timecourse of development of the wallaby trigeminal pathway. ii. brainstem to thalamus and the...

THE JOURNAL OF COMPARATIVE NEUROLOGY 364~494-514 (1996)

Timecourse of Development of the Wallaby Trigeminal Pathway. 11. Brainstem

to Thalamus and the Emergence of Cellular Aggregations

C.A. LEAMEY, L.R. MAKOTTE, AND P.M.E. WAITE School of Anatomy, University of New South Wales, Sydney, 2052, Australia

(C.A.L., P.M.E.W.), and Developmental Neurobiology Group, RSBS, Australian National University, Canberra, ACT, 2601, Australia (C.A.L., L.R.M.)

ABSTRACT This paper is the second in a series which makes use of the protracted postnatal maturation

of the wallaby to study the development of the trigeminal sensory system. Previous work has established similarities in the organisation of the trigeminal sensory system in the wallaby and in rodents. This study describes the structure and development of the ventroposteromedial nucleus in the wallaby in relationship to the arrival of afferents from the trigeminal nuclei, the formation of neuronal aggregations and naturally occurring cell death. Enzyme histochemistry, Nissl and myelin stains were used. Pathway development was followed using carbocyanine dyes.

In the adult wallaby the nucleus demonstrates evidence of a parcellated organisation. Cells are arranged in dorsoventrally aligned bands resembling fingers. In the horizontal plane, these appear as circular clusters which are encircled by fine myelinated bundles. The clusters of cells are believed to correspond to the mystacial vibrissae. The first afferents from the principal trigeminal nucleus arrive between 10 and 15 days postnatal. This is more than two weeks prior to the time at which the borders of the nucleus can be discerned cytoarchitecturally. The first hints of segmentation are visible around day 50, and discrete aggregations form over the ensuing 3-4 weeks. Coincident with the aggregation of the neurons is an increase in their level of reactivity for acetylcholinesterase. A high level of acetylcholinesterase reactivity is maintained for at least 4 months, but has disappeared in adult animals. The peak of cell death occurs subsequent to the appearance of aggregations in the thalamus, but coincident with the appearance of vibrissae related patches in the cortex at day 85 (Waite et al. 119911 Dev. Brain Res. 58:35-41). The timing of the appearance of the neuronal aggregations supports the hypothesis that pattern formation occurs sequentially at successive levels of the pathway, and suggests the importance of target maturation in pattern formation.

Indexing terms: marsupials, vibrissa, barrels, somatosensory, cell death

~S!tt i Wile>,-Liss. Inc

Understanding how maps of the periphery are established in the central nervous system is of focal interest in developmental studies. The discretely observable topography of the rodent trigeminal system presents many advan- tages for this type of study (reviewed by Killackey et al., 1990). The neuronal aggregations, or barrels, first observed by Woolsey and Van der Loos (1970) in the rodent cortex, correlate 1: 1 with the contralateral vibrissae, and thalamocortical afferents are clustered in these barrels (Bernard0 and Woolsey, 1987; Jensen and Killackey, 1987). Other studies have shown that the rodent vibrissal field is also represented in the brainstem trigeminal nuclei and somatosensory thalamus by similar patterns of afferent and cellular distributions, which are demonstrable using routine

histology and histochemistry and a variety of tracing techniques (Van der Loos, 1976; Belford and Killackey, 1979a, 1980; Ivy and Killackey, 1982; Erzurumlu and Killackey, 1983; Durham and Woolsey, 1984; Bates and Killackey, 1985; Land and Simons, 1985; Williams et al., 1994). The visible topography of the rodent trigeminal system, coupled with its responsiveness to experimental manipulation, has made it both useful and popular for studies of development, plasticity and structure-function

Accepted July 3, 1995. Address reprint requests to C.A. Leamey, Developmental Neurobiology

Group, Research School of Biological Sciences, Australian National Univer- sity, G.P.O. Box 475, Canberra, ACT, 2601, Australia.

1 1996 WILEY-LISS, INC.

DEVELOPMENT OF WALLABY SOMATOSENSORY THALAMUS 495

relationships (see reviews: Killackey et al., 1990; Woolsey, 1990; Kossut, 1992; Diamond, 1993).

In the rodent, the development of the trigeminal pathway is rapid and mainly prenatal (Killackey et al., 1990; Catalan0 et al., 1991; Erzurumlu and Jhaveri, 1992), with approximately 2 weeks between peripheral vibrissae development and cortical barrel formation (Yamakodo and Yohro, 1979; Killackey and Belford, 1979). Recent studies indicate that the system has achieved much of its organisation by the end of the first postnatal day (Henderson et al., 1994; Schlagger and O’Leary, 1994). This rapid, primarily intra-uterine developmental pattern hinders attempts to determine fac- tors important in the early establishment of topography. The slow, primarily extra-uterine marsupial maturational pattern is advantageous for developmental studies.

A number of studies have demonstrated similarities in the overall structure of the somatosensory system in marsupial and placental mammals (Pubols and Pubols, 1966; Sousa et al., 1971; Hazlett et al., 1972; Rockel et al., 1972; Hamilton and Johnson, 1973; Pubols et al., 1973; Haight and Neylon, 1978a,b; Gray et al., 1981). We have previously demonstrated that the structure and innervation of the sinus hair follicle of the wallaby is similar to that described in rodents (Marotte et al., 1992). A cortical representation of the vibrissae, comparable to that in rodents, has been described in two Australian marsupials, the tammar wallaby (Waite et al., 1991) and the possum (Weller, 1993). We have recently shown, in the wallaby, that the innervation of the facial periphery and the trigeminal brainstem nuclei is established by the day of birth. However, the first appearance of vibrissae-related aggregations associated with high levels of mitochondria1 enzyme reactivity in the brainstem nuclei does not occur until 40 days postnatal (Waite et al., 1994). This is approximately 45 days before the first appearance of vibrissae-related patches of high mitochon- drial enzyme reactivity in the cortex (Waite et al., 1991). This long delay between pattern formation in brainstem and cortex raises questions as to whether the intermediate relay, in the thalamus, shows aggregations and when these first develop.

This paper is the second in a series of the maturation of the wallaby trigeminal pathway. We report here on the structure and development of the ventroposteromedial nucleus (VPM) of the thalamus, with respect to the arrival of the first afferents from the principal trigeminal nucleus, the timecourse of naturally occurring cell death, and the formation of aggregations. The latter was assessed using

AChl? Aq Ch co em1 LGN Me MGN ml Po Pr5 s1 SDH VL VMO VP VPI, VPM

Abbreviations

acetylcholinesterase aqueduct cerebellum cytochrome oxidase external medullaly lamina lateral geniculate nucleus mesencephalon medial geniculate nucleus medial lemniscus posterior thalamic nucleus principal trigeminal nucleus primary somatosensory succinic dehydrogenase ventrolateral thalamic nucleus oral division of the ventromedial thalamic nucleus ventroposterior thalamic nucleus lateral division of the ventroposterior thalamic nucleus medial division of the ventroposterior thalamic nucleus

Nissl stains and reactivity for the metabolic markers cytochrome oxidase (CO) and succinic dehydogenase (SDH). These methods have been extensively used as markers for pattern formation in the trigeminal system and reflect pre- and/or postsynaptic components of the pattern (Van der Loos, 1976; Belford and Killackey, 1979a,b, 1980; Ivy and Killackey, 1982; Erzurumlu and Killackey, 1983; Durham and Woolsey, 1984). While SDH staining is reported to correlate with the distribution of afferent terminations (Bates and Killackey, 1985), there is evidence that GO staining may primarily reflect the distribution of postsynaptic cells and their processes (Chiaia et al., 1991). I t is probable that both pre- and postsynaptic elements may contribute to the staining produced by metabolic markers. Since some recent studies suggest that staining for acetylcholinesterase (AChE) provides an early marker for nuclear differentiation and pattern formation in rodents (Schlagger et al., 1993; Schlagger and O’Leary, 1994), AChE reactivity was also assessed in the wallaby. Some of this work has been presented previously in abstract form (Leamey et al., 1993,1995).

MATERIALS AND METHODS Experiments were performed on 54 wallabies (Macropus

eugenii 1 which were obtained from a breeding colony. Ages ranged from birth (postnatal day [PIO) to adult. Wallabies are seasonal breeders. During the breeding season removal of the pouch young results in reactivation of the dormant blastocyst, and birth occurs 26-28 days later. Out of the breeding season the reactivation of the blastocyst was induced following a single intramuscular injection of bromocriptine (Sandoz, Australia; Tyndale-Biscoe and Hinds, 1984). Young animals (PO-P20) were of known birth date. Older animals were either of known birth date, or ages were estimated from charts of mean head length of animals of known ages. These are accurate to within +2 days. Animals were deeply anaesthetised prior to perfusion by hypother- mia (PO-P35), intramuscular ketamine (0.05-3 mgi100 g body weight) andxylazine (0.1-0.2 mgi100 g; P4O-Pl60) or by intramuscular ketamine (0.2 mgikg) and xylazine (1.5 mg/kg) followed by intravenous 5%. sodium thiopentone (P 180-adult ).

Enzyme histochemistry Thirty-one animals (28 pouch young, PO-P201 and 3

adults) were used. Animals were perfused either with 10% glycerol, or with 0.9% saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4) for 20 minutes. In two animals (P33 and P73) 2% paraformaldehyde was used. In unfixed tissue the brain was dissected immediately and frozen in isopentane on dry ice. Fixed tissue was allowed to postfix for 1-2 hours, then cryoprotected in 30% sucrose in 0.1 M PB before freezing as above. In very young animals (PO-P20), the brain was left intact. In older animals the brainstem was removed, and the forebrain was halved sagittally. Sections were cut in the coronal plane (P0-35), or horizontal and coronal planes (P40-adult) on a cryostat at 30 pm (PO-P91) or 50 pm (P95-adult) and mounted directly onto gelatinised slides. In fixed tissue from older animals (P108-adult) the diencephalon was removed before placing it in sucrose to minimise the time required for cryoprotection; tissue was cut at 40 pm or 50 pm on a freezing microtome and collected in cold 0.1 M PB. In animals from PO to P20, alternate sections were Nissl-

A96

stained and reacted for either SDH or CO reactivity. In animals from P30 to adult, four series were prepared. In unfixed tissue the four series were reacted for SDH, CO, and AChE, and Nissl-stained. In fixed tissue two types of AChE reaction were carried out and the other series were used for CO and Nissl stains. In the adult only one AChE reaction was performed and the fourth series was stained for myelin.

Histochemistry for CO followed the method of Wong- Riley (1979). Histochemistry for SDH was performed accord- ing to the method of Nachlas et al. (1957). The reactions were monitored at regular intervals, and terminated with 0.1 M PB (CO) or with 10% neutral buffered formalin (SDH). The AChE reaction used in all animals from P30 to adult followed the modification of the procedure of Genesser- Jensen and Blakstad used by Kristt (1989). In addition, in some animals the modification of the method used by Bear et al. (1985) described by Schlagger et al. (1993) was used on an additional series. The method described by Kristt ( 1989) produced a clearer reaction product, with less arte- fact and background staining. To provide a control of the efficacy of the reaction, some tissue from PO-2 rats was included in the incubationireaction procedure. The myelin stain used followed the modification of the Heidenhain procedure described by Hutchins and Weber (1983).

Cell number and cell death This was assessed in 13 pouch young between P14 and

P208 and in the adult. Animals were perfused with 0.5-2% glutaraldehyde and 2-4% parafomaldehyde in 0.1 M PB. The diencephalon was removed, dehydrated and embedded in wax. Sections 10 pm or 20 pm (in 2 cases) thick were cut in the coronal plane, and stained with cresyl violet. The numbers of pyknotic cells were counted in every second section a t ~ 4 0 0 magnification. Counts were multiplied by 2 and corrected for double counting (Carriere and Patterson, 1962). Total neuron numbers were estimated at P47, P81 and in the adult. Counts of all neuronal nucleoli within a grid 0.34 x 0.34 mm2 at 4 0 0 ~ were performed at 2 sites on every fourth section. Neuronal density was determined using grid size and section thickness. The cross-sectional area of each section counted was determined using the Magellan programme (Halasz and Martin, 1985), and the volume calculated using Cavalieri’s basic estimator (Rosen and Harry, 1990). The number of dead cells per 1,000 live cells at the peak of cell death was then calculated to produce a pyknotic index.

Cell diameters were also measured at selected stages during development using the Magellan programme. Mean diameters (md) and standard deviations (sd) were then calculated.

Tracing studies Ten animals (1 at PO, 3 at P5 ,4 at P10 and 2 at P15) were

used to study the early development of the trigeminothalamic pathway. Animals were perfused through the ascending aorta with 0.9% saline followed by 4% paraformaldehyde in 0.1 M PB (pH 7.4). Previous work (Waite et al., 1994) had confirmed the position of the principal sensory trigeminal nucleus (Pr5) in animals of these ages, slightly dorsomedial to the entry of the trigeminal nerve root, adjacent to the pontine flexure. This region was exposed and a single crystal (diameter 50-75 pm) of 1,l‘ dioctadecyl- 3,3,3’,3’-tetramethylindocarbocyanine perchlorate (DiI) was applied to Pr5. In initial experiments (one animal at each

C.A. LEAMEY ET AL.

age studied) DiI was applied unilaterally. Since these indicated that the trigeminothalamic pathway projects exclusively contralaterally at this stage, the pathway was traced bilaterally in later experiments. In some cases 4-(4-dihexadecylaminostyryl)-N-methyl-pyridinium iodide (DiA) was used on one side and DiI on the other. Tissue was stored in darkness at room temperature for 4-6 weeks, before it was embedded in 6% agar, and 100 pm sections were cut on the Vibratome. In most cases these were cut in the plane perpendicular to the anterior-posterior axis of the brain a t this stage as illustrated in Figure 11. In one case a t P10 oblique sections were cut parallel to the fibre pathway through the rostral mesencephalon. Sections were counter- stained in 0.001% bisbenzimide (Sigma) in 0.1 M PB before they were viewed under epifluorescent illumination on the Zeiss axiophot. Excitationibarrier filters were: 5461590 nm for DiI; 3651420 nm, bisbenzimide; 450-4901520, DiI and DiA).

As part of another study looking at the development of interconnections between thalamus and cortex, DiI was applied to the developing somatosensory cortex of a separate series of animals. Since this permitted the visualisation of the details of dendritic arborisation in VPM, the results from one of these has been included here. Tissue was treated as above, and the fluorescent label in selected sections was photoconverted with diaminobenzidine to produce a photostable product (Maranto, 1982; Sandell and Masland, 1988).

RESULTS Adult structure

The terminology used in this description, and the delinea- tion of thalamic nuclei follows that of Mayner (1985, 1989). In the adult wallaby, VPM is a large, distinctive nucleus, clearly visible a t low power (Fig. la) . Its constituent neurons number around 48,600 (see later) and form a relatively homogeneous population; they are closely packed, with large (md = 21.9 pm; n = 54, sd = 1.71) somata with prominent nucleoli. Some smaller cells which are clearly neurons can be seen when scanning the nucleus at high power. These are fairly uncommon, with only a few present in a given section. The density of cells within VPM is higher than in surrounding nuclei (Fig. la,c).

The rostral pole of VPM starts approximately one-third of the way along the rostrocaudal axis of the thalamus, and in coronal sections is seen as a darkly staining oval region. In more caudal sections, VPM expands first medially and

Fig. 1. a: Low power view of a coronal Nissl-stained section showing the position of VPM, close to the ventrolateral border of the dorsal thalamus in an adult wallaby. Bar: 1 mm. b: Horizontal sections through VPM in the adult wallaby, reacted for CO. Rostra1 is to the top, and medial is to the left of the page. Overall CO reactivity is higher in VPM (arrow) than in surrounding structures, and circular clusters of high reactivity can be discerned. These are shown at higher power in the inset. Bar: 1 mm; inset: 500 pm. c,d: Adjacent horizontal sections stained for Nissl substance (c) and myelin (d). The cells in VPM are organised into rows which are separated by myelin bundles (arrows in d) which pass obliquely through the nucleus from anterolateral to posteromedial. The organisation of the cells into clusters (a few marked with a “c”) within these rows can be discerned ( c ) , with clusters surrounded by a fine plexus of myelinated fibres (d). The prominent fibre bundle which partially separates the rostral and main parts of VPM is indicated by an arrowhead in d. Bar: 400 pm.


Figure 1

498 C.A. LEAMEY ET AL.

then laterally. Around this level the cells of VPM are organised in dorsoventrally directed bands of cells which resemble fingers (Fig. 2a). Fine myelin bundles run parallel to, and separate the fingers (Fig. 2b).

In the caudal half of the nucleus, in coronal sections, VPM takes on a fan shape which is dorsally convex (Fig. 2c,d): and the oral division of the ventromedial nucleus (VMOI appears at the medial border of VPM (Fig. 2c). At this level the organisation within VPM is most obvious laterally. Here the fingers curve laterally as they proceed from the ventral to the dorsal borders of the nucleus (Fig. 2c). A fine bundle of myelinated fibres runs between each finger (Fig. 2d). Segments of larger fibre bundles which pass from ventrolateral to dorsomedial are also visible (Fig. 2d). These bundles are seen more clearly in horizontal sections and are described below. Caudally, VPM narrows ventrally and medially to become crescent-shaped, and terminates level with the rostra1 pole of the medial geniculate nucleus iMGN).

In the horizontal plane VPM is roughly kidney-shaped with its convex border facing laterally (Fig. lb-d). Some prominent fibre bundles traverse VPM from anterolateral to posteromedial (Fig. Id). These bundles can be followed into the internal capsule and represent the reciprocal connections between cortex and thalamus. As the bundles traverse the nucleus they separate rows of cells (Fig. lc). The rows of cells are characterised by the presence of distinct, roughly circular, clusters of cells within them. The clusters of cells are cross-sections of the fingers seen in the coronal plane, and each cluster is encircled by a fine plexus of myelinated fibres (Fig. 2d).

CO reactivity in VPM is somewhat higher than in surrounding nuclei (Figs. l b and 2e), although the degree of contrast is less than that seen in pouch young (see below). In coronal sections the lateral part of the nucleus exhibits segmentation into fingers of high reactivity (Fig. 2e), consistent with the organisation seen with Nissl (Fig. 2c) and myelin stains (Fig. 2d) at this level. In horizontal sections (Fig. lb, inset) clusters are seen as circles of reactivity surrounded by a thin band of low reactivity. The band of low reactivity corresponds to the myelin bundles which surround the clusters (Fig. Id), whereas the circle of reactivity corresponds to the clusters of cells seen in Nissl-stained material (Fig. lc). This gives a hint of the vibrissae related pattern clearly visible in pouch young.

As in other mammals, AChE reactivity is low throughout the whole of the ventral nuclear tier in the wallaby. In VPM, there is an extremely low level of background staining, and no cells demonstrate AChE reactivity (Fig. 2f).

Development of VPM Morphogenesis. The boundaries of VPM could not be

confidently identified on purely cytoarchitectural grounds until P47, although other evidence permitted identification of the region in younger animals. This included correlation with other species, positional information in comparison with slightly older animals, and subtle differences in enzyme reactivity. Whilst at very early stages identification is somewhat speculative, anterograde and retrograde tracing studies have confirmed results from P15 onwards.

During the first few postnatal days the diencephalon of the wallaby consists primarily of a thick neuroepithelial layer surrounded by a mantle zone (Fig. 3a). There is a thickening of the neuroepithelium just dorsal to the reticular protruberance in the intermediate

PO-26.

lobule of the developing thalamus. This region is believed to give rise to the cell line of the VP nucleus in the rat (Altman and Bayer, 1988,1989). By P10, the dorsal thalamus can be seen as a relatively dense cellular mass lateral to the narrower neuroepithelial layer of the intermediate thalamus (Fig. 3b). This region is presumed to contain the cells of the developing VP complex, either in migration from the neuroepithelial zone, or in position close to the ventral and lateral borders of this mass. The first signs of the external medullary lamina (em]) delineate the region, separating it from the developing lateral geniculate nucleus (LGN).

Up until P15, in Nissl stains, the cells comprising the putative VP complex appear as small (md = 4.19 pm; n =

57, sd = 0.88) undifferentiated, spherical structures which are packed extremely densely (Fig. 3c). This is due to the nuclei staining intensely and uniformly, with little or no staining of the cytoplasm or proximal processes. However, retrograde labelling of cells in VP from the cortex (thalamic axons have just reached the putative somatosensory cortex by this time; Leamey et al., 1993) reveals the cells are clearly multipolar, and have extended modest dendritic arbors at this stage (Fig. 3d).

By P20, VP is seen as a dense cellular mass medial to the now more obvious em1 (Fig. 3e). CO reactivity is marginally higher here, but this does not clearly define the region. The level of neuronal morphogenesis increases such that by P26, in Nissl stains, an intensely stained nucleolus is readily apparent within the nucleus (Fig. 3f). There is very little cytoplasm at this stage, but the proximal portions of processes can be seen.

By P33, VP can be more readily discerned in Nissl stains. The first hint of the fibre plexus which separates VPM and VPL in the adult can be seen as a narrow cell sparse band (Fig. 4a,b). CO reactivity is slightly higher in VP than in other thalamic nuclei (Fig. 4a). The cells of VPM show very weak AChE reactivity, which is indistinct and is not well localised to cell bodies (Fig. 4b, inset). Reactivity is somewhat higher in VPL, although this is primarily due to extracellular staining. Other thalamic nuclei (including MGN, LGN and the centrolateral, ventrolateral, and posterior nuclei) show similar low levels of AChE reactivity to that seen in VPM, suggesting that this degree of staining is nonspecific. The epithalamus and striatum demonstrate a significantly higher degree of AChE reactivity indicating that the low level of reaction product in VPM is not due to a failure of the reaction.

Subsequent to P33, there is a marked increase in the level of differentiation of the dorsal thalamus, and by P47 most of the nuclei seen in the adult are recognisable in Nissl stains at low power (Fig. 4c). In VPM, reactivity for SDH and CO rises relative to other nuclei but remains uniform.

By P52, the first signs of segmentation are apparent at the lateral borders of VPM; this is evident in both CO-reacted material (Fig. 5a) and in Nissl stains (Fig. 5b). AChE reactivity in VPM is higher than at P33, with many of the cells of VPM now clearly showing low levels of reactivity (Fig. 5c). The fibre lamina separating VPM and VPL shows a high level of reactivity.

At P59 the degree of contrast between the level of enzyme reactivity in VPM and surrounding structures has risen markedly to make VPM the most distinctive structure in the dorsal thalamus (Fig. 5d). The nucleus is ventrolaterally convex until this time, rather than dorsally convex as seen in the adult (Figs. la; 2c,d). The signs of segmentation first seen at P52 have become more obvious, with 3-4 bands

P30-47.

P50-60.


Fig. 2. Pairs of coronal sections through VPM in the adult. a,c: stained for Nisi substance; b,d: stained for myelin; e: reacted for CO; f: reacted for AChE. Medial is to the left, and dorsal to the top of the page. a,b: Adjacent sections from slightly behind the rostra1 pole. The organisation of the cells into dorsoventrally aligned rows (arrows in a ) , which are separated by very fine fibre bundles (arrows in b), can be seen. VPM is bordered dorsomedially by VL and ventrolaterally by VPL. Bar in a. 1 mm, b: as for a. c ,d At this level, VPM assumes its dorsally convex shape, and is characterised by the organisation of cells within curved bands or fingers (arrows in c). The fingers curve laterally as they pass ventrodorsally, and are separated by very fine myelinated bundles

(large arrows in d) . The ml is apparent as a thickening of the em1 and fibres pass dorsally from here to enter VPM ventrally. The fibres separating fingers appear to be continuous with those emanating from the ml. Prominent here are larger myelinated fibre bundles which pass obliquely through VPM (small arrows in d). Bar: as in a. e,f: These sections are taken from a similar level to those shown in c,d. CO reactivity (e) is somewhat higher in VPM than in surrounding structures. The segmentation of the nucleus into fingers of high reactivity is apparent laterally (arrows). AChE reactivity ifl is very low in W M , and in most of the surrounding nuclei; dorsally, the cells of Po demonstrate a moderate degree of reactivity. Bars: 500 Fm.

Figure 3

DEVELOPMENT OF WALLABY SOMATOSENSORY THALAMUS 50 1

of high reactivity clearly evident laterally in SDH and CO-reacted tissue (Fig. 5d). No clear pattern is visible in horizontal sections. At this stage the neurons are still immature in appearance, each characterised by a large nucleus surrounded by relatively little cytoplasm (Fig. 5e). They are also considerably smaller (md = 8.9 Fm; n = 56, sd = 1.09), and more densely packed than in mature animals.

By P73, the segmentation of the nucleus in CO-reacted material is obvious. At the rostral pole this takes the form of dorsoventrally directed bands (Fig. 6a) and though less obvious, this organisation can also be seen in Nissl stains (Fig. 6b). More caudally the segmentation is seen as the curved fingers (Fig. 6c) which characterise the adult nucleus. In some cases the fingers are broken up by the mediolaterally directed fibre bundles connecting thalamus and cortex (Fig. 6c). The organisation of VPM is most apparent in horizontal sections, where the nucleus is characterised by discrete circular clusters of cells. This is most obvious in material reacted for CO activity (Fig. 6d). In cross-section the clusters typically contain around 10-12 highly CO reactive cell bodies (Fig. 6d, inset) and CO is also high between the clustered cell bodies. Few cells are apparent between adjacent clusters, although there appears to be a cluster-free region in the midventral part of VPM. The clusters appear to be aligned in rows which pass from anterolateral to posteromedial (Fig. 6d) as seen in the adult (cf Fig. 4). AChE reactivity is now more intense in VPM, with the cells exhibiting fairly high levels of reactivity (Fig. 6e). An impression of the pattern seen clearly with CO is replicated in AChE reacted material.

The pattern formed by the discrete cluster- ing of cells seen at P75 is maintained until P108, but has lost a little of its clarity (Fig. 7a), and is less obvious still by P144. The organisation of the nucleus into curved fingers is still seen in coronal Nissl-stained sections, and clusters of cells can be discerned in horizontal sections, as in the adult (Fig. 7b). By P144 the general characteristics of the nucleus are now essentially adult-like. Neuron diameter (md = 20.4 Fm, sd = 1.4, n = 58) approximates that of adults and cells have a mature appearance (Fig. 7d), although they are more closely packed, and maintain the high levels of AChE reactivity seen at younger ages (Fig. 712). AChE reactivity is retained until at least P200.

This was examined in animals between P26 and P208 by counting pyknotic nuclei. Prior to P47 the boundaries of VPM had to be estimated, but since cell death is extremely low at early stages, this had little impact on the

P70-95.

P108-201.

Cell death.

Fig. 3. a: Coronal Nissl-stained section through the region believed to contain the cell line which will give rise to VP (arrow) in a P2 wallaby. Bar: 600 pm. b: Coronal Nissl-stained section through the putative VP nucleus [star) at P10. The em1 (arrow) can be seen separating the laterally developing LGN from the rest of the dorsal thalamus. Bar: 1 mm. c: High power view of a Nissl-stained section through the putative VP at P14. Note the low level of neuronal differentiation visible with this stain. Bar: 10 pm. d High power view of neurons in the developing VP at P15. These cells were photoconverted following retrograde labelling with DiI from the putative somatosensory cortex. In contrast to the apparent simplicity of the cells seen in Nissl stains (c), when visualised with this method the cells are seen to be relatively complex, and to have extended modest dendritic arbors. Bar: 20 pm. e: By P20 VP can be discerned as an area of increased cell density medial to the cml. Bar: 400 pm. fi High power view of a Nissl-stained section a t P26. A narrow ring of cytoplasm surrounds the nucleus and proximal dendritic processes are visible. Bar: 10 km.

results. Degenerating cells were seen as either single con- densed nuclei (Fig. 8b) or as 2-4 fragments of degenerating nuclei (Fig. 8a) sometimes encircled by clear cytoplasm. Fragments contained within a small radius (10 Fm) were treated as coming from a single nucleus.

The relative level of cell death at different ages is shown in Figure 8c. Until P61 the level of cell death is very low. Cell death began to increase by P76, and peaked a t P81. A relatively high level of cell death was maintained until P89, and returned to low levels by P108. The number of cells in VPM at the peak of cell death (P81) was found to be 65,953. This corresponds to a pyknotic index of 1.3 (pyknotic cells per 1,000 live cells). Neuron number decreases by 26.38 between P81 and the adult (48,586 neurons). Over the same period, cell packing density decreases by a factor of 11.5, while nuclear volume increases almost 9-fold.

Early development of the trigeminothalamic pathway. This was studied using carbocyanine dyes in animals between PO and P15. Results are summarised in Figure 11. In addition to the anterograde labelling of trigeminothalamic fibres described below, placement of DiI on Pr5 resulted in substantial retrograde labelling of the trigeminal tract, nerve root and ganglion. Intranuclear bundles of the trigeminal complex and the mesencephalic trigeminal nucleus were also labelled. There was also a varying degree of label present in the reticular formation both contralaterally and ipsilaterally. Where unilateral injections were made, no evidence of an ascending ipsilateral pathway was seen. The labelled fibres ascended solely towards the contralateral thalamus (e.g., see Fig. 9a).

There was no evidence of a rostrally directed pathway emanating from Pr5 a t PO (not shown, but see Fig. l l a ) . The label seen in the trigeminal tract and intranuclear bundles indicated that dye transport had been successful. Although a highly localised deposit of one small crystal was made, there was a considerable spread of label, particularly to radial glia, at this age. Labelling of the reticular formation was also seen.

By P5 the trigeminothalamic projection had begun to develop. A labelled band of fibres passed medially from Pr5 to cross the midline at the level of Pr5. This pathway is shown in a low power line drawing in Figure l l b . The fibres then turned dorsally to approach the mesencephalon. The most rostral fibres a t this stage terminated in growth cones in the rostral pons (Fig. 10a). No fibres entered the mesencephalon at this stage.

The trigeminothalamic fibres leaving Pr5 took the same course as that seen a t P5, but by this stage a larger number of fibres were labelled, and formed a more compact bundle (Fig. 9a). The growth cones of the fibres at the point where the path made an abrupt dorsal turn were particularly complex (Fig. 9b,c). At this stage the leading fibres were close to the rostral border of the mesencephalon and were approaching the diencephalon. The position of the fibres in this region was difficult to demonstrate in sections cut in the plane shown in Figure 11, as the fibres passed directly through the sections. However, an oblique section along the plane of the fibre path enabled them to be seen clearly (Fig. lob). The position of the leading edge at this stage is indicated in Figure l l c . The appearance of the growth cones on the most rostral fibres (Fig. lob) was notably simpler than those seen more caudally at the turning point (Fig. 9b,c).

At this age the pathway was traced bilaterally with carbocyanine dyes (Fig. 1Oc). The fibres took the same

PO-5.

P10.

P15.

C.A. LEAMEY ET AL.

Fig. 4. a,b: Adjacent CO-reacted (a) , and Nissl-stained (bJ coronal sections through rostra1 VP at P33. A narrow, cell sparse lamina (arrowheads) demarcates the border between VPM and VPL. CO reactivity la) is marginally higher in VP than in surrounding nuclei. Bar: 200 pm. Inset in b shows AChE reactivity a t this age. Reactivity is

weak and not well confined to cell bodies. In all panels medial is to the left and dorsal to the top. Bar: 100 pm. c: Low power view of a Nissl-stained coronal section through VPM in a P47 wallaby. Many of the nuclei of the dorsal thalamus may he readily identified, and the borders of VPM are apparent. Bar: 400 pm.

course described previously. By this stage a larger number

could be clearly seen Coursing through the ventrolateral tegmentum of this reDon (Fig. 1oc). A number of fibres continued rostrally towards the mesencephalic-diencephalic junction to enter the diencephalon. In the coronal plane of the putative ventrobasal complex the fibres turned abruptly dorsal and terminated (Fig. 10d). The reDon of termination is indicated in a low power line drawing in Figure :Id. The fibres appeared to extend terminal arborisations in this reDon (Fig. 10e).

of fibres had reached the rostra1 midbrain, and the bundles Fig. 5. a-c: Adjacent coronal sections through VPM in a p52

wallaby stained for CO reactivity (a), Nissl substance (b) and AChE (c). C 0 reactivity is higher in VPM than in surrounding structures (a), with a hint of segmentation appearingat the lateral border of VPM (arrows). This is also visible in Nissl stains (bJ. While AChE reactivity is still fairly low (c), it is now well localised to the somata of VPM cells (inset). In all panels medial is to the left and dorsal to the top. Bar: 400 pm, inset in c: 100 pm. d: Coronal CO-reacted section through VPM at P59. The relative level of CO reactivity has risen and the segmentation of the nucleus is becoming more pronounced (arrows). Bar: as for a. e: High power view of a Nissl-stained section through VPM at P59. Bar: 10 pm.

Figure 5

C.A. LEAMEY ET AL.

Figure 6


DISCUSSION This study has described the structure and development

of the VPM nucleus of the wallaby. It has been demonstrated that VPM displays evidence of a parcellated organisation. The segmentation is most easily visualised using enzyme reactivity in developing animals, but can also be seen with Nissl staining. It is less clear in the adult. The aggregations are obvious by P73, approximately 2 months after the first arrival of afferents from the brainstem, and at least a week prior to the peak of cell death in the nucleus.

Technical considerations Histochemical techniques, which assess relative levels of

endogenous mitochondria1 enzyme activity, have been extensively used as markers for pattern formation in the developing trigeminal system. Earlier studies used SDH reactivity (e.g., Belford and Killackey, 1979a,b, 1980; Killackey and Belford, 1979). However, recent work (Henderson et al., 1994), using CO reactivity, has demonstrated pattern formation earlier than previously reported. This suggests that CO may be a more sensitive marker than SDH. CO was found to be the method of choice in the present study as it produced a clearer reaction product and was compatible with the use of fixed tissue which greatly improved section quality. Although the use of fixed tissue decreased the reactivity of the enzyme, this could be effectively compen- sated for by an increase in reaction time.

Schlagger and O’Leary (1993) reported that AChE positiv- ity could be detected earlier in rodent VPM than previously found by Kristt (1989). However, in the present study, the methods used by both investigators revealed the development of AChE reactivity at the same time. The clearer reaction product produced by the method described by Kristt was preferable for photomicrographs.

The effectiveness of the lipophilic carbocyanine dyes as tracers in fixed tissue was first reported by Godement et al. ( 1987) and is now well established. Three questions need to be addressed here. Was Pr5 correctly targeted for dye insertion? Did the tracer label the full extent of the trigeminothalamic pathway at each stage? Did transcellular labelling occur? Firstly, the correct targeting of Pr5 is evidenced by the labelling of the trigeminal nerve root and tract, and of the intranuclear bundles between trigeminal subnuclei. Secondly, the transport of carbocyanine dyes in fixed tissue relies upon the time-dependent diffusion of the dye along the lipid membrane. Similar times were allowed for diffusion at each stage examined, and despite the

Fig. 6 . a,b: Adjacent coronal sections through the rostral pole of VPM a t P75 reacted for CO (a) and Nissl-stained (b) . The segmentation of this part of the nucleus is clearly visible in CO-reacted material (arrows in a) . Although less obvious this organisation can also be detected in Nissl-stains (arrows in b). No clear pattern is visible in VPL. Medial is to the left and dorsal to the top of a x . Bar: 200 ym. c: Coronal section reacted for CO through the body of VPM at P73. At this level the nucleus is clearly organised in arcuate hands or fingers which show a high level of CO reactivity. The fingers are broken up a little by the passage of fibre bundles (arrows) which pass obliquely through the nucleus to join the internal capsule. Bar: as for a. d Horizontal section reacted for CO a t P75. VPM is characterised by the presence of circular clusters of highly CO-positive cells. These are the fingers shown in a and c cut in cross-section. Bar: as for a. Inset: Higher power view of the cellular clusters. Note the absence of a cell sparse hollow and the lack of cells between adjacent clusters. Rostra1 is to the top and medial to the left. Bar: 100 pm. e: Horizontal section adjacent to d, reacted for AChE histochemistry Orientation as in d. Bar: as ford.

increase in brain size at each stage, labelled fibres were traced to progressively more rostral regions. The presence of growth cones on the tips of the leading fibres at P5 and P10 provides additional evidence as to the effective labelling over the length of the pathway. Finally, although some transcellular labelling did occur, this was confined to radial glial cells. No evidence of transneuronal labelling was seen.

Adult structure The observations made here on the structure of VPM in

the adult wallaby confirm and extend those of Mayner (1985,1989). As in most placental (reviewed in Jones, 1985) and Australian marsupial (Rockel et al., 1972; Haight and Neylon, 1978a,b, 1981; Mayner, 1985, 1989) mammalian species, the VPM nucleus of the wallaby is readily distin- guished using cytovrchitectural criteria, and is one of the most distinctive structures of the dorsal thalamus. This contrasts with the lack of nuclear differentiation in the American opposum (Oswaldo-Cruz and Rocha-Miranda, 1967; Donoghue and Ebner, 1981). In the wallaby, VPM is clearly separated from VPL by a fibre lamina, and exhibits extremely low levels of AChE reactivity. VPM shows a higher level of CO reactivity than surrounding nuclei, and exhibits considerable evidence of segmentation.

The segmentation of the VPM nucleus of the wallaby into bands (or fingers) of cells, is strongly suggestive of a modular organisation. The presence of functional modules which have anatomical correlates, parallel to those seen here in the wallaby, have been well described in the somatosensory thalamus of rodents (Van der Loos, 1976; Saporta and Kruger, 1977; Land and Simons, 1985; Hoogland et al., 1987) and primates (Jones et al., 1982, 1986; Rausell and Jones, 1991a,b). Electrophysiological studies have confirmed the representation of the vibrissae in VPM of the wallaby (Faulks and Mark, 1982). This, together with evidence of the correlation between vibrissae and patches seen in the primary somatosensory (Sl) cortex of the wallaby (Waite et al., 1991), strongly suggest a relationship between the fingers seen here in VPM and the mystacial vibrissae. Additional sup- port is provided by observations that a small injection of horseradish peroxidase into the electrophysiologically de- fined cortical representation of an individual mystacial vibrissa, yields retrograde labelling of VPM cells within a curved dorsoventral band which closely resembles the fingers described here (Marotte et al., unpublished). Similar observations (but without physiological confirmation) were made by Mayner, 1985. The presence of bands of cells believed to correspond to the rows of mystacial vibrissae has been noted in VPM of the adult possum (Jones, 1984).

In primates, groups of CO-positive neurons termed rods extend anteroposteriorly in VPM. They project to narrow radial domains within the S1 cortex, and demonstrate place- and modality-specific responses. The rods are the focus of input from Pr5; they are embedded in a CO-poor matrix which primarily receives input from Sp5 (Jones et al., 1982, 1986; Jones, 1985; Rausell and Jones, 1991a,b). In the rodent, the functional modularity of VPM is characterised by a similar array of CO-positive cylinders (Land and Simons, 1985) which correlate with the barreloids seen in Nissl stains (Van der Loos, 1976). The barreloids are similar to the rods seen in the monkey in that they are the focus of input from the contralateral Pr5, but there is no evidence of a matrix region within VPM of the rat (Williams et al., 1994). I t has

Significance of the parcellation of VPM.

Comparison with other species.


Fig. 7. a: Horizontal section reacted for CO through VPM in a P108 animal. The clusters are still visible but less clear than at P75 (see Fig. 81. A hint of segmentation can be seen in VPL. Medial is to the left and rostra1 to the top of a,b. Bar: 200 Fm. b Horizontal Nissl-stained section through VPM in a P I 4 8 animal. VPM is seen as a densely staining region, and circular clusters of cells (a few are indicated by arrows) can be discerned within it, as in the adult (see Fig. 4a). Bar:

250 km. c: Coronal section through VPM at P148 reacted for AChE histochemistry. The cells of W M maintain a high level of reactivity until this time. Medial is to the left and dorsal t o the top Bar: 500 km. d: High power view of a Nissl-stained section through VPM at P148. The neurons now appear essentially adultlike, but are more closely packed than in mature animals. Bar: 10 ym.


C 100

80

V .r( Y

20

0 0 100 200 Adult

Postnatal Age

Fig. 8. a,b: Examples of pyknotic cells (arrows) seen in wallaby VPM a t the peak of cell death. Bar: 10 pm. c: Graph illustrating the timecourse of developmental cell death in VF'M of the wallaby.

been suggested in the rodent, that each neuron belongs to a barreloid (Land and Simons, 1985; Diamond et al., 19921, with the narrow, CO-poor spaces between barreloids occu- pied by fibre bundles (Land and Simons, 1985; Diamond et al., 1992). The structural features of VPM in the wallaby (described above) thus appear to have more in common with the rodent system.

Rodent barreloids are described as a cell body-poor core surrounded by a ring of cells (Van der Loos, 1976; Land and Simons, 1985). Each barreloid extends from anterodorsome- dial to posteroventrolateral within VPM and corresponds to an individual vibrissa (Sugitani et al., 1990). The neuronal aggregations seen in the wallaby thalamus take the form of fingers. The fingers do not have the hollow centreldense surround organisation characteristic of rodents. Rather, the cells appear to be evenly distributed within individual fingers. Structural differences also exist in the vibrissae representation observed in the S1 cortex of marsupials (Waite et al., 1991; Weller, 1993) as compared to the mouse (Van der Loos, 1976).

Development Timing of afferent arriual. Trigeminothalamic fibres

were found to arrive in the putative VPM nucleus between

days P10 and P15. This is close to the time of completion of neurogenesis of VPM (neurogenesis of VPM in the tammar is beginning on P5, is in full swing at P8, and almost finished at P10; unpublished observations), and before the time that the nucleus can first be identified in Nissl stains. Interestingly, the arrival of brainstem afferents is after the first outgrowth of thalamocortical fibres, and coincident with their arrival below the cortical plate (Leamey et al., 1993). The arrival of afferents to the somatosensory thalamus at a comparably early stage of development has been noted in the opossum (Martin et al., 1987) and the clawed toad (Munoz et al., 1993).

Surprisingly, the time of arrival of trigeminal afferents to VPM has not been accurately determined in the rodents. Scheibel et al. (1976) note the presence of lemniscal afferents bearing growth cones in the vicinity of VP around E16, and Asunuma et al. (1986) report that projections from the dorsal column nuclei can be traced into VP at E17. These studies note the simplicity of the terminals of the ascending afferents at early stages, and it is likely that these would be overlooked without direct anterograde tracing methods. This may provide an explanation for the failure of recent studies using cytological criteria to detect lemniscal afferents in the VP nucleus before E l 9 (Kristt, 1989; Killackey, 1993). I t is probable that afferents from the trigeminal

508 C.A. LEAMEY E T AL.

Fig. 9. a: Section through Pr5 in a P10 wallaby. A crystal of DiI was inserted into Pr5. The developing trigeminothalamic tract is seen to pass medially from Pr5 and goes out of the plane of section. A lateral view of the pathway is illustrated diagramatically in the top panel of Figure 11. Section is taken at a similar level to that illustrated by the low power line drawing of the pathway in a P5 animal in Figure l l b . Dorsal is to the top in all panels. Bar: 400 pm. b: Higher power view of axons in the vicinity of the turning point arrowed in a, showing the complexity of the growth cones in this region. Bar: 100 pm. c: High power view of one of the axons with a growth cone near the turning point. Bar: 25 pm.

nuclei innervate VPM earlier in rodents than has been documented thus far for spinal inputs.

Growth cones of developing trigeminothalarnic fibres. The growth cones seen at P10 in the pons where the trigeminothalarnic fibres change direction were notably more complex than those seen more rostrally. An increase in growth cone complexity at points where decisions are being made has been noted in the development of various systems of both invertebrate (Raper et al., 1983) and vertebrate (Tosney and Landmesser, 1985; Bovolenta and Mason, 1987; Holt, 1989) species, and the importance of filopodia in normal axonal navigation has been demonstrated (Chien et al., 1993). The increase in the complexity of the growth cones presumably reflects their response to changes in the local environment which provides guidance clues at the turning point.

Whilst CO reactivity in VPM was higher than in surrounding nuclei at P40, this was uniform across the nucleus. The first hints of pattern formation were seen at P52 at the lateral border of VPM. These became more obvious by P59, and fingers of reactivity resembling those described above in the adult were clear by P73. The segmentation was most striking in the horizontal plane where discrete circular clusters of cells characterise the nucleus. Coincident with the formation of the aggregations was an increase in the relative level of CO reactivity. The pattern formed by the aggregations remained clear until at least P108, but then became less obvious, although the parcellation could still be discerned, as in adults.

The presence of vibrissae-related aggregates which are clearly visible in young animals using enzyme reactivity is a well known characteristic of the rodent somatosensory thalamus (Belford and Killackey, 1979a,b; Ivy and Killackey, 1982). There are striking parallels in the development of the 2 species. In both cases VPM initially demonstrates a higher level of enzyme reactivity which is fairly uniform over the nucleus (PO in the rat and P40 in the wallaby). The segmentation then gradually develops over the ensuing period to form a clear pattern. In the rat this occurs over a period of a few days (by P3 using SDH reactivity; Belford and Killackey, 1979b), but in the wallaby the same events take about a month (by P73). In rats, a clear pattern is retained for the first few postnatal weeks using SDH, after

Pattern formation.

Fig. 10. a: The developing trigeminothalamic pathway at P5. The pathway is illustrated in a low power line drawing in Figure l l b . The leading edge is shown in the rostral pons approaching the midbrain. Growth cones (arrows) are present on the tips of fibres. Bar: 100 pm. b: Leading edge of developing trigeminothalamic pathway a t P10. Note the simplicity of the growth cones (arrows) as compared to those at the turning point (Fig. 9b). The fibres are coursing through the ventrolat- era1 tegmentum of the rostral midbrain approaching the diencephalon. This section was taken in a plane parallel to the fibre pathway (see top panel of Figure 11 1. A line drawing indicating the position of the leading edge in the coronal plane is given in Figure l lc . Bar: 100 pm. c: Section through Pr5 in a P15 wallaby following the bilateral application of carbocyanine dyes to Pr5. The trigeminothalamic pathway is seen entering the Me. Bar: 400 pm. d: Coronal section through VPM in a P15 wallaby following the application of DiI to the contralateral Pr5. The photograph has been double exposed to reveal the presence of DiI-labelled fibres and the bisbenzimide counterstain. DiI labelled axons can be seen ventrolaterally (small arrow) forming the nascent ml. The terminal ends of these fibres (large arrow 1 are in the developing VP. A low power line drawing of the brain at this level is even in Figure l l d . Bar: 400 pn. e: High power view revealing detail of DiI-labelled fibres shown in d. The fibres appear to extend terminal arborisations in the region. Bar: 50 pm.


Figure 10

Dc

Vent rat

PO

sal L

Posterior t b Anterior

P I 0

P5

@

P15 Figure 11


which it is obscured, but can still be detected in adults using CO reactivity (Land and Simons, 1985). In the wallaby the pattern has become less obvious about 2 months after its initial appearance, but can still be detected in older animals.

The formation of discrete aggregations between P52 and P75 in the wallaby thalamus is in keeping with the sugges- tion that pattern formation follows a peripheral to central developmental sequence (Killackey, 1985). Vibrissae- associated patches of high enzyme reactivity appear in the brainstem nuclei between P30 and P40 (Waite et al., 1994) and in the cortex at P85-90 (Waite et al., 1991). The delay between the formation of aggregations at each level suggests that pattern formation is not triggered by a single peripherally activated signal.

At each level of the pathway there is a substantial delay between afferent arrival and the formation of aggregations. This is most pronounced in the cortex, where afferents first arrive in the putative somatosensory cortex over 2 months prior to the formation of vibrissae-associated patches (Waite et al., 1991; Leamey et al., 1993). However, the period separating these events at the cortical level may be ex- plained by the relatively late differentiation of layer IV from the cortical plate (layer IV can first be recognised at P70 and is clear by P76; unpublished observations). The period separating afferent arrival (P10-15) and the appearance of aggregations in the thalamus (P52-P75) is intriguing, and by analogy with the cortex, suggests the importance of target maturation in the formation of aggregations. It will be of interest to determine when the pathway becomes active, and given that CO is reported to primarily reflect postsynaptic structures (Wong-Riley, 1989; Chiaia et al., 1991), how the distribution of trigeminothalamic afferents changes during development. I t should be noted that the time at which the neurons of VPM become clustered into discrete aggregates (P73-75) is coincident with the appearance of their primary target, cortical layer IV.

The transient appearance of AChE reactivity has been described in the developing thalamocortical systems of rodents iKristt, 1979, 1983,1989; Hohmann and Ebner, 1985; Prusky et al., 1988; Robertson et al., 1989, 1991; Schlagger et al., 1993; Schlagger and O’Leary, 1994), cats (Bear et al., 1985; Heck and McKinley, 1990), and primates (Liu et al., 1989). Robertson et al. (1988) demonstrated that the reactivity seen in the developing cortex has its origin in the thalamocortical system, rather than in the basal forebrain cholinergic system.

AChE reactivity.

Fig. 11. Diagram summarising the development of the projection from the principal trigeminal nucleus to the diencephalon. The top panel illustrates a lateral view of the brain in a neonatal wallaby; note the prominence of the embryonic flexures at this stage of development. The resultant bending of the neuraxis causes sections cut perpendicular to the anterior-posterior axis to transect both rostral and caudal areas of the brainstem. The vertical lines a 4 show the position and plane of the sections illustrated in the bottom panel. The dotted line shows the course of the trigeminothalamic pathway. Triangles represent the position of the growth cones of the leading fibres at various postnatal ages. a: There was no evidence of the development of the trigeminotha- Iamic pathway at PO. b: By P5, the trigeminothalamic pathway had begun to develop, fibres passed medially to cross the midline, and then turned dorsally towards the Me but did not enter it at this stage. c: At P10 the leading fibres had reached the rostral midbrain and were approaching the diencephalon. The fibres are heading rostrally through the page towards the viewer. d By P15 trigeminothalamic fibres had reached the diencephalon, and appeared to extend terminal arborisations in the developing VP nucleus.

Overall, the ontogeny of the expression of AChE in VPM of the wallaby compares well with that described in rodents, where it is expressed intensely in the late embryonic period and during the first postnatal weeks, before declining to the very low levels seen in the adult (Hohmann and Ebner, 1985; Kristt, 1989; Schlagger et al., 1993). The development of AChE reactivity in the VPM nucleus of the rodent has attracted attention as AChE staining provides the earliest known marker for the development of periphery- related patterns in the S1 cortex (Schlagger et a]., 1993; Schlagger and O’Leary, 1994). The temporal coincidence of the increase in cholinesterase activity with the formation of aggregations in the wallaby is impressive given the protracted developmental period of this species.

The intensity of the reaction product seen in the wallaby tended to be less than has been reported in rodents. This appears to be a genuine interspecies difference, as neonatal rat tissue which was prepared in the same manner, and reacted with the wallaby tissue as a control, produced a more intense reaction product. The reason for the lower level of AChE reactivity observed here in the wallaby VPM is not clear. Schlagger et al. (1993) reported that AChE reactivity acts as a marker for the differentiation of the principal sensory thalamic nuclei, where it can be detected from very early in development, before VPM neurons have finished migrating to their settling position from the ven- tricular zone. At P33, the youngest age at which AChE reactivity was assessed in the wallaby, VPM can be discerned in Nissl stains, has received innervation from the principal trigeminal nucleus, and its cells have projected to the putative somatosensory cortex. However, the AChE reactivity displayed by the cells of VPM was at very low levels and this was nonspecific. This suggests that AChE does not serve as a marker of the principal sensory thalamic nuclei in the wallaby at this stage.

The significance of the transient expression of AChE in VPM is yet to be established. I t is unlikely that it is related to its conventional role at cholinergic synapses, as the more specific marker for cholinergic function, choline acetyltransferase (reviewed by Greenfield, 19841, is not expressed in significant levels in VP cells (Brownstein et al., 1975; Hoover et al., 1978; Armstrong et al., 1983; Houser et al., 19831, and the expression of choline acetyltransferase in S1 cortex has a different developmental timecourse to AChE (Hohmann and Ebner, 1985). Moreover, glutamate is believed to be the primary transmitter at thalamocortical synapses (LoTurco et al., 1991; Agmon and O’Dowd, 1992; Johnson and Burkhalter, 1992). Other workers have suggested that the AChE expressed in thalamocortical axons acts to decrease the influence of cholinergic forebrain inputs during developmentally significant periods (Hohmann and Ebner, 1985; Schlagger and O’Leary, 1994).

The peak of cell death seen here in VPM at P81 corresponds to a pyknotic index of 1.3 (dead cellsi1,OOO live). This is somewhat lower than found in the VP nucleus of the rat (3.2; Waite et al., 1992). The number of pyknotic cells seen at any one time is a function of their rate of production versus rate of clearance, and is at best a relative measure. Data from the developing rat visual system suggest that pyknotic profiles may be cleared within three hours (Harvey and Robertson, 1992). The low pyknotic index seen here is consistent with the protracted developmental pattern of the wallaby, where cell death occurs at relatively high levels for around a week. A possibly more accurate measure of cell death is the comparison of the

Cell death.


numbers of live cells in developing and adult animals. A decrease of 264 was found here between the number of cells at the peak of cell death and in the adult. This is almost identical to the figure found in the rat (27%; Waite et al., 1992).

The appearance of discrete neuronal aggregations in the thalamus of the wallaby (by P73) precedes the peak of cell death by at least a week. However, the time at which cell death is high (P81-89) correlates well with the time that SDH patches appear in the wallaby cortex (P85-90; Waite et al., 1991). This suggests that cell death in VPM may be related to events occurring in the cortex, rather than in the thalamus per se. Although speculative at this stage, it appears that the period of cell death in VPM is related to the elaboration of thalamocortical fibres within their primary target (layer IV), as reflected by the appearance of SDH patches (Waite et al., 1991), rather than with the aggregation of the thalamic cells themselves. This concurs with substantial evidence which links cell death to synapse formation within the target space (reviewed in Cowan et al., 1984; Oppenheim, 1991). Allendoerfer et al. (1994) have suggested that the differential expression of neurotrophin receptors by afferent fibres during development may be important in determining cell survival during this period.

Conclusions The VPM nucleus of the wallaby is characterised by the

presence of discrete neuronal aggregations which correlate with vibrissal representation. The timecourse of the appearance of the aggregations is consistent with a centripetal developmental sequence, with significant delays occurring between the pattern formation at each subsequent level of the pathway. This suggests that neuronal aggregation is not controlled by a single, peripherally activated signal, but rather is independently regulated at each level of the pathway. Furthermore, this indicates the importance of the maturation of the target tissue during pattern formation in the developing nervous system.

ACKNOWLEDGMENTS We thank Ms. Alicia Fritchle for the artistic work in

Figure 11, Professor R.F. Mark for his criticism of the manuscript, Ms. A. Devlin and Mr. K. Williams for the expert care of the wallaby colony, and Dr. R. Meischke for veterinary advice. Sandoz (Australia) generously donated the bromocriptine.

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