Early onset of the rat olfactory bulb projections

Neuroscience Vol. 70, No. 1, pp. 255-266, 1996

~ Pergamon

0306-4522(95)00360-6

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EARLY ONSET OF THE RAT OLFACTORY BULB PROJECTIONS L. LOPEZ-MASCARAQUE,* J. A. DE CARLOS and F. VALVERDE Instituto Cajal (CSIC), Avenida del Doctor Arce, 37, 28002 Madrid, Spain Abstract--Using the fluorescent carbocyanine tracer DiI, we examined in detail the early development of the projections emanating from the rat olfactory bulb. The study commenced at embryonic day 13 when the first fibres can be detected and ended at embryonic day 20, when all major fibre systems have been established. The first axons arising from the prospective olfactory bulb area are seen at embryonic day 13. Labelled fibres are provided with elaborate axonal growth cones advancing through the ventrolateral part of the telencephalic vesicle. At embryonic day 14, while the main fibre tract has not developed much further, some isolated fibres are located quite distally from the prospective olfactory bulb. These early fibres apparently course within a narrow cell-free space that extends caudally along the ventrolateral part of the telencephalic vesicle. At embryonic day 15, a number of labelled fibres form a compact bundle, corresponding to the lateral olfactory tract, that ultimately reaches the prospective primary olfactory cortex. The fibres do not stop growing, but continue to extend caudally at embryonic day 17. The results of this study provide new information on the development of axonal tracts in the olfactory system. We show that the olfactory tract projection develops earlier than the morphological appearance of the olfactory bulbs. This suggests that the early development of olfactory projections might not depend on the arrival of the olfactory epithelium axons and thus, could be governed by factors intrinsic to the neurons and/or cues present in the target environment. Key words : development, lateral olfactory tract, carbocyanine tracers, growth cones, olfactory projections.

The organization of fibre tracts within the CNS represents a major issue in developmental neurobiology. The mechanisms by which each neuronal pathway becomes established are the result of many factors that allow neurons to extend long distances to find their correct targets. As early as 1893, Caja112 postulated that target cells release some diffusible molecules that influence the direction and rate of neurite growth. This chemoattractant mechanism has been later confirmed by different a u t h o r s . 29'38'39"49'61 Since Harrison's28 studies, increasing evidence has supported the notion that axonal pathways are 'pioneered' early in development so that secondary fibres which develop later follow the paths laid down by the pioneers. More recent observations53-55'66have given rise to the "blueprint" hypothesis by means of which preformed extracellular channels may serve as guide paths for growing axons. Moreover, a variety of studies reveal that the guidance of axons to their targets is controlled by the interaction of growth cones with different cell adhesion molecules and cell surface components that promote or inhibit the axonal elongation. 18,26,58 The olfactory system has a precisely organized circuitry that is particularly suitable for the explo*To whom correspondence should be addressed. Abbreviations: E, embryonic day; P, postnatal day.

ration of some of these basic issues. Due to the external location of the olfactory bulb, it is easily accessible for the study of the development of its major afferent and efferent pathways. The relative simplicity of the primary olfactory projections can be appreciated in a transverse section passing through the olfactory peduncle (Fig. 1). Axons of internal tufted and mitral cells of the olfactory bulb (Fig. 1, OB) become grouped into fibre bundles forming the lateral olfactory tract (Fig. 1, LOT). The tract courses superficially along the surface of the basal telencephalon, first surrounding the anterior olfactory nucleus (Fig. 1, AON) and more caudally covering the primary olfactory cortex to end at the level of the entorhinal cortex. All along its course, the lateral olfactory tract provides terminal and collateral fibres to most olfactory centres, among which the anterior olfactory nucleus and primary olfactory cortex represent its major recipient areas. Lateral olfactory tract fibres make synaptic contacts with the superficial dendritic branches of cortical cells in the olfactory cortex, illustrated in Fig. 1 with various cell types in the anterior olfactory nucleus, which in turn send their axons to different parts of the basal telencephalon; and through the anterior commissure (Fig. 1, AC) to connect with homologous centres on the contralateral side. Direct projections have been shown to run from the olfactory bulb to the cortical and medial

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Abbreviations used in the figures A AC Aq AON BT bta e H LOT

Amydaloid area Anterior commissure Aqueduct (Silvius) Anterior olfactory nucleus Basal telencephalon Anterior basal telencephalon Eye cup Hippocampus Latral olfactory tract

LV OB OF SP TV VZ 4V POC

Lateral ventricle Olfactory bulb Olfactory fibres layer Septal area Telencephalicvesicle Ventricular zone 4th ventricle primary olfactory cortex

Fig. 1. Camera lucida drawing made from three consecutive transverse sections stained by the Golgi-Colonnier method at P8 passing through the olfactory peduncle. The sections passes through the most caudal part of the olfactory bulb. The olfactory bulb output neurons, external tufted and mitral cells, receive olfactory inputs from the olfactory fibres and send their axons through the caudolateral part of the olfactory bulb to leave this structure grouped into fibre bundles. These bundles course superficially along the basal telencephalon forming the lateral olfactory tract that surrounds and enter, in relation with the olfactory cortex represented here, by the anterior olfactory nucleus. The neuron population of the anterior olfactory nucleus project to the contralateral hemisphere through the anterior commissure, and to other olfactory structures in the basal telencephalon.

amygdaloid nuclei, including the nucleus of the lateral olfactory tract, the anterior and posterior amygdaloid cortical nuclei and the periamygdaloid and entorhinal cortices. In addition, fibres from the olfactory bulb pass through medial and basal regions to several structures including the septum, anterior hippocampal rudiment, olfactory tubercle and probably through the medial forebrain bundle to some diencephalic and mesencephalic regions. Detailed analysis of all these connections, which are beyond the scope of

the present work, have been reviewed elsewhere.S,45,~,51,60 Using the high resolution carbocyanine fluorescent tracer DiI, we set out to examine in detail the sequence of events during the early growth of the main fibre tracts emanating from the olfactory bulb output neurons. We focused this study mainly on the development of the axonal projections of these neurons from an immature state of differentiation. This work has been previously presented in abstract form. 37

Development of the olfactory bulb projections EXPERIMENTAL PROCEDURES

Litters and fetuses from timed-pregnant Wistar rats raised in the animal colony of the Institute were used in this study. The day of insemination was considered as embryonic day 0 (E0) and the first 24 h period after birth was designated postnatal day 0 (P0). Two litters were used for each embryonic age. All animals were handled in a humane manner to avoid major distress. Pregnant dams were deeply anaesthetized with Equithesin (3 ml/kg body weight) and the embryos were removed by caesarean section. Embryos younger than El4 were decapitated and the head fixed by immersion in 10% formalin in 0.1 M phosphate buffer (pH 7.4). Older embryos were anaesthetized by hypothermia and perfused transcardially with the same fixative. The fluorescent tracer used in these experiments was the lipophilic Carbocyanine dye DiI. 23'24After fixation, a small crystal of DiI was placed into the rostral tip of the telencephalicvesicle (prospective olfactory bulb area at EI2-EI4) or in the olfactory bulb of older embryos. When small injection sites were required, a 2% DiI solution in dimethylformamide was pressure injected using a Picospritzer, introducing a glass micropipette into the selected area. The brains were stored in 1% neutral buffered formalin for four to six weeks. All brains were vibratome-sectioned at I00 or 200/~m thicknesses, in the sagittal or coronal planes. Sections were counterstained by a brief exposure to a 0.01% solution of Bisbenzimide to allow the identification of different structures. The sections were studied in a Nikon fluorescence microscope equipped with the appropriate filter cubes (DiI: 560--610 rim; bisbenzimide: 340-380nm). Figure 1 is a camera lucida drawing from three consecutive sections of a P8 rat brain impregnated by the Golgi-Colonnier method. This material belongs to our large Golgi stained collection. RESULTS The most anterior region of the telencephalon contains a population of early generated cells that have been described in autoradiographic studies as the projecting neurons from the olfactory bulb (mouse, 3°'31 rat2'64). These are among the first maturing cells of the telencephalon, and have been identified as accessory olfactory bulb neurons. In the rat, this population has been identified at El2, and thus, we chose this as the earliest age to study the labelling of cells using carbocyanine injections. At this stage no labelled fibres were detected after carbocyanine injections in the most anterior part of the telencephalic vesicle. At El3, the olfactory bulb is not evident, therefore DiI injections were placed in the tip of the anterior pole of the telencephalic vesicle (Fig. 2C, star), a region that presumably corresponds to the area where the olfactory bulb will develop. Although few differentiated neurons are present in this region at this age, a few fibres with axonal growth cones were seen coursing down to the basal telencephalon (Fig. 2A and B). These labelled fibres are visible in the ventral part of sagittal sections and probably represent the initial appearance of the olfactory tract projections. Placement of the tracer in more dorsal regions did not label any fibres running into the ventral part at this age. This early labelling most probably corresponds to fibres originating largely in the accessory olfactory bulb that, as described above, are among the first

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generated neurons in the telencephalic vesicle. The fibres display features of active growth with elaborated growing tips (Fig. 2A). Their morphology changed as they grew further from the prospective olfactory bulb; growing tips become thinner, showing a simple morphology (Fig. 2A and B, arrowheads). Labelled fibres were seen at different locations as shown by the distance between their growth cone tips (Fig. 2A, arrowheads). This suggests that the most ventral growth cones correspond to leading, pioneer fibres. The axons grow within, or close to, a narrow cell-free space immediately under the pial surface in the ventral part of the telencephalic vesicle. This sagittally oriented slit is most clearly seen in doubleexposed Bisbenzimide treated sections (Fig. 2B, arrows). At El4, there is almost no trace of olfactory bulb formation, but a very light prominence at the tip of the telencephalic vesicle can be detected. After DiI injections into the prospective olfactory bulb (Fig. 3A and B, star), the labelling was similar to that obtained in El3 embryos. However, while the fibre tract has not extended much further, some isolated fibres are located more distally (Fig. 3D) in a region corresponding to the amygdaloid area. It is interesting to note several axons just emerging from the DiI injection site with elaborate growth cones (Fig. 3C, arrowheads). Their appearance may be related with the beginning of the main olfactory bulb cell projections. Retrograde-labelled cells located in the prospective septal area were also observed as well as sparselylabelled cells in the olfactory epithelium (data not shown). At E15, emerging olfactory bulbs can be seen in the brain (Fig. 4A, C and E). Tracer injections into the olfactory bulb demonstrated an explosive growth of the olfactory tract fibres (Figs 4D and 5). Photomicrographs corresponding to Figs 4 and 5 are taken from the same embryo, at different levels of the sagittal plane of the lateral olfactory tract, to show the full extent of the olfactory tract. At this stage, a dense group of labelled fibres were seen identified in the most lateral sections (Fig. 5). This corresponds to the ventrolateral part of the primary olfactory cortex, which is almost covered by the olfactory bulb axons. In sections adjacent to the injection site, some labelled neurons display significant morphological differentiation having features of mature mitral or tufted cells (Fig. 4E). Furthermore, a small number of layer 2 cells of the piriform cortex are retrogradely labelled (not shown). Growth cones were observed mainly in the primary olfactory cortex whilst some were seen throughout the different levels of the olfactory tract. Most growth cones were of simple morphology, with bulb-like forms at their tips (Fig. 5D). The development of the olfactory bulb between E16-E20 has been described previously in detail 2L5°'5~ and only a few additional points need to be made here. At El6 the embryos have well-developed

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E13 Fig. 2. Carbocyanine labelling at El3. (C) Figure displaying the localization of the DiI implant in the rostral prosencephalon (star). The box in the ventral border of the anterior basal telencephalon is enlarged in A to show, under fluorescent microscope, the early fibres exiting from the injection site. Arrowheads point to terminal growth cones showing the antero-caudal polarity of these fibres. (B) Magnification of the more caudal area of the photomicrograph in A, counterstained with Bisbenzimide to show the cell free scaffold (open arrows) that the pioneer olfactory fibres use to migrate. Arrowheads point to the same leading growth cones that in A. Sagittal sections. Scale bar = 50 #m.

olfactory bulbs. At this age, there is a tight bundle of olfactory fibres extending from the olfactory bulb similar to that shown at El5. Some fibres, mainly those located in the internal part of the lateral olfactory tract, displayed collateral branches innervating different regions of the olfactory cortex. In horizontal sections, the fibres coming out from the olfactory bulb descended past the piriform cortex towards the diencephalon into the medial forebrain bundle (data not shown). This later projection might be transitory since it was not observed in older animals. In the most ventral part of horizontal sections, the fibres originating in the olfactory bulb spread out in a fan-like fashion. Fibres running through the ventrolateral part of the cortex (lateral olfactory tract) organize into a tight bundle. Fibres in the region of the anterior commissure reach the midline, but do not cross it at this age. The labelling of anterior commissural fibres results from invasion of the tracer into the anterior olfactory nucleus. At El7, the fan-like radiation of fibres from the olfactory bulb can no longer be observed. The fibre systems labelled when large injections of the tracer were deposited in the olfactory bulb were grouped into distinct bundles: (i) directed medially, toward the septal area, (ii) running to the contralateral hemisphere, via the anterior commissure, (iii) toward the

amygdaloid nuclei and (iv) covering the primary olfactory cortex forming the lateral olfactory tract. From El7 to E20, the size and complexity of the different tracts increased as a function of age. As shown in a horizontal section at E20 (Fig. 6), the lateral olfactory tract forms a compact bundle bordering the primary olfactory cortex. Retrograde- and anterograde-labelled cells and fibres extended medially toward the amygdaloid area. The pattern of fibres originating in the olfactory bulb at E20 almost corresponds to the adult pattern. Figure 7 summarizes the main findings of the present study. At El3, pioneering fibres began to grow through the ventral part of the telencephalic vesicle following a cell-free subpial space. At El4 and El5, these fibres soon reached distant parts in the basal and lateral parts of the telencephalic vesicle establishing the first connections with the prospective amygdaloid area and primary olfactory cortex, while others appeared extending towards the medial side. At El6, fibres coming from the olfactory bulb ran caudally in a fan-like fashion, the lateral olfactory tract increased considerably in size, while other fibres coursed along the anterior commissure but do not cross to the contralateral side. At El7, the major fibre systems appeared to be established providing the substrate for the subsequent increase in size and complexity to form the adult pattern.

Development of the olfactory bulb projections DISCUSSION The adult olfactory projections are well characterized in many different species 7'15'27'4°'45'46'6°'65and the development of the central olfactory projections in fetal rats has been the matter of several studies. 2'48,5°'5~,56However, none of these studies have focussed on the development of the olfactory tracts at ages prior to El5, The olfactory bulb can be recognized at El5, therefore we considered it of interest to study the earliest onset of projections from its prospective area in the most anterior tip of the telen-

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cephalic vesicle. Thus, this study extends from E13, when the first fibres were detected, to E20, when all major fibre systems had been established.

The morphological appearance of the olfactory bulb is not required for the early development of the olfactory tract projections Several studies have shown that cells in the accessory olfactory bulb are generated significantly earlier than the output neurons of the main olfactory bulb (mouse, 3°,31 rat2,64). According to Bayer2 a significant population of output neurons in both the accessory

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E14 Fig. 3. Carbocyanine labelling at El4. Sagittal section in A is represented in a camera lucida drawing in B, displaying the exact position of the DiI injection site in the rostral prosencephalon (star), and two boxes in the anterior basal telencephalon. Boxes C and D are enlarged in photomicrographs C and D to show two different gradients of emerging fibres. The growth cone morphologies are also shown (arrowheads). Proximal fibres to the injection site display complicated morphologies (C) and distal fibres display elongated and simpler morphologies (D). Scale bars: A, B = 500 itm; C, D = 50 pro.

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Fig. 4. Carbocyanine labelling at El5. Sagittal sections. The localization of the DiI injection site in the nascent olfactory bulb is shown in A (star) and B, and in neighbouring sections in B and D. Sections in A, C and E are counterstained with Bisbenzimide displaying the cytoarchitecture of the olfactory bulb, anterior basal telencephalon and teleneephalic vesicle. In E some output olfactory bulb neurons are labelled in an adjacent section to the injection site. D shows labelled fibres exiting the injection area and forming the lateral olfactory tract at this embryonic age. Scale bar: A, C = 300 pro; B, D, E = 200/~m.

Development of the olfactory bulb projections and main olfactory bulbs have nearly completed neurogenesis at El4 (this author considered the day of vaginal plug as El, while for us it is E0). Within the amygdaloid area, target neurons, receiving fibres from the accessory olfactory bulb, mature earlier than those coming from the main olfactory bulb. 1 Based on these data, we consider that the first fibres seen at El3 correspond to the axons of these early generated cells in the prospective olfactory bulb. Furthermore, injections at El3 in dorsal regions of telencephalic vesicles did not label any fibres. This is consistent with the fact that the first cortical fibres enter the basal telencephalon at El4 in the ratJ 6

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However, it is not possible to differentiate if the early olfactory fibres belong to either the accessory or main olfactory bulb because both cells and axons develop before the anatomical formation of the olfactory bulb. Hence, the beginning of the olfactory tract projection coincides with the gradients of maturation of the accessory- and main-olfactory bulb neurons. Early pioneer fibres may serve as guides to late follower axons. We presume that these early fibres emerged from the accessory olfactory bulb, as has been suggested by Schwob and Price. 5~ Moreover, after carbocyanine injections at El3 into the prospective olfactory bulb, only a few fibres were seen

Fig. 5. Carbocyanine labelling at El5. Same animal as in Fig. 4 showing labelled olfactory tract fibres in more lateral sections. Rostral is up; dorsal is to the left. (A) section counterstained with Bismenzimide showing labelled fibres of the lateral olfactory tract extending through the basal telencephalon. B shows the same group of fibres in an enlarged view. (C) section counterstained with Bisbenzimidepassing almost tangentially through the telencephalic vesicle. Labelled fibres occupy the ventral part extending through the prospective primary olfactory cortex. D shows a group of axonal growing tips located in the most ventral part of the prospective olfactory cortex. Scale bar: A, C = 300/lm; B = 500 #m; D = 100/~m.

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Fig. 6. DiI implant in the olfactory bulb (star) at E20. Horizontal sections. The figure shows in part, the basic pattern of olfactory bulb projections found in the adult. The lateral olfactory tract forms a compact bundle surrounding the primary olfactory cortex. A group of fibres course in a deeper stratum to the amygdaloid area where several retrogradely-labelled cells can be seen. Other medial projections to the septal area are barely visible at the top right side. Scale bar = 200 pm.

running from the injection site, with other axons running ahead and located close to the presumptive amygdaloid area. The first neurons generated in the prospective piriform cortex appear at E l 2 in the rat. 3'64 It has been suggested that these early cells may be involved in the release of specific guidance cues for the olfactory tract fibres, s'36 In addition, the transitory expression (from E l 4 to E 17) of neurotensin m R N A in cells of the olfactory bulb primordium, appears to be

involved in axonal growth. 35 Therefore, even in absence of morphologically-defined olfactory bulbs, early olfactory projections follow a predictable course that may depend not only on the early maturation of their target areas but also on the possible existence of preformed channels laid down in the basal telencephalon. Thus, early olfactory bulb projections appear to follow precise pathways as has also been demonstrated for other parts as in the mouse entorhinal cortex. 56

Development of the olfactory bulb projections

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Fig. 7. Schematic representation that summarizes the main findings of the present study in two different planes, sagittal (S) and horizontal (H). At El3, pioneering fibres begin to grow through the ventral part of the teleucephalic vesicle. At E 14 and E 15, these fibres reach distant parts in the basal and lateral parts of the telencephalic vesicle establishing the first connections with the prospective amygdaloid area and primary olfactory cortex. At El6, fibres coming from the olfactory bulb run caudally in a fan-like fashion, the lateral olfactory tract increases considerably, while some fibres course along the anterior commissure. At El7, the major fibre systems appear already established providing the substrate for the adult pattern.

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necessary for the growth of axons. Moreover, recent observations 5 have shown the relationship between the disposition of early generated neurons in There are several pieces of evidence demonstrating the rat brain stem and, the presence of cell-free spaces that axons from the olfactory receptor neurons influence the development of the olfactory bulb. 9 11,52.59 in the marginal layer, with the pathways followed by axonal tracts. It is also known that many different One could also imagine that the development of the molecules can inhibit or promote the axonal outcentral olfactory projections might be directly ingrowth (for review see Refs 18 and 26). During duced by the arrival from the axons of the olfactory axonal development neural cell adhesion molecules epithelium. Thus, a point requiring some discussion mediate adhesive interactions between axons and is the apparent independence of the early growing cells? Thus, the guidance cues along these pathways axons from the afferent input of the olfactory epican be provided by both neural cell adhesion molthelium. According to our study, whilst at El3 the ecules and molecules of the extracellular matrix. 47 In mitral/tufted cells are not clearly defined morphologithis way, the growing axons appear to follow specific cally, it is at this age when these cells begin to send pathways to reach their target areas. their axons through the olfactory tract. However, In recent studies, Pini 43'44suggests that chemorepulsynapses between primary olfactory axons and bulbar neurons cannot be observed until after E17-E18, two sion might contribute to the early patterning of the days after the central olfactory projections reach lateral olfactory tract by creating exclusion zones for their targets (mouse, 33 rat19'2°'22'63). In the mouse, the developing axons. The chemorepulsive effect is mediated by a diffusible chemorepellant released by septal first axodendritic synapses in the olfactory bulb occur explants cocultured with olfactory bulb explants. at El4, the age when mitral cell axons have grown into the basal telencephalon. ~7'32It has been shown in These studies raise the possibility of secreted factors acting at a distance to inhibit axonal growth. This several studies that the axons of olfactory receptor possibility has also been suggested in a recent work neurons reach the bulb before their olfactory receptor neuron dendrites have matured. ~4'2°'41 Therefore, it of the developing optic chiasm where the possibility of co-existence of both growth-promoting and seems likely that a similar mechanism might occur growth-inhibiting cues on the same cell surface. 5v with respect to the mitral cells. This suggests that the In support of these findings, our results reveal at olfactory bulb efferent cells begin to send their axons very early stages of development, the presence of a through the lateral olfactory tract before they receive cell-free space where the olfactory tract fibres course. any synaptic information from the olfactory epiAt El6, the fibres arising from the olfactory bulb thelium. However, it is interesting to mention that grow in a fan-like distribution and it is not until after some olfactory pit fibres reach the anterior part of the telencephalic vesicle at El2 (mouse, 32 rat 25'48) El7 when the different olfactory tracts form specific fibre bundles directed to their target areas. This fact reaching the germinative ventricular zone of the suggests that the final shaping into the different neuroepithelium. Those fibres may induce the tracts may be controlled by local cues, either molecumitotic activity of cells that may differentiate into lar and/or structural, which develop in the basal mitral cells. We think that this type of induction in telencephalon. the absence of synaptic contacts is not enough to initiate the development of the olfactory bulb efferent projections, although other authors postulate that Growth cone morphology through the lateral olfactory synaptic functions may be present despite the lack tract of morphological features of mature synapses? z In According to different reports, growth cones are summary, we propose that neurons in the rat quite simple in morphology when they grow through olfactory bulbs develop their target projections established pathways, but they become more complex well before receiving synaptic input from olfactory at "decision points" in their pathways. 6'~4'62 It has receptor cells. Probably, the complete dendritic matubeen proposed that these changes are in relation to ration of mitral and tufted cells may be related to the specific cues present within the axonal tract. Our arrival of olfactory fibres at the glomeruli. In fact, the results show that the first growing tips observed close effect of the olfactory bulb on the maturation of to the olfactory bulb, displayed features of very active olfactory receptor neurons seems to be a contact growth cones with flattened regions (lamellipodia) mediated response between olfactory axons and buland many fillopodia. Nevertheless, when the fibres bar tissue, rather than one induced by a diffusible are running through the cell-free channel, the growth factor.13 cones become thinner and do not show any elaborate structure. These observations agree with O'Leary et al. 42 who postulate that the primary growth cone Possible mechanisms for initial olfactory axonal plays no role in target selection or axon branching. projections into the basal telencephalon Probably, the process for target recognition could be The "blueprint" hypothesis 55 suggests that the due to other cues derived from the target areas as presence of cell free spaces organized in channels are occurs in the corticopontine projection. 29'49 Do the olfactory bulb projections depend on the arrival o f the primary olfactory fibres?

Development of the olfactory bulb projections CONCLUSIONS The results of the present study provide new data regarding the early d e v e l o p m e n t a l stages o f axonal tracts in the olfactory system. O u r d a t a can be s u m m a r i z e d as follows: (i) the olfactory tract projection develops earlier t h a n the a n a t o m i c a l f o r m a t i o n o f the olfactory bulbs, (ii) the early d e v e l o p m e n t of the olfactory tract projections seems to proceed

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independently o f extrinsic influences (the olfactory epithelium) a n d thus, m i g h t be governed by factors intrinsic to the olfactory n e u r o n s a n d / o r local signals present in the central targets. Acknowledgements--We would like to thank Maria Luisa

Poves for the technical and photographic work. We also want to thank Nieves Salvador for animal facilities. This work was supported by DGICYT Research grant PB910066 from the Ministerio de Educaci6n y Ciencia.

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