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.l.]: [S.n.].

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35

Dorsal border PAG neurons project to the central canal area

Chapter 2

Dorsal border Periaqueductal Gray Neurons project tothe Area directly adjacent to the Central Canal Ependyma

of the C4-T8 Spinal Cord in the Cat

Leonora J. Mouton, Lenka Kerstens, Johannes Van der Want and Gert Holstege

Exp. Brain Res. 112:11-23 (1996)

AbstractIn a previous study horseradish peroxidase (HRP) injections in the upper thoracic and cervi-cal spinal cord revealed some faintly labeled small neurons at the dorsal border of theperiaqueductal gray (PAG). The present light microscopic and electronmicroscopic tracingstudy describes the precise location of these dorsal border PAG-spinal neurons and their ter-minal organization. Wheat germ Agglutinin-conjugated HRP (WGA-HRP) injections intocervical and upper thoracic spinal segments resulted in several hundreds of small retrogradelylabeled neurons at the dorsal border of the ipsilateral caudal PAG. These neurons were notfound after injections in more caudal segments.WGA-HRP injections in the dorsal border PAG region surprisingly resulted in anterogradelylabeled fibers terminating in the area dorsally and laterally adjoining the central canal ependymaof the C4-T8 spinal cord. No anterogradely labeled fibers were found more caudal in thespinal cord. The labeled fibers found in the upper cervical cord were not located in the areaimmediately adjoining the ependymal layer of the central canal, but in the lateral part oflaminae VI, VII and VIII and in area X bilaterally.Electronmicroscopic results of one case show that the dorsal border PAG-spinal neurons ter-minate in the neuropil of the subependymal area and in the vicinity of the basal membranes ofcapillaries located laterally to the central canal. The terminal profiles contain electron-lucentand dense cored vesicles, suggesting a heterogeneity of possible transmitters. A striking ob-servation was the lack of synaptic contacts, suggesting nonsynaptic release from the profiles.The function of the dorsal border PAG-spinal projection is unknown, but considering thetermination pattern of the dorsal border PAG neurons on the capillaries the intriguing similar-ity between this projection system and the hypothalamohypophysial system is discussed.

IntroductionThe periaqueductal gray (PAG) is the largearea of gray matter surrounding the cerebralaqueduct in the mesencephalon. The PAG inrat, cat, monkey and human has long beenknown to play an important role innociception control (see Besson and Chaouch1987 for review). However, physiologicalstudies in rat and cat made clear that electri-cal and chemical stimulation in the PAG canalso elicit motor activities such as cardiovas-cular changes, vocalization, micturition, lor-dosis, pupil dilatation, and movements ofneck, back and hind limbs (Abrahams et al.1960; Kanai and Wang 1962; Skultety 1963;

Mayer et al. 1971; Liebeskind et al. 1973;Sakuma and Pfaff 1979; Fardin et al. 1984;Bandler 1988; Bandler et al. 1991; Lovick1993). In the cat, stimulation of different PAGareas elicits different types of defensivebehavior; in the caudal PAG the ventrolateralpart mediates immobility and the lateral partelicits flight. In the rostrocaudally intermedi-ate one third of the PAG the ventrolateral andventral parts mediate threat display (Bandlerand Carrive 1988; Zhang et al. 1990). To ac-complish nociception control and the men-tioned motor activities, the PAG and adjoin-ing tegmental field project to a large number

36

Chapter 2

of structures in the caudal brainstem, such asthe nucleus raphe magnus and adjacentreticular formation (Abols and Basbaum1981), Barrington’s nucleus (Blok andHolstege 1994), the subretrofacial nucleus(Carrive et al. 1988) and the nucleusretroambiguus (Holstege 1989), and to dif-ferent areas of the spinal cord, such as theintermediolateral cell column at the segmentsT1 and T2 (Holstege 1988). Mouton andHolstege (1994) showed that the PAG alsohas direct projections to premotorinterneurons in lamina VIII and the medialpart of lamina VII throughout the length ofthe spinal cord. These authors observed alsoa few small neurons located at the dorsal bor-der of the dorsomedial and dorsolateral cau-dal PAG that projected directly to the cervi-cal and upper thoracic cord.The present anterograde and retrograde trac-ing study in the cat determines for the firsttime the location of these dorsal border PAG-spinal neurons and their projection pattern inthe spinal cord. The ultrastructural study inone case provides evidence for the locationof the PAG-spinal terminals and their post-synaptic elements.

Materials and methodsSurgical proceduresA total of 11 adult cats was used. The surgi-cal procedures, pre- and postoperative care,handling and housing of the animals followedprotocols approved by the Faculty of Medi-cine of the University of Groningen.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 (approx. 0.2ml/h). During surgery the elecrrocardiogramand body temperature were monitored. Fol-lowing a survival time of 3 days the animalswere initially anesthetized with ketamin(Nimatek, 0.1 ml/kg) and xylazine (Sedamun,

0.1 ml/kg) intramuscularly, followed by 6 ml6% pentobarbital sodium intraperitoneally.The cats were perfused transcardially withtwo liters of 0.9% saline at 37o C, directlyfollowed by two liters of 0.1M phosphatebuffer, containing 4% sucrose, 1%paraformaldehyde and 2% glutaraldehyde.

Retrograde tracing studyIn seven cats after laminectomy approxi-mately 2-4 µl 2.5% wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) in saline was injected into a particularsegment of the spinal cord through glass mi-cro pipettes, using a pneumatic picopump(World Precision Instruments PV830). In fivecases the injections were made bilaterally. Toverify whether the dorsal border PAG-spinalpathway descends ipsi- or contralaterally, inthese cases prior to the injection a hemisec-tion was made some segments rostral to theinjection site. In two cases (2244, 2272) theWGA-HRP was injected unilaterally and nohemisection was made.After perfusion the brains and spinal cordswere removed, postfixed for two hours andstored overnight in 20% sucrose in phosphatebuffer (0.1M, pH 7.4) at 4o C. Subsequently,the brainstem was cut in 40-µm frozen trans-verse sections of which every fourth sectionwas incubated according to thetetramethylbenzidine (TMB) method, dehy-drated and coverslipped. The spinal segmentscontaining the injection site or hemisectionwere cut in 40-µm sections and every fourthsection was processed with diaminobenzidine(DAB). The results showed the area of injec-tion, and the precise extent of the hemisec-tion. The sections were studied with a Zeissdarkfield stereomicroscope and a ZeissAxioskop light microscope. In each case allprocessed transverse sections between thethird and fourth ventricle were studied andlabeled neurons in the PAG and the dorsallyadjoining area were plotted. For plotting adrawing tube projecting directly on a digitizer,connected to a computer, was used.

37

Dorsal border PAG neurons project to the central canal area

case

2244

2272

2292

2295

2315

2329

injection hemisection

C2/C3

C5/C6

C7/C8

T1/T3

L6/L7

S1/S3

number of labeled dorsal border PAG neurons

ipsi midarea contra total

55 17 6 78

68 29 22 119

38 26 15 79

49 19 4 72

1 0 0 1

0 0 0 0

2330 T7/T9

2

4 0 6

---

---

C1

C6

L4

L5

T6

Fig. 1. Schematic drawings of the location of the injections and hemisections at different spinal cordlevels of the cat, and the number of labeled dorsal border PAG neurons found ipsilaterally, contralaterallyand in the midline area of the processed section of the mesencephalon (which was one out of 4 cutsections). The midline area was defined as an area of 125 µm left and right from the midline.

Anterograde tracing studyTo determine where the dorsal border PAG-spinal neurons terminate, in three cats injec-tions of approximately 40 nl 2.5% WGA-HRPin saline were made at the area of the dorsalborder of the dorsomedial and dorsolateralPAG. This area, according to the retrogradetracing study, contained the PAG-spinal neu-rons. Two of these injections were placedstereotaxically, approaching the target areaeither through the cerebellum (2262) orthrough the inferior colliculus (2297). Oneinjection (2316) was made after suction of thecortical areas overlying the mesencephalon.In one control case (2293) an injection of ap-proximately 40 nl 2.5% WGA-HRP in salinewas stereotaxically placed in the aqueduct ofSilvius.All surgical and histological procedures were

similar to those in the retrograde tracing studyand every fourth transverse section of thebrainstem and up to 31 segments were stud-ied. An extra series of every fourth section ofthe mesencephalon was processed with DABto determine the area of injection. Spinal cordsections were studied with a Zeiss Axioplanmicroscope using darkfield polarized illumi-nation. The labeled fibers and neurons wereplotted using the same equipment as in theretrograde study.

Electronmicroscopic studyTo localize precisely the PAG-spinal fiberswithin the central canal area and to identifytheir terminals and postsynaptic elements,from the C6 and T3 segments of case 2316an extra series of 40-µm transverse microtome

38

Chapter 2

SC

2244

A 3.0

2315

2292

2330

Aq

A 0.6

P 1.5

C6 T2C2injection

case

T8

Aq

SC

PAG

T6---

SC

PAG

Aq

IC

hemisection C1 C6

Fig. 2 Schematic drawings of various levels of the cat mesencephalic PAG, with labeled neurons afteran WGA-HRP injection in the spinal cord. Each drawing represents four 40 µm thick section. Abbrevia-tions: Aq: aqueduct of Silvius, IC: inferior colliculus, PAG: periaqueductal gray, SC: superior colliculus.

39

Dorsal border PAG neurons project to the central canal area

sections (one out of four) was stored in phos-phate buffered saline (0.01M, pH 7.4) forelectronmicroscopic evaluation. The sectionswere incubated with TMB and ammoniumheptamolybdate overnight (Olucha et al.1985), after which they were osmicated over-night in 1% OSO4/cacodylate buffer (pH 5.5)at 4o C. (Henry et al. 1985). After rinsing inbidest the sections were stained en bloc in 1%uranyl acetate in bidest, dehydrated in agraded series of alcohol and flat embeddedin Epon between dimethyldichlorosilane-coated glass slides (Vinores et al. 1984). Sec-tions, that showed light microscopicallyanterogradely labeled fibers in the area sur-rounding the central canal were selected forultrastructural study. The area surrounding the

SC

Aq PAG

Aq PAG

Fig. 3. Schematic drawings and darkfield polarized photomicrographs of a caudal (bottom) and a rostral(top) section of the dorsal mesencephalon. In the drawings on the left the boundaries of the dorsal borderregion have been indicated by dark and light gray. Of this region the dark gray represents the midlinearea. The photomicrographs show the areas indicated by the dashed lines seen in the drawings. Note thelabeled dorsal border PAG-spinal neurons after a WGA-HRP injection in the upper thoracic spinal cord.In the caudal section labeled neurons are located ipsilaterally along the dorsal border of the PAG and inthe rostral section they have been shifted to the midline area. Abbreviations: Aq: aqueduct of Silvius,PAG: periaqueductal gray, SC: superior colliculus.

central canal was cut out and mounted on po-lymerized Epon-stubs. From the mountedblocks ultrathin sections were cut and, afterstaining in uranylacetate and lead citrate(Reynolds 1963), studied with an electron mi-croscope.

ResultsRetrograde tracing studyLocation of injection sites and hemisectionsFigure 1 shows the location of the injectionsand hemisections. Rostrocaudally all injectionsinclude at least one and at most three spinalsegments. In the midcervical cord (case 2244and 2272) unilateral injections were made.These injections both involved the entire leftgray matter. In all other cases with a bilateral

40

Chapter 2

injection the total gray matter of both sideswas filled with WGA-HRP. In the bilaterallyinjected cases a left sided hemisection wasmade which involved the entire half of thecord. Only in case 2292 was a small portionof the ventrolateral funiculus not removed andin case 2315 a small part of the mediodorsalfuniculus was intact.

Labeled neurons in the PAGRetrogradely labeled neurons were found atthe dorsal border of the PAG after injectionsin the cervical spinal cord or the upper partof the thoracic cord (Fig. 2), but not after in-jections in the lower thoracic and lumbosac-ral cord (Figs 1, 2). The dorsal border PAG-spinal neurons were oval or spindle shapedand relatively small. They measured 15-45

Fig. 4. Histograms showing the rostrocaudal location of the labeled dorsal border PAG-spinal neuronsafter a WGA-HRP injection in C5/C6 (case 2244) and T1/T3 (case 2292). Note that most labeled dorsalborder PAG neurons were found ipsilaterally in the subtentorial PAG. Rostrally the location of thelabeled neurons gradually shifted to the midline area (area of 125 µm left and right from the midline),and to the contralateral side.

0

5

1 0

1 5

2 0

P 1.5 A 0.6 A 4.1caudal rostral

num

ber

of l

abel

ed n

euro

ns

ipsilateralmidline areacontralateral

C5/C6 injection (case 2244)

0

5

1 0

1 5

2 0

P 1.5 A 0.6 A 4.1caudal rostral

num

ber

of l

abel

ed n

euro

ns

ipsilateralmidline areacontralateral

T1/T3 injection ( 2 2 9 2 )

41

Dorsal border PAG neurons project to the central canal area

µm (mean ≈ 30 µm) in their largest dimen-sion, and 10-25 µm (mean ≈ 17 µm) in theirsmallest dimension (Fig. 3). The largest di-ameter was mostly oriented along the borderof the PAG, except for the cells located in themidline area, being defined as the area meas-uring ≈125 µm left and right from the mid-line (Fig. 3.), which were oriented dorsoven-trally .Except for the C2/C3 injected case, mostlabeled dorsal border PAG-spinal neuronswere found ipsilaterally at the level of thecaudal one third, or subtentorial, PAG (Fig.4). Rostrally, the location of the labeled neu-rons gradually shifted to the midline area(Figs. 2-4). In each processed section (one outof four) the number of labeled dorsal borderPAG-spinal neurons was counted. The high-est number in one section was 13 (case 2244),after an injection in C5/C6, the highest totalnumber was 119 in this same case (Fig. 1). Inall cases only a small portion (less than 20%) of the total number of labeled neurons waslocated contralaterally (Fig. 1).In all cases other labeled neurons were foundin the ventrolateral and lateral PAG. In the

upper cervical injected cases labeled neuronswere also found in the deep layers of the su-perior colliculus, in the mesencephalictrigeminal nucleus and in the dorsomedialPAG. In addition, in all cases anterogradelylabeled fibers were present in the PAG.

Anterograde tracing studyInjection sitesIn 3 cases WGA-HRP injections involved thearea between the commissure of the inferiorcolliculus and the PAG (Fig. 5). In case 2262the injection involved a small part of thedorsomedial subtentorial PAG and the com-missure of the inferior colliculus and in case2316 the injection was located in this sameregion but more laterally. In case 2297 theinjection was placed more rostrally and in-volved the dorsolateral pretentorial PAG anda small part of the superior colliculus. In con-trol case 2293 the injection was placed in theaqueduct and did not involve the dorsal bor-der PAG region.

Descending pathwaysFrom the injection sites in the subtentorial

Fig. 5. Schematic drawings of the WGA-HRP injection sites in the dorsomedial and dorsolateral PAGborder region in cases 2262, 2297 and 2316. Case 2293 is not included in the figure as this injectionmissed the aimed area and was placed in the aqueduct of Silvius. Abbreviations: Aq: aqueduct of Silvius,IC: inferior colliculus, PAG: periaqueductal gray, NR: nucleus ruber, SC: superior colliculus, III: oculo-motor nucleus, IV: trochlear nucleus.

PAG

2297

NR

SC

P0.2

A1.6

P0.9

A0.6

A2.5

III

2297SC

2297

PAG

A3.3

23162262

IV

2316

PAG

2262

IC2316 2262

Aq

42

Chapter 2

dorsal border PAG area (cases 2262 and 2316)two strongly labeled descending pathwayswere found. One stream of labeled fiberspassed laterally, dorsal to the central nucleusof the inferior colliculus, turning ventrally todescend through the lateral tegmental field ofcaudal mesencephalon and rostral pons. An-other stream of labeled fibers passed ventrally

through the lateral and ventrolateral PAG,turning ventrolaterally via the cuneiform nu-cleus to descend in the lateral tegmental field.In both cases the projections were observedbilaterally because the injection sites extendedacross the midline. In case 2316, with an al-most unilateral injection, the bulk of thelabeled fibers was on the ipsilateral side.

Fig. 6. Schematic drawing of the anterogradely labeled fibers and retrogradely labeled neurons (dots) atvarious levels of the spinal cord after a WGA-HRP injection in the dorsal border PAG region of the cat(case 2316). It must be emphasized that each drawing of a spinal cord segment represents two 40 µmthick sections.

case 2316C1 C2

L7

S1

S2

C4

C6

C8

T2T4

T6 T8T10

L1L5

43

Dorsal border PAG neurons project to the central canal area

Fig. 7. Darkfield polarized photomicrographs of various levels of the spinal cord of the cat, after aWGA-HRP injection in the dorsal border PAG region (case 2316). Bar represents 100 µm.

C1

C6

T2

T6 T10

T3

C8

C2

44

Chapter 2

A B

C D

Fig. 8 A. Electronmicrograph showing ependymal cells bordering the central canal with transverselycut cilia (asterisks) and filamentous microvilli extend into the central canal. In the apical part of thecytoplasm basal bodies of the cilia (arrows) can be seen. In the subependymal area a dense spot oflabeling (arrowhead) is found between the glial elements, next to a myelinated axon. Bar represents 2.5µm. B. High magnification of the labeling observed in Fig. 8A. In the area adjacent to the labeling,

45

Dorsal border PAG neurons project to the central canal area

in the cuneiform nucleus, but descendingfibers or terminals were not observed caudalto the mesencephalon.Distribution pattern in the spinal gray mat-terIn the caudal cervical and thoracic cord thinlabeled fibers were, unexpectedly, found toterminate in the area dorsally and laterally ad-joining the central canal ependymal layer ofthe segments C4 to T8 (C6-T6 in Fig. 7). Noanterogradely labeled fibers were found be-yond T8 (T10 in Fig. 7). In the upper cervicalcord labeled fibers were found to terminatein area X bilaterally, but virtually not in thearea immediately adjoining the ependymallayer of the central canal (C1 and C2 in Fig.7). At upper cervical levels terminating fiberswere also found in the lateral part of laminaeVI, VII and VIII. These fibers are probablyfrom contralaterally projecting tectospinalneurons and from neurons located in thedorsomedial PAG (Fig. 2 case 2315).In the more rostrally injected case (2297) afew labeled fibers were observed in the lat-eral part of the intermediate zone of the up-per cervical cord, but none in area X. In thecontrol case (2293), no labeled fibers werefound to terminate in area X.

Electronmicroscopic studyUltrastructureIn the electronmicroscopic study on the C6and T3 segments of case 2316 labeled pro-files were recognized on the basis of a densecrystalline WGA-HRP reaction product.Sometimes details were obscured by the in-tensity of the labeling. Labeled axons and ter-minals were found in the neuropil of thesubependymal area around the central canal

At upper medullary levels many labeled fibersgradually shifted ventromedially just dorsaland dorsomedial to the superior olivary com-plex and ventromedial to the facial nucleus.Most labeled fibers terminated in the ventro-medial tegmental field, at the level of the fa-cial nucleus and the inferior olive. In the cau-dal medulla some fibers were found to termi-nate in the dorsal vagal and solitary nuclei aswell as in the nucleus retroambiguus. In thecaudal medulla and spinal cord the stream oflabeled descending fibers was hard to local-ize. At upper cervical levels some thin labeledfibers were found in the lateral funiculus,while at lower cervical levels they were lo-cated in the dorsolateral funiculus (Fig. 6).At more caudal levels no labeled fibers werefound in this dorsolateral funiculus, althoughin the upper thoracic cord a few labeled fiberswere found running from the dorsolateral fu-niculus through the lateral portion of the graymatter to the central canal area (Fig. 6, T2-T8, Fig 7, C8 and T2). At thoracic, lumbarand sacral levels labeled fibers were found inthe ventrolateral, ventral and ventromedialfuniculi, respectively. It is assumed that thesefibers represent retrogradely labeled ascend-ing fibers, originating from retrogradelylabeled neurons in the lower spinal cord.In the more rostrally injected case (2297),similar to the two caudal cases, two labeled,ipsilateral, descending pathways were found.However, in the rostral case almost all labeledfibers terminated in the upper medulla andvirtually no labeled fibers were found beyondthe level of the inferior olive. In the controlcase (2293) with the injection in the aque-duct of Silvius, anterogradely labeled fiberswere found in the central part of the PAG and

dense cored vesicles (arrows) and translucent spherical vesicles are dispersed between filamentous glialprocesses. Bar represents 0.5 µm. C. Electronmicrograph depicting WGA-HRP labeling (arrowhead)that is continuos with a terminal profile filled with dense cored vesicles (arrows). Glial cells form adher-ent junctions (asterisks). Bar represents 0.5 µm. D. Electronmicrograph of a labeled terminal (arrow-head) that forms an asymmetric synapse (arrows). The terminal profile is enclosed between glial cells,and adherent junctions (asterisks) are present. Bar represents 0.5 µm. Abbreviation: CC: Central canal.

46

Chapter 2

(Fig. 8). Often labeled profiles were presentadjacent to capillaries, located lateral to thecentral canal (Figs. 9, 10).

NeuropilRelatively large labeled terminals were oc-casionally found directly apposed to a soma,however more often in synaptic contact withdendrites. The labeled terminals formedasymmetric synapses and contained eitherspherical electron lucent vesicles with somedense-cored vesicles clustered, or pleomor-phic vesicles (Figs. 8A, B, C). Both types ofterminals showed mitochondria of variablesizes. Although special attention was paid onthe presence of synapses at the margins of aterminal, hardly any was found. It was onlyafter careful screening that some asymmetri-cal contacts were detected (Fig. 8D).

CapillariesA striking electronmicroscopic observation

was the finding of labeling around capillar-ies. Terminal profiles containing clear spheri-cal vesicles and some dense-cored vesicles,were observed (Fig. 9). The terminals wereopposed to the basal lamina extensions in thearea around the central canal. No synapticcontacts with the basal lamina were found,but apposition of membranes of pre- and postterminal profiles was frequently observed(Fig. 10). Due to the filamentous and granu-lar matrix, synapses were hard to identify,although in some cases asymmetric densitiesof free nerve endings were found. Occasion-ally a fusion between the terminal vesiclesand the basement membrane was observed.

DiscussionThe present study in the cat demonstrates theexistence of small neurons at the dorsal bor-der of the PAG, projecting to the central ca-nal area of the C4-T8 spinal cord. Almost

BA

Fig. 9 A. Survey of a capillary surrounded by an endothelial cell. Adjacent to the basal lamina thatseparates the endothelial cell from the glial profiles, WGA-HRP labeling is found in dense deposits(arrowhead). Bar represents 2.5 µm.B. Higher magnification of the area shown in Fig. 8A. The labeling (arrowhead) is located in the laby-rinth of the basal lamina surrounding the endothelial cell. Bar represents 0.5 µm. Abbreviation: cap:capillary.

47

Dorsal border PAG neurons project to the central canal area

nothing is known about this pathway.Holstege and Kuypers (1982), after an HRPinjection in T2 of the cat, plotted someretrogradely labeled dorsal border PAG-spi-nal neurons, but they did not pursue this find-ing. In the HRP retrograde tracing study ofMouton and Holstege (1994) a few dorsalborder PAG-spinal neurons were observedalso. The present results, using WGA-HRPas a retrograde tracer, demonstrate for the firsttime that there exist at least several hundredsof these neurons. Other studies have not men-tioned any dorsal border PAG-spinal neurons.A termination pattern in the area surroundingthe C4-T8 central canal ependyma has neverbeen shown before, neither from the dorsalborder PAG-neurons nor from any other cellgroup. The projection pattern is clearly dif-

ferent from all other brainstem-spinal cordprojections. For instance, the nucleus raphemagnus (Holstege and Kuypers 1982; Mar-tin et al. 1982), nucleus raphe obscurus (Mar-tin et al. 1982), nucleus raphe pallidus(Holstege and Kuypers 1982; Martin et al.1982), the locus coeruleus and subcoeruleus(Holstege et al. 1969; Westlund and Coulter1980), the nucleus paragigantocellularis(Martin et al. 1979) and the hypothalamus(Holstege 1987) are known to project to areaX, but not to its very central part, surround-ing the central canal ependymal layer.Histochemical studies in rat, cat and monkeydemonstrated that neurons, axons and termi-nals within area X contain serotonin and manydifferent neuropeptides, for instance sub-stance P, enkephalin, neurophysin, neuroten-

Fig. 10. Electronmicrograph demonstrating WGA-HRP labeling (arrowheads) next to the basal laminaof a capillary and glial processes. Between the extensions of the basal lamina dense cored (arrows) andelectron lucent vesicles are present. In some cases fusion of vesicles with the basal lamina (asterisks)seem to occur, which might represent examples of exocytotic activity and release of vesicle contents.Bar represents 0.5 µm. Abbreviations: CAP: capillary.

48

Chapter 2

sin, vasoactive intestinal polypeptide andcholecystokinin (Swanson and McKellar1979; Krukoff et al. 1985; LaMotte 1988).Of these, only serotonin, enkephalin,neurotensin and cholecystokinin were alsofound in the area directly surrounding thecentral canal (Swanson and McKellar 1979;Krukoff et al. 1985; LaMotte 1988).The functional implication of the presence ofthese peptides in the central canal area arenot clear. Neuropeptides have been shown toaffect neurons, glial cells, smooth muscles,glands and vascular cells, but no specificpeptides have yet been linked selectively to agiven functional system in the brain (Cooperet al. 1991).The present light microscopic results, show-ing the projection pattern dorsally and later-ally to the central canal, does not give insightin whether the dorsal border PAG-spinal neu-rons project to neurons located within thisarea, or terminate on other structures presenthere. The ultrastructural study, however,shows many labeled terminals of dorsal bor-der PAG-spinal neurons near the labyrinth ofbasal lamina of the laterally located capillar-ies, and some subependymal terminals.No labeled fibers were found to terminate oncell bodies, but only on distal parts ofdendrites. The terminals demonstrated a richvariety of vesicular profiles. One might ex-pect that in accordance with these differencesin size and shape, the contents of the vesicleswill also differ. This suggests that severaltransmitter-like substances and/or neuropep-tides may be released from these terminals.Another intriguing aspect of this ultrastruc-tural study is the relative scarcity of synapticcontacts. Even after inspection of large areasof the spinal cord central canal area of thestudied segments, the number of identifiedsynapses was small. This relative scarcity ofsynapses suggests that nonsynaptic release islikely to occur. This will result in a gradual,prolonged effect as a result of the slow diffu-sion and transport of the transmitter-like con-tents of the vesicles.

Possible functional implicationsThe function of the PAG-spinal neurons, ter-minating in the subependymal area of the cen-tral canal and next to the basal membranes ofthe nearby located capillaries, at the C4-T8spinal levels, is completely unknown. One canonly speculate about this function.Axons terminating in the subependymal areaof the spinal cord central canal give the im-pression that they might release transmitteror peptide hormones from their terminals intothe cerebrospinal fluid. These transmitter orpeptide hormones could be released directlyinto the cerebrospinal fluid, or stimulate theependymal cells of the central canal, whichcells in turn release peptides into the cerebro-spinal fluid. However, such a function ofependymal cells lining the central canal andthe ventricular system has not yet been dem-onstrated. Gee et al. (1993) have demon-strated, in in vitro experiments, that ependy-mal cells of the rat brain contain a set of en-zymes sufficient for neuropeptide biosynthe-sis, but if these cells actually produce thesepeptides has not been shown. Consideringthis, it might be possible that the dorsal bor-der PAG-spinal neurons are involved in re-leasing transmitter or peptide hormones intothe cerebrospinal fluid, but the question bywhich mechanism this might occur, remainsunanswered.Axons terminating in the vicinity of bloodvessels suggest a role of the dorsal borderPAG-spinal neurons in blood pressure regu-lation as it is known that stimulation in thePAG can elicit blood pressure changes. How-ever, stimulation experiments in the cat haveshown that these changes are elicited mainlyfrom the lateral and ventrolateral and not fromthe dorsal PAG (Carrive et al. 1987; Carriveand Bandler 1991). Furthermore, blood pres-sure changes can only be elicited by vaso-constriction or vasodilatation of the aorta andof arteries and arterioles, as these have a layerof smooth muscle and elastic tissue. Theaxons of the dorsal border PAG-spinal neu-rons are found to terminate in the vicinity of

49

Dorsal border PAG neurons project to the central canal area

capillaries and no arteries or arterioles wereobserved in this area. So it is unlikely that thedorsal border PAG-spinal neurons are in-volved in blood pressure regulation.Another possible function of axons terminat-ing in the perivascular spaces of capillariesmight be the secretion of transmitter or pep-tide hormones into the blood circulation.Axons terminating in the perivascular spacessurrounding capillaries are also known to bepresent in the hypothalamohypophysial pro-jection system (see Haymaker et al. 1969,Daniel and Pritchard 1975, McKinley andOldfeld 1990 for reviews). In the magno-cel-lular hypothalamic neurosecretory systemaxons of the paraventricular and supraopticneurons terminate into the perivascular spacessurrounding systemic capillaries in the neu-rohypophysis. Within the hypothalamicparvocellular neurosecretory system, whichis the connection between the paraventricularnucleus or other hypothalamic areas and theadenohypophysis, axons terminate into theperivascular spaces, surrounding the portalcapillaries, located in the median eminenceIf one compares the functions of the hypo-

thalamus and those of the PAG one sees strik-ing similarities. Both can be considered as partof the emotional motor system, see Holstegeet al. (1995) for review. The hypothalamuscontains an integrative system for basic emo-tional responses, including blood pressure,food ingestion and energy metabolism, ther-moregulation, immune response and matingbehavior. The PAG is known to play an es-sential role in emotional behavior (Fig. 11).Possibly, the dorsal border PAG-spinal neu-rons are comparable with neurons in thehypothalamic paraventricular nucleus. Bothcell groups are located near the ventricularsystem, but not immediately bordering it. Thecentral canal area would then have a similarfunction as the median eminence or the pos-terior lobe of the hypophysis, both borderingthe ventricular system and receiving projec-tions to the capillaries in their immediate vi-cinity.Which of the mentioned functions the dorsalborder PAG-spinal neurons might have, it isnot understood why these neurons do not havetheir projection throughout the spinal cord,but only to the C4-T8 spinal segments.

Fig. 11. Schematic overview of the descending projections from the PAG to different regions of thecaudal brainstem and spinal cord. The functions in which each of the projections might be involved arealso indicated.

Ventral 1/3 of caudal pontine and medullary

medial tegmentum

Periaqueductal gray(PAG)

lordosisnociceptioncontrol

nucleusretroambiguus

lam. VIII + med. VIIspinal cord

arching of the back

pupil dilation

subretrofacialnucleus

bloodpressure

Barrington'snucleus

micturition

T1-T2 IML

vocalization

C4-T8 central canal

bordering cells

?

50

Chapter 2

ConclusionThe PAG, as part of the emotional motor sys-tem, is known to project to several brainstemareas, to lamina VIII and the medial part oflamina VII of the entire spinal cord, and tothe T1-T2 intermediolateral cell collumn. Thepresent results show that neurons located atthe dorsal border of the PAG project to thearea dorsally and laterally adjoining the cen-tral canal ependyma of the C4-T8 spinal cord.

Ultrastructural results show that the main tar-get of this projection system is the basal mem-brane of the capillaries located laterally to thecentral canal, but also some subependymalterminals are found. Considering these capil-lary-projections, the similarity between thedorsal border PAG-central canal projectionand the hypothalamohypophysial system isdiscussed.