pentobarbital sodium (≈ 0.2 ml perhour). During surgery, ECG and body tem-perature were monitored. Following a sur-vival time of 3 days the animals were initiallyanesthetized with intramuscular ketamin(Nimatek, 0.1 ml/kg) and xylazine (Sedamun,0.1 ml/kg), followed by an overdose of intra-peritoneal 6% pentobarbital sodium. The catswere perfused transcardially with two litersof 0.9% saline at 37oC, immediately followedby two liters of 0.1M phosphate buffer, con-taining 4% sucrose, 1% paraformaldehydeand 2% glutaraldehyde.
Histological proceduresIn 11 female cats 20-100 nl 2.5% wheat germagglutinin-conjugated horseradish peroxidase(WGA-HRP) in saline was injected into thePAG either with a Hamilton syringe, or witha glass micro pipette using a pneumaticpicopump (World Precision InstrumentsPV830). In one control case an injection wasmade in the tectal area lateral to the PAG. Intwo other control cases the injections were
71
Five spino-PAG systems
nal cords were cut into 33 separate segments(C1-Coc2). Each segment was cut into 40 µmfrozen transverse sections of which everyfourth (cases 2300, 2316, 2338, 2367, 2385,2390, 2395, 2401 and 2471) or every fifth(cases 2155, 2159 and 2182) section was in-cubated according to the tetramethyl benzi-dine method, dehydrated, and coverslipped.The brainstem area, containing the injectionsite, was cut in 40 µm sections and everyfourth section was processed with diamino-benzidine (DAB). The injection sites wereplotted with the aid of a drawing tube con-nected to a Zeiss brightfield stereomicro-
placed into the cerebrospinal fluid of the aq-ueduct. Except for case 2316, all injectionswere made stereotaxically, using Berman’satlas (1968) and approaching the PAGdorsally through the superior colliculus,dorsolaterally through the inferior colliculus,or caudally through the cerebellum and fourthventricle (case 2471). In case 2316 the corti-cal areas overlying the PAG were removedand the injection was made under visual guid-ance. After perfusion the brains and spinalcords were removed, post-fixed for two hoursand stored overnight in 20% sucrose in phos-phate buffer at 4o C. Subsequently, the spi-
2300
2155
2338
2471
Fig. 1. Photographs and drawings showing the injections sites of four cases. On the left: darkfield pho-tographs and in the middle: brightfield photographs of the injection areas (scale bar, 1000 µm). On theright: drawings of the injection sites in gray. The core of the injections is indicated in black.
72
Chapter 6
scope, and were described in terms of dorso-medial, dorsolateral, lateral and ventrolateralparts of the rostral, intermediate or caudalPAG.
Quantification of retrogradely labeled neu-ronsTo enable counting the spino-PAG neuronsin the various spinal segments, in each casethe retrogradely labeled neurons in all proc-essed transverse sections of the C1-Coc2 spi-nal cord were plotted in drawings, using adrawing tube connected to a Zeiss Axioskopwith darkfield polarized illumination, adigitizer, and a Macintosh computer. Fromthese drawings the numbers of ipsilaterallyand contralaterally located PAG projectingneurons per segment were counted. It shouldbe emphasized that it was not the aim of thisstudy to determine the absolute number ofspino-PAG projecting neurons.In seven of the PAG and/or tectum injectedcases one out of every four sections was proc-essed, in three cases one out of five. In orderto compare the numbers from these twogroups the 1:5 processed cases were corrected
into 1:4 cases by multiplying the countednumbers by 1.25. Such a correction is allowedbecause in both groups an unbiased samplewas taken and double counting of cells couldnot occur in a 1: 5 series of 40µm sections.The corrected numbers were used in all fur-ther analysis.In order to describe the laminar location ofthe spinal neurons projecting to the PAG, inall drawings the laminae of Rexed (1954)were depicted as well as a line dividing lami-nae VI and VII into a medial and lateral part.This line was set at half the distance betweenthe lateral border of lamina X and the lateralborder of the gray matter, dorsoventrally atthe level of the central canal (Fig. 3). Thelabeled neurons were counted in eight differ-ent areas, i.e.: lamina I, laminae II-IV, laminaV, the medial part of laminae VI-VII, the lat-eral part of laminae VI-VII, including the lat-eral motoneuronal cell groups, lamina VIIIwith the medial motoneuonal cell groups,lamina X, and the white matter. Results ofthese countings per segment are presented inhistograms. To enable reliable comparisonsbetween the different histograms, the magni-
Table 1. Numbers of labeled neurons (counted in 1:4 series) after WGA-HRP injections in different parts of the PAG and adjacent deep tectum.
case total ipsilateral contralateral
2367 2141 675 31.5% 1466 68.5%2159 2257 789 35.0% 1468 65.0%2385 5268 1462 27.8% 3806 72.2%2155 7468 2398 32.1% 5070 67.9%2316 551 211 38.3% 340 61.7%2395 539 122 22.6% 417 77.4%2300 1924 522 27.1% 1402 72.9%2390 1731 386 22.3% 1345 77.7%2401 1100 320 29.1% 780 70.9%2471 696 107 15.4% 589 84.6%2182 2649 643 24.3% 2006 75.7%2338 1633 646 39.6% 987 60.4%
73
Five spino-PAG systems
tude of the y-scale of all histograms is keptstandard, except for the histograms showingthe total numbers per segment and thoseshowing the numbers of neurons per segmentin lamina X. The y-scale of the histogramsshowing the total numbers of neurons per seg-ment, due to the high numbers per segment,is set at half the standard size. The y-scale ofthe histograms showing the numbers ofneurons in lamina X, due to the small numbersof labeled neurons per segment, is set at threetimes the standard size.
ResultsInjection sitesFig. 1 contains photomicrographs of four ofthe injection sites and the corresponding sche-matic drawings. In the drawings the core ofthe injections, as observed with brightfieldillumination, is shown in black. Fig. 3 con-tains schematic representations of all injec-tion sites in the PAG and adjacent deep tectalareas. In cases 2367 and 2159 the large injec-tions involved the rostral PAG and in cases2385 and 2155 the intermediate and caudalPAG. The other, smaller, injections wereplaced in the intermediate and/or caudal PAG,involving the dorsomedial and/or dorsolateral(cases 2316 and 2395) and the lateral and/orventrolateral parts (cases 2300, 2390, 2401,2471 and 2182). In control case 2338 theinjection involved the deep layers of thesuperior colliculus at the level of the rostraland intermediate PAG, and extended to onlya very limited extent into the lateral part ofthe PAG (Fig. 1).In cases 2270 and 2293 the WGA-HRP wasinjected in the cerebrospinal fluid of the aq-ueduct. These cases showed labeling of theependymal layer surrounding the aqueduct aswell as of the ependymal layer around thecentral canal of the spinal cord. No labeledneurons were found in these two cases, nei-ther in the brainstem nor in the spinal cord.In all other cases, except for case 2471,labeling of the ependyma was not observed.In case 2471, in which the injection was made
approaching the PAG caudally through thecerebellum and fourth ventricle, a smallamount of tracer leaked into the aqueduct,resulting in labeling of the ependyma aroundthe central canal throughout the length of thespinal cord.
Labeled neurons in the lateral cervical nu-cleus and dorsal column nucleiIn the upper cervical spinal cord labeled neu-rons were found in the cuneate and gracilenuclei and in the lateral cervical nucleus.These neurons are not included in this study.
Total number of labeled spino-PAG neuronsIn all cases many labeled neurons werepresent throughout the length of the spinalcord (Figs. 2-3). In cases 2385 and 2155, withlarge injections involving the intermediate andcaudal PAG and a small part of the tectum,but not the rostral and most caudal PAG, atotal number of 5268 and 7468 labeledneurons respectively were found. Taking intoaccount that these numbers are from a seriesof 1:4 transverse sections, but also consideringthat multiplication by four would lead to anoverestimation (Coggeshall and Lekan,1996), a conservative estimation is that thereare at least 15,000 spino-PAG neurons.The number of labeled neurons varied con-siderably from case to case. In case 2395, withan injection in the dorsolateral PAG, 539labeled cells were counted, in contrast to the7468 in case 2155, with a large injection in-volving the intermediate and caudal PAG (Ta-ble 1). Obviously, differences in numbers ofneurons per case largely reflect the differencesin injection size. On the other hand, the verysmall injection in case 2471 did not result inthe lowest numbers of labeled neurons, andthe slightly larger, but still limited injectionsin cases 2300, 2316, 2390, 2395, and 2401showed large variations in the numbers ofneurons, from 539 neurons in case 2395 to1924 neurons in cases 2300. Thisdemonstrates that certain regions of the PAGreceive more spinal afferents than others. For
74
Chapter 6
Fig. 2. left and right: Schematic drawings of the WGA-HRP injection sites in the different parts of thePAG. The AP coordinates are according to the atlas of Berman (1968). The black areas are consideredthe core of the injection sites. The histograms show the total numbers of retrogradely labeled neurons
0
2 0 0
C1
C3
C5
C7
T1
T3
T5
T7
T9
T1
1
T1
3
L2
L4
L6
S1
S3
Co
2
0
2 0 0
C1
C3
C5
C7
T1
T3
T5
T7
T9
T1
1
T1
3
L2
L4
L6
S1
S3
Co
2
0
2 0 0
C1
C3
C5
C7
T1
T3
T5
T7
T9
T1
1
T1
3
L2
L4
L6
S1
S3
Co
2
0
2 0 0
4 0 0
6 0 0
C1
C3
C5
C7
T1
T3
T5
T7
T9
T1
1
T1
3
L2
L4
L6
S1
S3
Co
2
0
2 0 0
4 0 0
C1
C3
C5
C7
T1
T3
T5
T7
T9
T1
1
T1
3
L2
L4
L6
S1
S3
Co
2
21822182218221822182
24012401240124012401
23002300230023002300
23162316231623162316
23852385238523852385
23672367236723672367
totaltotaltotaltotaltotal ipsilateralcontralateralA2.5
P0.9 A0.6P4.2
0
2 0 0
C1
C3
C5
C7
T1
T3
T5
T7
T9
T1
1
T1
3
L2
L4
L6
S1
S3
Co
2
75
Five spino-PAG systems
per segment, in a series of one out of four 40 µm transverse sections. The labeled neurons in the lateralcervical nucleus and in the dorsal column nuclei are not included.
0
2 0 0
C1
C3
C5
C7
T1
T3
T5
T7
T9
T1
1
T1
3
L2
L4
L6
S1
S3
Co
2
0
2 0 0
C1
C3
C5
C7
T1
T3
T5
T7
T9
T1
1
T1
3
L2
L4
L6
S1
S3
Co
2
0
2 0 0
C1
C3
C5
C7
T1
T3
T5
T7
T9
T1
1
T1
3
L2
L4
L6
S1
S3
Co
2
0
2 0 0
C1
C3
C5
C7
T1
T3
T5
T7
T9
T1
1
T1
3
L2
L4
L6
S1
S3
Co
2
0
2 0 0
4 0 0
6 0 0
C1
C3
C5
C7
T1
T3
T5
T7
T9
T1
1
T1
3
L2
L4
L6
S1
S3
Co
2
0
2 0 0
4 0 0
C1
C3
C5
C7
T1
T3
T5
T7
T9
T1
1
T1
3
L2
L4
L6
S1
S3
Co
2
A2.5 P4.2totaltotaltotaltotaltotal ipsilateral
contralateralP0.9 A0.6
23382338233823382338
24712471247124712471
23902390239023902390
23952395239523952395
21552155215521552155
21592159215921592159
76
Chapter 6
Tabl
e 2A
. Num
bers
of i
psila
tera
lly lo
cate
d la
bele
d ne
uron
s (c
ount
ed in
1:4
ser
ies)
per
sub
area
thro
ugho
ut th
e co
rd, a
fter
WG
A-H
RP
inje
c-tio
ns i
n di
ffere
nt p
arts
of
the
PA
G a
nd a
djac
ent
deep
tec
tal
laye
rs.
The
per
cent
ages
rel
ate
to t
he t
otal
of
the
ipsi
late
rally
loc
ated
lab
eled
neur
ons
thro
ugho
ut th
e le
ngth
of t
he s
pina
l cor
d.
wm
, whi
te m
atte
r.
case
tota
l
III-
IVV
VI-
VII
VI-
VII
VIII
Xw
mla
tm
ed
2367
675
81.
2%1
0.1%
147
21.8
%33
649
.8%
8011
.9%
8813
.0%
111.
6%4
0.6%
2159
789
172.
2%3
0.4%
133
16.9
%52
366
.3%
8811
.2%
91.
1%16
2.0%
00.
0%23
8514
6218
512
.7%
171.
2%43
229
.5%
538
36.8
%20
113
.7%
533.
6%28
1.9%
80.
5%21
5523
9829
212
.2%
552.
3%58
824
.5%
835
34.8
%35
014
.6%
176
7.3%
883.
7%14
0.6%
2316
211
4722
.3%
10.
5%11
152
.6%
3215
.2%
188.
5%0
0.0%
20.
9%0
0.0%
2395
122
21.
6%0
0.0%
6150
.0%
3629
.5%
108.
2%13
10.7
%0
0.0%
00.
0%23
0052
273
14.0
%29
5.6%
133
25.5
%19
337
.0%
6312
.1%
224.
2%7
1.3%
20.
4%23
9038
634
8.8%
61.
6%16
643
.0%
115
29.8
%42
10.9
%14
3.6%
92.
3%0
0.0%
2401
320
175.
3%3
0.9%
105
32.8
%14
244
.4%
185.
6%29
9.1%
10.
3%5
1.6%
2471
107
2220
.6%
00.
0%24
22.4
%36
33.6
%21
19.6
%2
1.9%
21.
9%0
0.0%
2182
643
164
25.5
%28
4.4%
211
32.8
%94
14.6
%96
14.9
%33
5.1%
132.
0%4
0.6%
2338
646
223.
4%2
0.3%
164
25.4
%34
653
.6%
507.
7%44
6.81
%11
1.7%
71.
1%
77
Five spino-PAG systems
Tabl
e 2
B. N
umbe
rs o
f co
ntra
late
rally
loca
ted
labe
led
neur
ons
(cou
nted
in 1
:4 s
erie
s) p
er s
ubar
ea t
hrou
ghou
t th
e co
rd,
afte
r W
GA
-HR
Pin
ject
ions
in
diffe
rent
par
ts o
f th
e P
AG
and
adj
acen
t de
ep t
ecta
l la
yers
. T
he p
erce
ntag
es r
elat
e to
the
tot
al o
f th
e co
ntra
late
rally
loc
ated
labe
led
neur
ons
thro
ugho
ut th
e le
ngth
of t
he s
pina
l cor
d.
wm
, whi
te m
atte
r.
case
tota
lI
II-IV
VV
I-V
IIV
I-V
IIV
IIIX
wm
lat
med
2367
1466
222
15.1
%8
0.5%
406
27.7
%23
115
.8%
313
21.4
%25
517
.4%
251.
7%6
0.4%
2159
1468
213
14.5
%8
0.5%
410
27.9
%34
023
.2%
404
27.5
%53
3.6%
352.
4%5
0.3%
2385
3806
1076
28.3
%60
1.6%
1070
28.1
%62
316
.4%
693
18.2
%21
45.
6%46
1.2%
240.
6%21
5550
7013
2326
.1%
104
2.1%
1168
23.0
%81
116
.0%
1050
20.7
%37
47.
4%19
43.
8%46
0.9%
2316
340
118
34.7
%0
0.0%
157
46.2
%28
8.2%
319.
1%3
0.9%
20.
6%1
0.3%
2395
417
4611
.0%
20.
5%22
854
.7%
358.
4%28
6.7%
6716
.1%
102.
4%1
0.2%
2300
1402
364
26.0
%68
4.9%
463
33.0
%18
713
.3%
243
17.3
%32
2.3%
342.
4%11
0.8%
2390
1345
324
24.1
%11
0.8%
498
37.0
%18
113
.5%
241
17.9
%57
4.2%
211.
6%12
0.9%
2401
780
134
17.2
%5
0.6%
333
42.7
%11
715
.0%
104
13.3
%64
8.2%
101.
3%13
1.7%
2471
589
234
39.7
%0
0.0%
7913
.4%
117
19.9
%12
621
.4%
274.
6%6
1.0%
00.
0%21
8220
0681
040
.4%
502.
5%49
524
.7%
175
8.7%
306
15.3
%10
05.
0%44
2.2%
261.
3%23
3898
732
3.2%
80.
8%41
141
.6%
246
24.9
%18
418
.6%
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Fig. 3. Schematic drawings of the labeled neurons in the C1-Coc2 spinal cord after a WGA-HRP injec-tion in the lateral intermediate and caudal PAG (case 2390). Each drawing represents twelve 40 µmthick sections. wm, white matter; cun, cuneate nucleus; grac, gracile nucleus.
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cal (40-60%) and lumbosacral cord (10-30%).In cases 2316 and 2471 the majority of labeledneurons was found in the lumbosacral cord.A moderate number of afferents originatedfrom the thoracic cord, mainly from the up-per thoracic segments, and a few from thecoccygeal segments. With the exception ofC1, most labeled neurons were presentcontralaterally. In C1, a large number of ipsi-laterally located neurons was found, some-times even exceeding the number of contral-ateral cells at this level (cases 2159 and 2401).
Lamina IThroughout the cord approximately 1600 and1200 labeled lamina I neurons were found incases 2155 and 2385 respectively, with largeinjections in the caudal and intermediate PAG(Table 2). Only 230 labeled lamina I neuronswere found in cases with large injections inthe rostral PAG (2367 and 2159), and only54 after the injection in the deep tectal layers(case 2338).With the exception of the sacral cord, alllamina I-PAG projecting neurons were lo-cated in the dorsal and dorsolateral parts oflamina I, and not in its medial or ventrola-teral parts. In the sacral cord labeled lamina Ineurons were found in the dorsal and dorso-lateral parts, but also more ventrolaterally(Figs. 3 and 9). With the exception of the sac-ral cord, the contralateral lamina I-PAG pro-jection was five times stronger than the ipsi-lateral one.Fig. 4 shows the numbers of lamina I neu-rons per segment in each case. The resultsindicate that all spinal segments containlamina I neurons projecting to the PAG, andthat they are most numerous in the cervicaland lumbosacral enlargements. Another find-ing is that labeled lamina I neurons werepresent in both the rostral and caudal PAGinjected cases (cases 2367, 2159, 2385 and2155), indicating that the lamina I-PAG pro-jections target the entire rostro-caudal extentof the PAG. However, independent of the spi-nal level, the lamina I projection was always
example, many more spinal neurons projectedto the lateral and ventrolateral PAG (cases2300, 2390, 2401 and 2471) than to thedorsomedial and dorsolateral PAG (cases2316 and 2395). The central part of the vent-rolateral and lateral PAG (cases 2300 and2471) received the strongest projections.Although the spino-PAG projections werebilateral, about 70% of the PAG projectingspinal neurons was located contralaterally(Table 1). In the control case with an injec-tion in the deep tectal layers (case 2338) 60% of the labeled neurons was found on thecontralateral side.
Total number of labeled neurons per laminaDifferent injection sites resulted in differentnumbers of labeled neurons per laminathroughout the cord. The largest numbers oflabeled neurons were found contralaterally inlaminae I, V and lateral VI-VII, and ipsilater-ally in lamina V and the lateral part of lami-nae VI-VII (Table 2). Smaller numbers werefound contralaterally in laminae VIII and Xand ipsilaterally in lamina I, the medial partof laminae VI-VII and in laminae VIII andX. Relatively few labeled neurons werepresent in laminae II-IV and no labeled neu-rons were present in the motoneuronal cellgroups. A few labeled neurons were locatedin the white matter lateral to the intermediatezone and ventral horn, mainly contralaterally.In case 2338 with an injection in the deeptectal layers most of the labeled neurons werefound in lamina V contralaterally, and in lami-nae VI-VII ipsilaterally. In contrast to mostPAG injected cases, this tectum injected caseshowed almost no labeled cells in lamina I.
Distribution per segmentGeneral overviewFig. 2 shows the total number of labeled neu-rons per segment. Large segmental differ-ences were found in the numbers of spino-PAG projecting neurons, even between adja-cent segments. In most cases the spinalafferents came mainly from the upper cervi-
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lamina Ilamina Ilamina Ilamina Ilamina I
Fig. 4. Histograms showing the segmental distribution of the labeled neurons in lamina I in the C1-Coc2spinal cord. The numbers represent the labeled neurons in a series of 1:4 transverse sections.
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lamina Vlamina Vlamina Vlamina Vlamina V
Fig. 5. Histograms showing the segmental organization of the labeled neurons in lamina V in the C1-Coc2 spinal cord. The numbers represent the labeled neurons in a series of 1:4 transverse sections.
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stronger to the intermediate and caudal PAGthan to its rostral part. In other words, thecaudal PAG injected case 2385, compared tothe rostral PAG injected case 2367, not onlyshowed more labeled lamina I neurons in thelumbosacral cord, but also in the cervical cord.Apparently, all lamina I neurons have a simi-lar projection pattern in the PAG, which isindependent of their segmental location.The results also revealed that the ventrola-teral and lateral parts of the intermediate PAG,and especially the medial area adjacent to theaqueduct, received strong afferent projectionsfrom the lateral part of lamina I of the L6-S2segments (cases 2155, 2385, 2300, 2390,2471 and 2182). These neurons are part ofthe earlier defined specific lumbosacral-PAGprojection, which also involves neurons in thelateral part of laminae V, VI and VII (seeVanderHorst et al., 1996; and system V in thediscussion)
Laminae II, III and IVOnly very few labeled neurons were found inlaminae II, III and IV (Table 2). More thanhalf of these labeled laminae II-IV neuronswas located in the C1 segment.
Lamina VWithin lamina V, most PAG projecting neu-rons were located laterally (Figs. 3 and 9),and only in the C1-C2 and sacral segmentsalso medially (Fig. 3). The contralaterallamina V-PAG projection was about twice asstrong as the ipsilateral one (Fig. 5).The rostrocaudal distribution of the laminaV-PAG neurons resembled that of the laminaI neurons, except that in the upper cervicalcord, and especially in the C1 segment, manymore lamina V-PAG neurons were found (Fig.5). These higher numbers of labeled laminaV cells in the upper cervical cord were notpresent in cases 2316 and 2471. Another dif-ference between the distributions of labeledneurons in lamina I and V was in whichlumbosacral segments the large numbers oflabeled neurons were found. They were not
found in the L6-S2 segments, as in lamina I,but in the S1-S3 segments. However, bothgroups of cells belong to the same distinctlumbosacral cell group projecting to the PAG(see system V in the discussion).The deep tectal layers received much strongerprojections from lamina V than from laminaI, but mainly from the upper cervical cord(control case 2338).
Lateral part of laminae VI and VIIOnly a limited number of labeled neuronswere found in the lateral part of laminae VIand VII, with the exception of the cervicaland sacral cord. In the upper cervical cord,and especially in C1, in all cases, except 2316,2395 and 2471, large numbers of labeled neu-rons were found in this area (Fig. 6). The greatmajority was located in the ventrolateral por-tion of lamina VII (Figs. 3 and 9). In contrastto all other spino-PAG projections, this up-per cervical laminae VI-VII projection to thePAG and deep tectal layers was stronger ipsi-laterally than contralaterally.Another group of labeled laminae VI-VII neu-rons was found laterally in laminae VI-VII inthe sacral segments, but only in the cases withan injection involving the lateral or ventrola-teral intermediate and caudal PAG (cases2385, 2155, 2300, 2390, 2401, 2471 and2182). This group of neurons also takes partin the earlier mentioned lumbosacral cellgroup (see system V of the discussion).
Medial part of laminae VI and VIIThroughout the length of the spinal cord threeseparate populations of labeled neurons wereobserved in the medial part of laminae VI-VII (Fig. 7). The first population was locatedin the C1-C2 segments, between the cuneatenucleus and the medial border of lamina Vbilaterally, with a contralateral preponder-ance. The second population consisted of neu-rons scattered throughout the medial part oflaminae VI and VII in the cervical and upperthoracic segments, mainly contralaterally.These two populations were present in all
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cases in which the injections involved the tec-tum (cases 2155, 2159, 2182, 2338, 2367 and2385), but were almost absent when the tec-tum was not (case 2471), or only slightly in-volved in the injection site (cases 2300, 2316,2390, 2395, and 2401).The third population was located mainlycontralaterally in the L5-S3 segments (Figs.3 and 9). This group was most prominent inthe cases with injections involving the lateraland ventrolateral parts of the intermediate andcaudal PAG, and also takes part in the dis-tinct lumbosacral-PAG projection (see systemV of the discussion)
Lamina VIIIIn all cases most labeled lamina VIII neuronswere observed in the cervical, and a few inthe lumbosacral cord (Fig. 8). Within laminaVIII most labeled neurons were locateddorsolaterally, some dorsomedially and onlya few in other parts (Fig. 3).
Lamina XIn all cases labeled neurons were found inlamina X, mainly contralaterally. In contrastto all other groups of retrogradely labeledneurons, most of the labeled lamina X neu-rons were located in the thoracic and upperlumbar cord, and virtually none in the cervi-cal cord (Fig. 9). Especially in cases 2155 and2300 relatively many labeled neurons werealso found in lamina X of the sacral cord, butthe impression was gained that also these neu-rons belonged to the distinct cell group lo-cated in laminae I and VI-VIII of the lum-bosacral cord.
DiscussionTechnical considerationsOne major concern in tracing studies is thepossibility of uptake of tracer by fibers ofpassage. In the present study the retrogradetracer used is WGA-HRP, of which is knownthat the problem of uptake of tracer by fibersof passage is very small compared to manyother tracers. Furthermore, a comparison be-
tween the results from different cases revealedthat uptake of tracer did not cause a majorproblem. In case 2300 a relatively small in-jection of WGA-HRP was made in the cen-tral part of the lateral PAG, involving only asmall part of the laterally adjacent tegmen-tum. This injection resulted in many labeledneurons in the upper cervical and lumbosac-ral cord. In cases 2390 and 2401 WGA-HRPinjections were placed more laterally in thelateral PAG, and in case 2338 the injectionalmost exclusively involved the deep tectalarea laterally adjoining the lateral PAG. Thespinal afferent fibers always enter the PAGventrolaterally and laterally (Mehler, 1969;Björkeland and Boivie, 1984; Yezierski,1988). An injection in the lateral PAG and/orthe laterally adjacent deep tectal area, involvesthe fibers terminating in the lateral PAG, aswell as the fibers passing to the medial partof the lateral PAG. On the other hand, an in-jection in the medial part of the lateral PAGwill exclusively involve the medially locatedterminals. If a substantial uptake of WGA-HRP occurs by fibers of passage, it wouldresult in more labeled spinal neurons in thelaterally injected cases 2390, 2401, and 2338than in the medially injected case 2300. Theresults, however, show exactly the opposite.Thus, although one can never rule out thepossibility of some uptake of tracer by fibersof passage, the results demonstrate that in thepresent study this has not been a majorproblem.
Comparison with earlier spino-PAG studiesIn the present study as many as 5000 to 7500labeled neurons were found in cases with largeinjections in the intermediate and caudal PAG,counted in a 1:4 series of sections. Such alarge number of spino-PAG neurons has neverbeen reported before, but in none of the ear-lier studies on the spino-PAG projections (rat:Keay et al, 1997, Yezierski and Mendez, 1991;cat: Wiberg and Blomqvist, 1984; Keay andBandler, 1992; monkey: Wiberg et al., 1987;Zhang et al, 1990) were injections made in-
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lateral part of lamina VI/VIIlateral part of lamina VI/VIIlateral part of lamina VI/VIIlateral part of lamina VI/VIIlateral part of lamina VI/VII ipsilateralcontralateral
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Fig. 6. left and right: Histograms showing the segmental organization of the labeled neurons in thelateral part of laminae VI-VII in the C1-Coc2 spinal cord. The numbers represent the labeled neurons ina series of 1:4 transverse sections.
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Fig. 7. Histograms showing the segmental organization of the labeled neurons in the medial part oflaminae VI-VII in the C1-Coc2 spinal cord. The numbers represent the labeled neurons in a series of 1:4transverse sections.
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Five spino-PAG systems
0
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2
Fig. 8. Histograms showing the segmental organization of the labeled neurons in lamina VIII in the C1-Coc2 spinal cord. The numbers represent the labeled neurons in a series of 1:4 transverse sections.
ipsilateralcontralateral
lamina VII Ilamina VII Ilamina VII Ilamina VII Ilamina VII I
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23852385238523852385
23672367236723672367
23162316231623162316
21552155215521552155
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23902390239023902390
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0
2 0
4 0
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C3
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2
Fig. 9. Histograms showing the segmental organization of the labeled neurons in lamina X in the C1-Coc2 spinal cord. The numbers represent the labeled neurons in a series of 1:4 transverse sections. Notethat the y-scaling is three times larger than that of the histograms shown in figures 3-8.
lamina Xlamina Xlamina Xlamina Xlamina X ipsilateralcontralateral
0
2 0
4 0
C1
C3
C5
C7
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23852385238523852385
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Five spino-PAG systems
volving such a large rostrocaudal part of thePAG. It should be emphasized, however, thata precise comparison between the numbersof spino-PAG neurons (totals, numbers perlaminae, or numbers per segment) observedin the present study, and the numbers pre-sented in other studies, is difficult. There arethree reasons that the numbers in all earlierstudies are much less precise. First, in sev-eral of these studies these numbers are theresult of large injections primarily targettingthe intercollicular area and not the PAG(Wiberg and Blomqvist, 1984; Wiberg et al,1987; Yezierski and Mendez, 1991). Second,often the numbers of labeled neurons werederived from blocks consisting of several seg-ments, and were “averaged”. For instance, thetotal number of neurons was taken from 25sections out of a block containing the C1-C4segments (Yezierski and Mendez, 1991).Third, some of the earlier studies give num-bers that result from averaging the numbersobtained from several cases with differentinjection sites, even when there were largedifferences between the numbers of neuronsper case (Keay et al., 1997).The segmental and laminar distribution of thespino-PAG neurons observed in the presentstudy are generally in good agreement withthose found in earlier studies. Similar to theearlier studies, the present results show manylabeled neurons bilaterally in the cervicalcord, contralaterally in the sacral cord, and inlamina I of the cervical and lumbar enlarge-ments. However, the present results do notshow a different input from C1-C3 to the ven-trolateral PAG compared to the lateral PAG,as reported by Keay and Bandler (1992) inthe cat and by Keay et al. (1997) in the rat.Instead, it was found that the upper cervicalprojection to the central part of both the lat-eral and ventrolateral PAG was much strongerthan to the more lateral parts of these sameregions. Differences in the spinal projectionsto the medial versus the lateral PAG were alsoreported by Mantyh (1982) in the primate, butthis study did not report precisely which seg-
ments were involved, which prohibits a reli-able comparison with the present results.The same is true in respect to comparing thesegmental and laminar distributions of thespino-PAG neurons observed in the presentstudy and those in other earlier studies(Mehler, 1969; Hazlett et al., 1972; Beitz,1982; Björkeland and Boivie, 1984; Wibergand Blomqvist, 1984; Meller and Dennis,1986; Wiberg et al., 1987; Harmann et al.,1988; Lima and Coimbra, 1989; Zhang et al.,1990; Yezierski, 1991; Keay and Bandler,1992; Bernard et al., 1995; Craig, 1995;VanderHorst et al., 1996; Keay et al., 1997),mainly because none of them has investigatedthe different parts of the PAG on one handand all separate spinal segments on the otherhand, as was done in the present study.
The existence of five separate spino-PAGsystemsComparing the different laminar and segmen-tal distributions of the spino-PAG neuronsafter injections in different parts of the PAG,gives the impression that the spino-PAG pro-jection is not one general ascending system,but consists of different groups of spinal neu-rons that project to different parts of the PAG.In all cases in all segments labeled lamina Iand V neurons were found, which suggeststhe existence of one particular system origi-nating in laminae I and V throughout thelength of the spinal cord and projects to allparts of the entire rostrocaudal PAG, butstronger to the intermediate and caudal PAG.However, besides this rather diffuse system,at least four other specific systems can be dis-tinguished on the basis of the laminar andsegmental locations of their neurons. It hasled to the hypothesis that there are at leastfive separate spino-PAG systems (Figs. 10and 11):System I (indicated in red in Fig. 11) ispredominantly contralateral and originates inthe dorsolateral part of lamina I and the lateralpart of V (Fig. 10) throughout the length ofthe spinal cord. About 50 % of all spino-PAG
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neurons belong to this system.System II (indicated in gray in Fig. 11)originates exclusively from the upper cervicalcord (C1-C3) and most strongly from C1. Thissystem is bilateral, but predominantlyipsilateral, and originates from the lateral partof laminae VI-VII and the dorsolateral partof lamina VIII (Fig. 10). System II projectsto the ventrolateral and lateral parts of theentire rostrocaudal PAG and to the adjacentdeep tectal layers. Almost 20 % of all spino-PAG neurons belong to this system.System III (indicated in green in Fig. 11) ispredominantly contralateral, originates inlamina X of the thoracic and upper lumbarspinal cord (Fig 10) and projects to the PAGas well as to the adjacent deep tectal area.About 2% of all spino-PAG neurons belongto this system.System IV (indicated in yellow in Fig. 11) ispredominantly contralateral and originates inthe medial part of laminae VI-VII of the L5-S3 segments (Fig. 10). It terminates in thelateral and ventrolateral parts of therostrocaudal PAG, but mainly in itsintermediate and caudal region. About 10 %of all spino-PAG neurons take part in thissystem.System V (indicated in blue in Fig. 11) hasbeen described in detail in an earlier paper ofour department (VanderHorst et al., 1996) asa system that is predominantly contralateraland originates in the lateral part of lamina Iof the L6-S2 segments and the lateral parts oflaminae V-VII and X at the S1-S3 levels (Fig.10). The present results show that there arealso some neurons located in lamina X of thesacral cord that may belong to this system.System V projects to the lateral, ventrolateraland dorsomedial parts of the intermediate andcaudal PAG, but most strongly to its centralportion (see cases 2300 and 2471). About 10%of all spino-PAG neurons belong to systemV.
Possible functions of the 5 systemsAlthough physiological data are scarce, it is
tempting to predict that each of the 5 systemssubserves a different function. Consideringthe locations of the cells of origin of each ofthe different systems the following functionalconsiderations can be made:System I contains laminae I and V neuronsthroughout the length of the cord that projectto all parts of the PAG. Lamina I is known toreceive afferent fibers from nociceptors andthermoreceptors from the skin as well as frommuscles, joints and viscera. Lamina V re-ceives afferent fibers from mechanoreceptorsin the skin, joints and viscera (Willis andCoggeshall, 1991). Apparently, the entirerostro-caudal PAG, but mainly its lateral andventrolateral parts, receive nociceptive, ther-mic, mechanical and/or visceral informationfrom all parts of the body.System II contains neurons in the lateral partof laminae VI-VIII of the C1-3 segmentswhich project to the lateral and ventrolateralPAG and to the deep tectum. Keay andBandler (1992) and Keay et al. (1997) alsofound many laminae VI-VII neurons in thesesegments projecting to the PAG and suggestedthis projection to play a role in conveyinginformation from the deep neck muscles tothe ventrolateral PAG. The results of thepresent study, however, do not support thishypothesis, because the location of the largenumbers of C1-C3 laminae VI-VII neuronsprojecting to the PAG does not coincide withthe area where the deep neck muscle afferentsterminate. The former terminate medially inthe upper cervical intermediate zone (Hiraiet al., 1984; Bakker et al., 1984; Abrahams etal., 1984; Nyberg and Blomqvist, 1984; Roseand Keirstead, 1986), while the PAGprojecting neurons were found more laterally.The medial region also contained retrogradelylabeled neurons when the injection involvedthe deep tectum, suggesting that the neckmuscle information is not conveyed primarilyto the PAG, but to the deep tectum.The lateral part of the intermediate zone, withthe many PAG projecting neurons, receivesmany afferents from the ventrolateral and lat-
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Fig. 10. Darkfield polarized photomicrographs showing labeled neurons of each of the five spino-PAGsystems, after WGA-HRP injections in the PAG (case 2385). Abbreviation: CC: central canal.
SYSTEM I
C6 contra L6 contra
C1 ipsi C1 contra
L5 contra L7 contra
S1 contra S2 contra
SYSTEM II
SYSTEM V
SYSTEM IV
SYSTEM III
T4 contra L1 contra
CCCC
CC
CC
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Fig. 11. Schematic representation of the five spino-PAG systems. For each system the location of theneurons is indicated by color and their termination area is described on top. dm, dorsomedial; dl, dorso-lateral; l, lateral; vl, ventrolateral. Areas between parentheses receive much fewer spinal projectionsthan the other areas mentioned.
SYSTEM IV (rostro)caudal (dm, dl), l, vl PAG (deep tectal layers)
mainly contralateral
SYSTEM II rostrocaudal l, vl PAG deep tectal layers bilateral, stronger ipsilateral
SYSTEM V caudal PAG dm, l, vl PAG strongly to central (ventro)lateral PAG mainly contralateral
SYSTEM I rostrocaudal (dm, dl), l, vl PAG
lamina V cervical to deep tectal layers
mainly contralateral
C1
C8
T5 T7
T11 T12 L1
L4L7
S1 S2 S3 Coc 1
T6 T8
T10T9
T2 T3 T4
L5
L2
L6
T13
Coc 2
C2 C3 C4 C5
C6 C7 T1
L3
SYSTEM III (rostro)caudal dl, l, vl PAG deep tectal layers
mainly contralateral
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eral PAG and deep tectum (Mouton andHolstege, 1994). Furthermore, it has beenshown that cells in this same region sendmany fibers to the motoneuronal cell groupsof the cervical enlargement (Holstege, 1988).It is not yet determined whether these mo-toneuron projecting cells receive afferentsfrom the PAG, but one might speculate thatvia this projection the PAG has access to fore-limb motoneurons. Such a projection mightbe important for eliciting the motor activitiesin for example the framework of aggressivebehavior.System III contains neurons in lamina X ofthe thoracic and upper lumbar spinal cord thatproject to the PAG as well as to the deep tectalarea. Anatomical and physiological studies incat and monkey (Light and Perl, 1979; Hondaand Perl, 1985; Honda, 1985) showed thatlamina X plays a role in nociception, as thinmyelinated nociceptive primary afferentsfrom both cutaneous, subcutaneous andvisceral structures terminate in this area.However, as these studies were confined tothe lower lumbar and sacral cord, it isunknown whether this is also true for thethoracic lamina X-PAG projecting neurons.On the other hand, a striking feature is thatthe lamina X cells are exclusively located atthe level of the sympathetic preganglionicmotoneurons. Perhaps the lamina X cells atthis level relay some specific visceral affer-ent feedback information to the PAG concern-ing the total output activity of the sympatheticsystem.System IV contains neurons in the medial partof laminae VI-VII of the L5-S3 segments thatproject to the lateral and ventrolateral partsof the contralateral PAG. The function ofsystem IV is not known, but, unlike all other
spino-PAG cells in the lumbosacral cord,several of its neurons contain estrogenreceptors (VanderHorst et al., 1997). Possi-bly, they are involved in relaying informationof tactile stimuli to the PAG, for example inthe context of the receptive behavior inducedby tactile stimuli of the flanks of an estrousfemale (Kow et al., 1979, 1980).System V contains neurons in the lateral partof lamina I of the L6-S1 and laminae V-VIIand X at the S1-S3 segments. In this area theafferents from the pelvic and pudendal nerves(Morgan et al., 1981) terminate. Theseafferents convey information from the blad-der, perineum, vagina and cervix. In all like-lihood, system V plays an important role inthe relay of specific information from thepelvic organs to the PAG concerning mictu-rition and mating behavior (Blok et al., 1995;VanderHorst et al., 1996).
ConclusionThe PAG is known to elicit analgesia, but alsomany motor functions related to survival andreproduction. To accomplish this it needsmany afferents from rostrally as well as fromcaudally located structures. The present studyshows that the PAG receives a strong, direct,projection from the spinal cord. Resultsshowed the existence of at least 15,000 spi-nal cord neurons projecting to the PAG, lo-cated throughout the entire length of the cord.The different laminar and segmental distri-butions of labeled neurons suggest that thespino-PAG projection is not one general as-cending system of which the main functionis the relay of nociceptive information, butconsists of at least five separate systems, eachof which might have a separate function.
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