Neuroscience Research, 11 (1991) 41-54 41 © 1991 Elsevier Scientific Publishers Ireland, Ltd. 0168-0102/91/$03.50

NEURES 00439

Divergent axon collaterals from fastigial oculomotor region to mesodiencephalic junction and paramedian

pontine reticular formation in macaques

Hitoshi Sato and H i r o h a r u N o d a

Department of Visual Science, School of Optometry, lndiana University, Bloomington, IN (U.S.A.)

(Received 30 October 1990; Revised version received 26 December 1990; Accepted 18 January 1991)

Key words: Saccade-related nuclei; Cerebellum; Double-labeling; Horizontal saccades; Vertical saccades; Fastigial nucleus

SUMMARY

Collateralization of efferent fibers from the fastigial ocuiomotor region (FOR) to the paramedian pontine reticular formation (PPRF) and the mesodiencephalic junction (MDJ) was studied in macaque monkeys using a fluorescent double-labeling technique. Retrogradely-labeled neurons in the contralateral FOR were examined following injections of fast blue (FB) into the MDJ and diamidino yellow (DY) into the PPRF, or vice versa. Some FOR neurons were labeled with FB, while some other FOR neurons were labeled with DY and intermingled within the FOR. While single-labeled cells in the FOR projected either to the PPRF or to the MDJ, the presence of double-labeled cells indicated that the FOR contains neurons whose axons collateralize to project to both the MDJ and PPRF. These are regarded as the preoculomotor nuclei responsible for vertical and horizontal saccades, respectively.

INTRODUCTION

Because paralysis of vertical or horizontal eye movements can occur independently, it has been suggested that there are separate anatomical sites for the generation of vertical and horizontal eye movements. However, since many eye movements have both vertical and horizontal components, the anatomical sites must be functionally coupled. The paramedian pontine reticular formation (PPRF) is regarded as the supranuclear structure responsible for conjugate horizontal eye movements 9,22,23,27. Physiological recordings of unit activity related to vertical eye movements have identified a site at the transition between the mesencephalon and diencephalon (mesodiencephalic junction, MDJ) as the supranuclear structure concerned with the generation of vertical eye movements 4. Cells concerned with vertical eye movements lie ventral to the nucleus of Darkschewitsch, rostral to the interstitial nucleus of Cajal, and are situated among fibers of the medial longitudinal fasciculus (MLF) rostral to the oculomotor complex. This locus has been referred to as the rostral interstitial nucleus of the medial longitudinal fasciculus (riMLF) 4.

Correspondence." Dr. Hiroharu Noda, School of Optometry, Indiana University, 800 E Atwater, Bloomington, IN 47405, U.S.A.

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Saccadic eye movements, evoked by microstimulation of the cerebellum of macaque monkeys, are usually oblique with both horizontal and vertical components. The duration and peak velocity of the oblique saccades, observed at many sites in the oculomotor vermis, increased monotonically with eye movement amplitude 35. In contrast, the hori- zontal and vertical components of saccades evoked from some other vermal sites did not show linear ampl i tude-durat ion and ampli tude-veloci ty relations 11. The time courses of the horizontal and vertical components were not identical in these saccades; they exhibited asynchronous onset and offset times, as well as difference in the time to peak velocity. The trajectory of such an oblique saccade was curved or had a hooked shape when plotted in two-dimensional space. When these vermal sites were stimulated at different intensities, the trajectories of the evoked saccades were modified. For example, a hooked diagonal saccade evoked with a threshold stimulation became nearly straight and more inclined when a stimulus 10 × the threshold was applied. The increase in stimulus current caused an extension of the duration of the horizontal component without having much effect on the vertical component. The finding indicates that the cerebellar output influences the horizontal and vertical components of saccades independently 11

Microstimulation of fastigial sites produces contralateral saccades in different oblique directions, suggesting that each fastigial site contains a mixture of neurons projecting either to the PPRF or to the MDJ. The proportion of these neurons may be different from site to site because oblique saccades were evoked in different directions. The physiological data 40 that some neurons of the fastigial oculomotor region (FOR) show most robust presaccadic activity when the eyes moved diagonally would indicate that these neurons may send divergent axon collaterals to both the MDJ and the PPRF. The present study was initiated to provide the anatomical basis to explain these physiological phenomena.

MATERIALS AND METHODS

The data were obtained from 3 adolescent pig-tailed monkeys (Macaca nemestrina), weighing 4.5-5.0 kg. Each animal was intubated under Ketalar sedation and was deeply anesthetized with a gas mixture of N 2 0 / O 2 plus a varying amount of methoxyflurane. After mounting the animal in a stereotaxic head holder, trephined holes were made in the skull over the MDJ and PPRF for injections of the fluorescent dyes stereotaxically. The MDJ is indicated here as the mesencephalic tegmentum anterior to the fasciculus retroflexus including the posterior part of the diencephalon. Injection sites were chosen from the locations where anterogradely-labeled axon terminals aggregated following wheatgerm agglutinin-horseradish peroxidase (WGA-HRP) injections into the F O R 30. The stereotaxic coordinates of these locations were determined with reference to photomi- crographs of parasagittal sections of the brains of the other pig-tailed monkeys, stained with cresyl violet, and the stereotaxic atlas of Macaca nemestrina 46. A fluorescent-dye combination of 2% fast blue (FB) in distilled water and diamidino yellow (DY) in 2% dimethyl sulfoxide solution was used. The combination of FB-DY fluorescent dyes was chosen because effective retrograde DY labeling requires a survival time comparable to that of FB 26 and the same excitation wavelength can be used to detect labe led cells. Because of relatively slow diffusion of the substances, it was necessary to make multiple injections in order to cover the entire target area. The maximum amount of injection at each injection site was limited to 0.6 /~1 to minimize necrosis around the injection site. Monkey Db received a total of 2.5/~1 of DY in the MDJ at 5 sites along two penetrations of 1 m m apart and 3/~1 of FB in the PPRF at 6 similarly separated sites. Monkey Sk was

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given 6/~1 of FB at 10 MDJ sites and 5 .0 /d of DY at 9 PPRF sites along 3 penetrations. Monkey KI was injected with 2.0 ~1 of FB at 4 MDJ sites and 7.2 ~1 of DY at 12 PPRF sites. After recovery from the anesthesia, the monkey returned to its cage. Monkeys Db and K1 survived 10 days and monkey Sk survived 6 days after injections; they behaved normally during the survival period. Despite multiple microlesions found later in the injection areas, no obvious oculomotor deficit was observed.

After post-injection survival periods, the monkeys were again deeply anesthetized with N 2 0 / O 2 plus a maximum evaporation rate of methoxyflurane and were perfused trans- cardially by fluid delivered under controlled pressure through a cannula inserted into the ascending aorta. We perfused each monkey with 5 liters of warm (36 o C) Ringer solution followed by 4 liters of 4% paraformaldehyde in a 0.1 M phosphate buffer (pH 7.4) and then by 4 liters of 10% sucrose in the same fixative solution. The brain was removed from the skull and was stored overnight in the same fixative with 30% sucrose (pH 7.4) at 4 o C. The following day the brain was sliced into 40-#m parasagittal sections on a freezing microtome and the serial sections were collected in compartmentalized trays.

The fluorescent material was studied under epi-illumination on a Nikon microphoto-FX microscope using a barr ier / f i l te r combination delivering an excitation wavelength of 365 nm at which both FB and DY fluoresce. After identifying sections which contained the fastigial nucleus (FN), 16-20 pictures were taken from each section at 100 x magnifica- tion by systematically shifting the object so that the entire FN was included. A large map (50 x 60 cm) of the FN was composed for each section, which included the FOR, and the locations of fluorescent-positive cells in the FN were marked by projecting from the 35-ram negative films. Each cell was re-examined under the same illumination, but this time at 400 x magnification and we determined whether the cell was single-labeled with either DY or FB, or double-labeled. After examining fluorescent labelings, the cover glass was removed and each section was stained with cresyl violet. Four to 6 pictures were taken from the same area of each section and a map of the FN for Nissl-stained cells was made on paper of the same size. Retrogradely-labeled cells were then identified by superimposing the identified fluorescent-labeled cells on the map of Nissl-stained cells. Quantitative evaluation of labeled neurons was made only for the FOR which was identified in each monkey with reference to the map of the FOR drawn from ante- rogradely-labeled terminals in the FN following W G A - H R P injection into the oculomo- tor vermis of macaque monkeys 49

RESULTS

Injection sites Monkeys Sk and KI received FB injections into the MDJ and DY injections into the

PPRF, while FB was injected into the PPRF and DY was injected into the MDJ in monkey Db. The FB injection area around the needle tracks in MDJ and PPRF showed 3 concentric zones with different characteristics. Immediately around the needle track, a brilliantly white-blue fluorescent zone occurred which contained few cellular elements and many orange fluorescent granules (zone 1). This zone was surrounded by a second zone which was characterized by a dense accumulation of blue fluorescent glial cells with a blue fluorescent nucleus (zone 2). A third zone contained fluorescent glial nuclei and neurons as well as blue fluorescent fibers. Towards the periphery of the third zone the number of fluorescent glial nuclei progressively diminished and the zone thus faded into an area of normal tissue with little fluorescence (zone 3). The DY injection area also showed 3 similar concentric zones. The center contained a mass of brown-yellow

44

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Fig. 1. Camera - luc ida d rawings of parasag i t t a l sect ions of the b ra ins tem of 3 monkeys , ind ica t ing f luorescent-dye in jec t ion sites in the r i M L F (A, C, E) and the P P R F (B, D, F). Rt = right; Lt = left; F R = fasciculus re t rof lexus ,

G = genu; IC = infer ior col l iculus; M D = dor somed ia l tha lamic nucleus; M L F = media l long i tud ina l fasciculus; N I V = t rochlear nucleus; n I I I = ocu lomotor nerve; N V1 = abducens nucleus; n I V = t rochlear nerve; n

VII = facial nerve; N R T P = nucleus re t icular is t egment i pont is ; P A G = pe r i aqueduc ta l gray; PC = pos te r io r

commissure ; PN = pon t i ne nucleus; Py = p y r a m i d a l tract; R N = red nucleus; SC = super ior col l iculus; SCP = super ior cerebel lar pedunc le ; SN = subs tan t i a nigra. Bold let ters at the r ight lower corner ind ica te monkeys '

names.

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45

Fig. 2. Photomicrographs of retrogradely-labeled FOR neurons of macaque monkeys. (A) A cell in the left FOR of monkey Db, FB-labeled from the right PPRF. (B) Monkey Db, DY-labeled from the fight MDJ. (C) Monkey K1, FB-labeled (B) from right MDJ and DY-labeled (Y) from right PPRF. (D) Monkey Db, FB-labeled (B) from PPRF and DY-labeled (Y) from MDJ. (E) Monkey K1, FB-labeled (B) from MDJ and DY-labeled (Y) from PPRF. (F) Monkey K1, the same as E. (G) Monkey K1, DY-labeled (Y) from PPRF and double-labeled (D). (H)

Monkey Sk, double-labeled (D). Calibration bar for A-H: 30 #m.

46

fluorescent material, presumably consisting of DY combined with some necrotic tissue (zone 1). This area was surrounded by a zone which displayed blue tissue fluorescence (at 365 nm excitation wavelength) and contained a dense accumulation of fluorescent glial nuclei and neurons with fluorescent nuclei (zone 2). This zone in turn was surrounded by a zone containing only fluorescent glial and neuronal nuclei (zone 3).

Camera-lucida drawings of the injection sites (zones 1 and 2) found in 3 monkeys are shown in Figure 1. In monkey Db, zones 1 and 2 of DY injection were confined to the right riMLF, while zone 3 included a medial part of Forel's H-field and a rostral part of mesencephalic reticular formation (MRF). Zone 1 of FB was found in the fight PPRF in the same monkey, while zones 2 and 3 also involved the pontine raphe. The FB injection in monkey Sk was centered at the left r iMLF but also included the nucleus of Darksche- witsch (ND), Forel's H-field, MRF, periaqueductal grey (PAG), and fasciculus retroflexus (FR). The zone 3 in the same monkey involved parts of the dorsomedial (MD), parafascicular (PF), centromedian (CM), ventral posteromedial (VPM), and ventral posterolateral (VPL) thalamic nuclei. The DY injection in monkey Sk was confined to the left PPRF, but zone 3 included also the PR and a marginal part of the nucleus reticularis tegmenti pontis (NRTP). In monkey K1, the FB injection was confined to the r iMLF and the DY injection site included the PPRF and pontine raphe (PR).

Retrogradely-labeled F O R neurons Photomicrographs in Figure 2 show examples of retrogradely-labeled FOR neurons

after DY and FB injections into the MDJ and PPRF. respectively (monkey Db), or vice versa (monkeys Sk and K1). When excited at a wavelength of 365 nm. neurons single- labeled retrogradely with FB display a blue fluorescence of the cytoplasm of cell body and dendrites (Fig. 2A, C). They show very little nuclear labeling (Fig. 2A. C. E. F). Retrograde transport of DY produces mainly fluorescence of neuronal nuclei (Fig. 2C, D, E) and gives some fluorescence of the cytoplasm (Fig. 2B, F. G). The advantage of using DY is the relatively slow migration of DY out of the retrogradely-labeled neurons. However, the relatively pronounced DY cytoplasmic labeling could be mistaken for the blue FB cytoplasmic fluorescence in double-labeled neurons (Fig. 2B, G). The blue FB cytoplasmic fluorescence in double-labeled neurons can be identified only when the color in the cytoplasm was comparable to that in FB single-labeled neurons located in the vicinity. Double-labeled neurons show a blue fluorescence of the cytoplasm and a yellow fluorescence of nuclei at an excitation wavelength of 365 nm (Fig. 2G, H). It Is important to note that neurons single-labeled either from the PPRF or the MDJ intermingled and were frequently located close to each other (Fig. 2C. D, E. F).

The locations of FB and DY single-labeled neurons and FB-DY double-labeled neurons in the FOR, found in monkey Sk, are illustrated in Figure 3. The ellipsoidal region in the caudal FN is the FOR which receives Purkinje-cell projections from the oculomotor vermis. Although the boundaries of the FOR are not easily distinguishable from the other portions of the FN, the region encircled by broken lines is the area where anterogradely-labeled axon terminals always appear when W G A -H RP is injected into the oculomotor vermis 49 and presaccadic neuronal activity can be recorded 40. Neurons retrogradely-labeled from the MDJ (encircled) and from the PPRF (filled) intermingle not only within the FOR but also in the entire FN. There was a higher incidence of FB-DY double-labeled neurons (6 /8 cells) in the FOR in these particular parasagittal sections.

The numbers of retrogradely-labeled neurons in the FOR in each of two consecutive 40-/~m sections are shown for 3 monkeys in Figure 4. Open columns represent the numbers of FOR neurons retrogradely-labeled from the PPRF, stippled columns repre-

47

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(encircled), and double-labeled (encircled and filled) neurons in the right FOR of monkey Sk. Open ce l l s are

Nissl-stained ce l l s . T h e l e v e l s of parasaglttal sections are indicated at t h e l e f t - l o w e r comer. Ellipsoidal r e g i o n s

e n c i r c l e d w i t h b r o k e n l i n e s correspond to the FOR.

sent those from the MDJ, and filled co lumns represent double- labeled neurons from both structures. The percentages of retrogradely-labeled F O R neurons are summarized in Table I. Approx imate ly 29% of F O R neurons were retrogradely-labeled fo l lowing D Y injections into the P P R F in m o n k e y s K1 and Sk, whi le 20.5% of F O R neurons were labeled when FB was injected into the P P R F in m o n k e y Db. The distribution of F O R neurons labeled from the P P R F was slightly shifted media l ly in m o n k e y K1 (Fig. 4C). In

48

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Fig. 4. Mediolateral distributions of PB- and DY-smgle-labeled neurons and double-labeled neurons in the FORs of 3 monkeys. Labeled neurons were counted on the left F O R for monkeys Db and KI and on the right

F O R for monkey Sk.

general, the distributions were similar in the 3 monkeys, despite the fact that the locations of fluorescent injections were reversed in monkey Db. Only about 3% of F O R neurons were retrogradely labeled from the MDJ.

Conclusion Figure 5 gives a summary of the present study. When fluorescent substances were

injected into the M D J and PPRF, neurons in the contralateral F O R were retrogradely- labeled. Some neurons were labeled only from the MDJ, while other neurons were labeled

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TABLE I

N U M B E R S A N D PERCENTAGES OF FOR N E U R O N S R E T R O G R A D E L Y LABELED F R O M M D J A N D PPRF F L U O R E S C E N T - D Y E INJECTIONS

Animals Total F OR F OR neurons F O R neurons FOR neurons neurons labeled from labeled from labeled from

PPRF MDJ PPRF and M D J

Monkey KI 1162 333 (28.7%) 38 (3.3%) 12 (1%) Monkey Sk 941 271 (28.8%) 25 (2.7%) 19 (2%) Monkey Db 1262 259 (20.5%) 22 (1.7%) 8 (0.6%)

from the PPRF. These single-labeled neurons intermingled within the FOR. There were neurons which were double-labeled from both the MDJ and PPRF. The percentages of double-labeled FOR neurons were low in all monkeys (Table I); they were only 0.6-2.0% of the FOR neurons. The FOR neurons labeled from the MDJ were also few (1.7-3.3% of the entire FOR population). Nonetheless, the present study has provided the 'anatomical evidence that some FOR neurons supply both the horizontal and vertical preoculomotor nuclei with divergent collaterals and control eye movements in oblique directions in the macaque monkey.

MD

~iiiy'

FN

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Fig. 5. Summary diagram of the present experiment. Note that the cerebellum represents the vermis approxi- mately 1.0 m m left f rom the midline, while the brainstem represents a parasagittal plane approximately 1.5 m m to the right. The structures indicated with broken lines appear at approximately 0.7 m m to the right. MB = mammilary body; r i M L F = rostral interstitial nucleus of the MLF; N D = nucleus of Darkschewitsch; INC = interstitial nucleus of Cajal; III = oculomotor nucleus; IV = trochlear nucleus. For further explanation

see legend to Fig. 1.

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DISCUSSION

The present study has demonstrated that some neurons in the FOR have divergent axon collaterals which terminate in both the MDJ and PPRF in the macaque. Neurons in the MDJ and the PPRF will receive identical impulses from the same F O R neurons almost simultaneously, or with a short time-lag which corresponds to the difference in conduction times from the F O R to these structures. On the other hand, there were neurons which projected only to the MDJ or to the PPRF and these two groups of neurons intermingled within the FOR. These anatomical findings are in good agreement with the results from physiological recording of unit activity from the F O R of macaque monkeys. Neurons in the FOR discharged in burst when monkeys made saccades to a visual target presented in the contralateral hemifield. The optimal direction of a large number of saccadic-burst neurons was horizontal, but some neurons showed either diagonal or vertical preferred directions 40

Separate pools of medium lead-burst neurons located in the PPRF and in the MDJ, particularly in the riMLF, are thought to produce the excitatory burst of neural discharge that generates the horizontal and vertical components of saccades 4,22,23,28. It is known from anterograde H R P transport in macaque monkeys that the majority of F O R fibers decussate within the cerebellum and terminate in brainstem regions which are involved in the control of saccades 39. These include, but are not limited to, the PPRF and r iMLF of the contralateral side. Saccade-related signals of the cerebellum are transmitted to these preoculomotor nuclei almost exclusively through the uncinate fasciculus (UF) of the contralateral side. Microstimulation within the UF, but not the juxtarest iform body, can produce saccades and the direction of the evoked movements is almost purely horizontal when the descending limb of the UF is stimulated 36. Contralateral saccades could be evoked when fastigial neurons are stimulated 38, but ipsilateral saccades were elicited from microstimulation of the UF because the UF is composed of the decussated fastigial axons. In contrast, when the ascending limb of the UF is stimulated near the superior cerebellar peduncle, the direction of evoked saccades was predominantly vertical 37. These physiological observations indicate that the information about the saccade direction has already been separated into horizontal and vertical components in the fastigiofugal fibers.

On the other hand, neurons projecting to the M D J and PPRF intermingled within the F O R and the proportion of these neurons varied from site to site. This anatomical finding implies that different numbers of horizontal and vertical neurons will be activated when a microstimulation is applied to a F O R site. The direction of evoked saccades may then depend on the proport ion of the horizontal and vertical neurons at the site. As described in the Introduction, increasing the stimulus current applied to the same cerebellar site resulted in changes in the direction of evoked saccades 11. This would be explained by the spread of current which activated different proportions of horizontal and vertical neurons in the vicinity. If there are more horizontal (or vertical) neurons in the vicinity, the direction of evoked saccades would be more horizontal (or vertical) as the stimulus current is increased. Thus, the present study has provided a firm anatomical ground for explaining the variability in the direction of saccades evoked from different cerebellar sites as well as those evoked from the same site but stimulated at different intensities ]1,35

The r iMLF was outlined in the monkey 6 and in the cat 19, with the aid of neuro- anatomical markers. Only this cell group and not the interstitial nucleus of Cajal ( INC) or the surrounding M R F receives a terminal input from the midline structures in the caudal pons 6,19. Lesions involving the r iMLF are associated primarily with downward gaze paralysis 4], indicating the important role of the r iMLF in vertical gaze control. The INC

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is also considered to contain premotor neurons which directly activate the vertical extraocular eye muscle motoneurons 12. The role of the INC in oculomotor function, however, appears to be different from that of the riMLF. This was suggested by an anatomical study showing that when HRP injection was confined to the FOR, the INC was almost totally devoid of anterogradely-labeled axon terminals 39

Direct connections of the r iMLF with the vertical oculomotoneurons are suggested by HRP transport 5,6,19,45 and by the presence of monosynaptic EPSPs and IPSPs evoked in trochlear motoneurons by stimulation of the r iMLF 34. Nakao and Shiraishi 33,34 have shown that stimulation of this region evokes EPSPs in the motoneurons directly innervat- ing the inferior rectus muscle. Afferents to the r iMLF have been described from the PPRF 6, the omnipause region of the pontine raphe 29, the superior colliculus 17,21, the vestibular nuclei 6,7, and the frontal eye fields 31,32. However, the projection from the FOR was discovered only recently 39 and none of the above authors has described fastigial projections to the riMLF. The present study has confirmed our preceding report

39 using anterograde HRP transport that the r iMLF receives FOR axons The present study has also confirmed the finding that the FOR sends a significant

number of axons to the PPRF 39. The PPRF in the monkey has been considered to be involved primarily in horizontal saccadic eye movements 9,22,23 The cells in the PPRF are active immediately before a saccade. The size and the direction of the subsequent saccade could be exactly predicted from the burst of activity 10. Earlier observations on the fastigial projection to the PPRF 2,8,47,48 have been supported by autoradiographic studies 1,18,19 and an HRP study 13.14 and by the present fluorescent-marking study.

The PPRF receives afferent fibers from the frontal eye fields 24,43, the superior colliculus 20, the MRF 30,42,44 the vestibular nuclei 25,42,48, the perihypoglossal

16 30 42 44 nuclei 30,42,44, and the nucleus of the posterior commissure • ' ' . These anatomical connections indicate that both the r iMLF and PPRF receive mutual projections from these saccade-related structures. The possible convergence of fastigial projections with these inputs to the vertical and horizontal preoculomotor nuclei suggests the significance of the FOR in parallel processing of oculomotor control signals.

FOR neurons double-labeled retrogradely from both the r iMLF and the PPRF were discovered consistently in all 3 monkeys, demonstrating that some FOR cells have divergent axon collaterals and control both the vertical and horizontal preoculomotor nuclei. The fastigial nucleus seems to contain abundant neurons which send axons to more than one distant target region. Collateralization of fastigiofugal fibers to the paraoculomotor region, superior colliculus, and medial pontine reticular formation has been demonstrated in the rat ~5. The medial pontine reticular formation of the rat corresponds to the monkey's PPRF and the paraoculomotor region includes the riMLF, INC, ND, the medial accessory nucleus of Bechterew, and the dorsomedial parvicellular red nucleus 14. Following fluorescent marker injections into both regions, double-labeled cells were predominantly found in the middle and caudal fastigial nucleus in the rat 15 Many rat fastigial neurons have divergent axon collaterals to the diencephalon and the superior colliculus, to the diencephalon and the spinal cord, or to the diencephalon and the medulla oblongata, as demonstrated by double-labeling methods 3. As these double- labeling experiments have been conducted in rats, the relative sizes of the injection sites were extremely large compared to those in the monkey. It is difficult, therefore, to make a quantitative comparison between the results from the rat and the present study. The abundant double-labeled neurons in the caudal part of the FN in the rat 345 and the consistent discovery of double-labeled FOR neurons in all 3 monkeys would indicate the

52

e x i s t e n c e o f c o l l a t e r a l i z a t i o n of f a s t ig i a l n e u r o n s to t he v e r t i c a l a n d h o r i z o n t a l p r e o c -

u l o m o t o r nuc le i .

ACKNOWLEDGEMENTS

W e are g r a t e f u l to M r . J a c q u e K u b l e y fo r p h o t o g r a p h i c a s s i s t a n c e a n d to Ms . R i t s u k o

N o d a for h e r h e l p a t v a r i o u s s t ages o f t h e i n v e s t i g a t i o n . T h i s w o r k w a s s u p p o r t e d b y

N a t i o n a l I n s t i t u t e s o f H e a l t h g r a n t E Y 0 4 0 6 3 .

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