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The use of a novel and simple method of revealing neural fibers to show the regression of the lateral geniculate nucleus in the naked mole-rat (Heterocephalus glaber)
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Research Report
The use of a novel and simple method of revealing neural fibers to show the regression of the lateral geniculate nucleus in the naked mole-rat (Heterocephalus glaber ) Jun Xiao ⁎, Jonathan B. Levitt, Rochelle Buffenstein Biology Department, The City College of New York, 138th Street and Convent Avenue, New York, NY 10031, USA
A R T I C LE I N FO
AB S T R A C T
Article history:
The lateral geniculate nucleus (LG) is an important subcortical nucleus in the visual system.
Accepted 6 January 2006
It receives primary projections from the retina and relays these to central visual structures.
Available online 14 February 2006
Although there are studies on the retina and visual cortex of animals with regressed vision, little is known about the LG in such animals. The strictly subterranean naked mole-rat
Keywords:
(Heterocephalus glaber) has markedly reduced visual acuity with concomitant pronounced
Thalamus
changes in the visual cortex. We used a novel method to reveal myelinated neural fibers in a
Neuroanatomy
histological study assessing if the LG shows regressive changes commensurate with the
Histology
level of reliance on vision by this rodent. Myelin detection here relies on significant
Visual system
differences in visible light reflection between neural fibers and the gray matter. Moreover,
Myelin staining
this simple method does not interfere with further staining for additional analyses. This method reveals that the contribution of the LG to brain volume in the naked mole-rat is less than a third of that of the rat. This shows that the retinogeniculocortical system in the naked mole-rat is considerably smaller than that of rodents that rely heavily on their visual system, but is nevertheless less regressed than that of the extensively studied blind molerat; this may facilitate limited responses to visual stimuli. © 2005 Elsevier B.V. All rights reserved.
1.
Introduction
Naked mole-rats (Heterocephalus glaber) are strictly subterranean rodents that spend almost their entire lives below ground. Occasional forays onto the surface, although uncommon, may occur when animals excavate tunnels and kick loose soil onto the surface, or when a burrow system is breached (Braude, 2000). Living in this dark and sandy milieu, it is not surprising that these mammals rely heavily upon tactile stimuli and mechanosensory hairs scattered over the body (Crish et al., 2003). Naked mole-rats' eyes are markedly reduced in size, and generally these rodents move about with
their eyes closed, suggesting that these animals do not rely on vision. Nevertheless, they have retained most of the wellcharacterized mammalian cell types found in the eye, although their eye structural organization is considerably less regular than that in more sighted mammals (Mills and Catania, 2004; Nikitina et al., 2004). The naked mole-rat reportedly cannot perceive images but can detect amorphous light (Hetling et al., 2005). Similarly, although the blind molerat (Spalax ehrenbergi) has a subcutaneous eye and atrophied structures involved with image formation, non-image forming visual pathways involved in photoperiod perception are well developed (Cooper et al., 1993a,b). In contrast to the exten-
⁎ Corresponding author. Fax: +1 212 650 8585. E-mail address: [email protected] (J. Xiao). 0006-8993/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.01.021
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sively studied visual system of the Israeli blind mole-rat, the visual system of Bathyergid mole-rats has received much less attention (Bronchti et al., 1991; Catania and Remple, 2002; Cooper et al., 1993a,b; Nikitina et al., 2004; Rehkamper et al., 1994), even though from a comparative evolutionary perspective, it would be both interesting and informative to study this regressed visual system as a viable model for the study of evolutionary remodeling. Most of the studies to date assessing the visual system of subterranean rodents have focused on eye morphology (Nevo, 1979; Nikitina et al., 2004), retinal structure (Cernuda-Cernuda et al., 2003; Mills and Catania, 2004), and the visual cortex (Bronchti et al., 2002; Heil et al., 1991; Necker et al., 1992). Although the lateral geniculate nucleus (LG) is an important sensory nucleus governing the flow of visual information from retina to visual cortex, little is known about this region in subterranean rodents. The primary purpose of this paper is to assess if this structure, like the eye and visual cortex, differs from that of above ground dwelling animals. The LG in rodents can be easily identified using myelin staining to identify the superior thalamic radiation (str: a bundle of myelinated neural fibers) and the dorsolateral edge of the thalamus as landmarks. Although there are a variety of myelin staining methods, most of these are labor intensive, involve many reagents, and each method requires specific and/or different modes of section preparation. To facilitate experiments in this study, we introduce a simple and reliable method that can reveal neural fibers using a novel mechanism. This new method reveals myelinated axons using differences in visible light reflection of the myelinated axons and the gray matter. The main advantages of this method are that it is simple and reliable, and does not interfere with other staining techniques.
2.
Results
We first demonstrate the validity of our new method, and then use it to document and compare the extent of the LG in naked mole-rats and laboratory rats.
2.1.
Assessment of our method for revealing neural fibers
The new method works well on brain sections from both naked mole-rats and common laboratory rats (Fig. 1) as well as carnivorous species like ferrets (data not shown). The black background plate gives excellent contrast of the neural fibers that are in white. The sections in Figs. 1A and B show myelinated fibers revealed by the new method from naked mole-rat forebrain 1.90 mm posterior to Bregma. This section had previously been reacted to reveal cytochrome oxidase (CO). The sections in Figs. 1D and E show myelinated fibers of the laboratory rat forebrain 4.16 mm posterior to Bregma. This method reveals both fiber bundles and single axons in either frozen or paraffin sections. Figs. 2A–C shows that our method of revealing myelinated fibers in the naked mole-rat forebrain gives comparable results to that obtained from the widely used Gallyas (1979) method (Fig. 2D), confirming that our novel method does reveal myelinated neural fibers.
A significant advantage of this method is that sections processed by this method first (e.g. Figs. 1D and E) can be stained later by any other method (Fig. 1F, see Discussion). The section in Fig. 1C was stained by cresyl violet with no other processing. There are no differences between the staining quality of the section in Fig. 1F and that in Fig. 1C. Alternatively, sections may be stained first by other methods, dried, coverslipped, and then processed some time later to reveal myelin by our method. CO staining did not essentially affect myelinated fibers observation (Figs. 1A, B). However, when cresyl violet was used first on a section and then subsequently used for our method, we noted lower resolution. This was most likely due to the dehydration and hydration process, for although the fibers could be seen clearly with the new method (Fig. 3B), the resolution was lower than that of CO-stained or unstained sections. Myelin may have been disrupted by the dehydration process. Thinner sections provided better resolution. Fig. 3C shows a 10 μm thick section. Single fibers can be identified under high amplification. Because the neural fibers are not confined to a strict 10 μm thick plane, the fiber bundles seem to be cut off periodically. In contrast, the fiber bundles in thicker sections were less cut off (Fig. 3D, 100 μm thick). Therefore, while thinner sections provide better single fiber observations, thicker sections support better global observations.
2.2. Regression of the naked mole-rat lateral geniculate nucleus The size of naked mole-rat LG was reduced significantly compared with that of the laboratory rat (Fig. 1). The naked mole-rat LG also appeared to be cytoarchitecturally underdeveloped for there is no obvious division into laminae or divisions (Figs. 1C vs. F, 4B1 and B2 vs. D1 and D2). Nevertheless, typical LG subdivisions of the naked mole-rat dorsal lateral geniculate nucleus (LGd), ventral lateral geniculate nucleus (LGv), and intergeniculate leaflet (IGL) were architectonically distinct entities. However, the boundary between the medial and lateral divisions of the LGv (LGvm and LGvl) was not clear in naked mole-rat brain sections (Figs. 1C, 4B1 and B2). In contrast, the rat LGv could be divided into LGvm (parvicellular part) and LGvl (magnocellular part) (Figs. 1F, 4D1 and D2). The cells in LGvm were smaller than cells in LGvl (Fig. 1F). The naked mole-rat LG was found to typically extend from 0.90 mm to 2.36 mm posterior to Bregma (Fig. 4A). The overall shape of the naked mole-rat LG was like a bending sheet with an average thickness of 81 μm, occupying roughly 3.4% of the width of the thalamus (from the midline to the lateral edge of the thalamus). The thickness reached its maximum (198 μm) at roughly 1.23 mm posterior to Bregma (Fig. 4B). The length dimension along the curved band in the lateroventral– mediodorsal direction reached its maximum (864 μm) at roughly 1.23 mm posterior to Bregma. The volume of naked mole-rat LG was roughly 0.075 mm3, occupying roughly 0.019% of the entire brain volume. In contrast, the average thickness of the rat LG was 311 μm, occupying roughly 7.8% of the width of the thalamus. The thickness reached its maximum (537 μm) at roughly 4.32 mm posterior to Bregma (Fig. 4D). The dimension along the curved band in the lateroventral–mediodorsal
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Fig. 1 – Neural fibers revealed by the new method in naked mole-rat (A and B) and laboratory rat (D and E). Panel B is the inset in panel A; panel E is the inset in panel D. Panel C illustrates a section adjacent to that in panels A and B, showing a region corresponding to the inset in panel B, and was stained with cresyl violet without any other processing. Panel F is the same section as in panel E, showing the region indicated by the inset in panel E. It was processed by the new method first and then was stained with cresyl violet. ml, medial lemniscus; opt, optic tract; str, superior thalamic radiation; IGL, intergeniculate leaflet; LGd, dorsal lateral geniculate nucleus; LGv(m,l), ventral lateral geniculate nucleus (medial/lateral divisions). All sections are 40 μm thick frozen sections. The number at the upper right corner in each panel indicates the A–P distance from Bregma.
direction reached its maximum (1677 μm) at roughly 4.08 mm posterior to Bregma. The volume of rat LG was roughly 0.83 mm3, occupying roughly 0.059% of the entire brain volume. Thus, the relative thickness (percent of the thalamus width) of the naked mole-rat LG was less than half that of the laboratory rat, while the relative volume (percent of the entire brain) of the naked mole-rat LG was roughly one-third that of the laboratory rat. Indeed, if the relative volumes of the LG were scaled to body mass0.67, as is commonly done in allometric based biological studies (Schmidt-Nielsen, 1984), the naked molerat LG would be predicted to be over three times larger. The diminished relative size of this nucleus in naked mole-rats
suggests that the LG in this subterranean species is indeed markedly regressed.
3.
Discussion
This paper set out to describe both a novel histological method for assessing myelinated regions in neural tissue, and the use of this method to compare among naked mole-rats and laboratory rats the histology and size of the LG. This easy myelin-specific method relies upon physical properties of the myelinated axon and yields similar results to those of
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Fig. 2 – Neural fibers revealed by the new method. (A) Frozen section (40 μm) 0.65 mm posterior to Bregma. cc, corpus callosum; dhc, dorsal hippocampal commissure; ec, external capsule; fi, fimbria of the hippocampus; ic, internal capsule. (B) High amplification view of the inset in panel A. (C) A paraffin-embedded section (10 μm) from another brain of approximately the same position as the inset in panel A. (D) A frozen section (40 μm) of similar location as the inset in panel A from another brain stained by the silver method (Gallyas, 1979).
conventional methods (Gallyas, 1979). Using this method, we have noted that differences in the LG between rats and naked mole-rats are commensurate with reported differences in eye morphology and visual cortex (Bronchti et al., 2002; CernudaCernuda et al., 2003; Francescoli, 2001; Heil et al., 1991; Mills and Catania, 2004; Necker et al., 1992; Nevo, 1979; Nikitina et al., 2004). Our data confirm that the entire visual system of naked mole-rats has regressed in a similar manner, suggesting a reduced ability to perceive images. Myelin staining is a fundamental technique employed in many areas of neuroscience. Although myelin staining has been employed for over a century (Cohnheim, 1866; Weigert, 1884), new less complex and reliable methods are continuously being sought after (Ciaccio, 1909; Fuentes, 1960; Gallyas, 1979; Kaatz et al., 1992; Kutscher et al., 1987; Larsen et al., 2003; McNally and Peters, 1998; Schmued, 1990). A common feature of the many previous complex myelin-staining procedures is that they exploit chemical or biological properties of the histological section: either using oil-soluble dyes to stain lipophilic components of the fibers (including the lipid phase of the myelin, complex phospholipids, oligodendrocytes), or binding metal particles to some components of myelin (for review, see Schmued, 1990). Each method has its drawbacks or limitations: some are suitable for only one type of section (e.g. frozen or paraffin sections), others work best on sections cut at a specific thickness (Gallyas, 1979; Mahon, 1937), and still others require that tissues are postfixed for prolonged periods
(Larsen et al., 2003) and/or kept at high temperatures (e.g. 50 °C in Waiss et al., 1971). These latter harsh conditions not only result in the loss of stainable phospholipid (for review, see Smith and Dunbar, 1966) but also inactivate enzymes (Namba et al., 1967), thereby preventing the use of myelin-stained sections in multiple co-staining procedures, especially those based upon enzymatic procedures. These shortcomings thus curb the amount of information obtained in neurohistological studies. A key advantage of our method is that it does not change chemical and biological properties of the section. This allows additional section staining with both enzyme and dye based techniques (e.g. cytochrome oxidase and cresyl violet). Furthermore, our method can be used with both fresh sections and dehydrated sections (even those that have been dehydrated for prolonged, ∼N1 month, periods) that cannot be stained for myelin using conventional protocols (Schmued, 1990). Our simple reliable technique is quick and does not require postfixing. In contrast, conventional methods not only involve numerous steps and chemicals but also typically require meticulous care to maintain a metal-free environment. Unlike traditional methods that work well only on one type of section (for example, Klüver and Barrera (1953) technique only on paraffin sections), this method can be used on different kinds of sections (frozen, paraffin, and celloidin) and on sections that vary in thickness (10–100 μm and up). We used this method to examine the cyto- and
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Fig. 3 – The new method reveals neural fibers in sections that had been stained by other methods (A and B) and in sections of different thickness (C and D). The section in panel A had been stained for CO first; that in panel B had been stained by cresyl violet first. Sections in panels A and B are 40 μm thick. (C and D) Effect of section thickness on resolution. Thin section (C: 10 μm) provides better resolution than thick section (D: 100 μm). Conventions as in Fig. 1.
myeloarchitecture of the LG so as to assess if the visual thalamic relay nuclei are regressed in a highly eusocial, strictly subterranean rodent, the naked mole-rat. Subterranean animals have received considerable attention as a viable model for the study of mosaic evolutionary remodeling. Like other subterranean mammals, the Bathyergid family of mole-rats (which include naked mole-rats) shows a suite of morphological and physiological adaptations to the underground habitat they have inhabited since the early Miocene (Buffenstein, 2001). Although the naked mole-rat eye retains a typical mammalian organization, eyes are markedly reduced in size, and the optic nerve cross sectional area is extremely small with a very low fiber density (Hetling et al., 2005). Furthermore Catania and Remple (2002) have reported that the somatosensory cortex in these animals encompasses large regions of the neocortex normally devoted to vision in above-ground inhabitant. The LG is a key nucleus in the central nervous system to receive projections from retina and to relay the information received to the cerebral cortex. In the blind mole-rat S. ehrenbergi, the LG has regressed resulting in a very small superficial thalamic area (Cooper et al., 1993a,b; Rehkamper et al., 1994). Similarly, the LG of naked mole-rats is also markedly attenuated and cytoarchitecturally underdeveloped when compared to laboratory rats, but nevertheless substantially bigger than that of the blind mole-rat (compare Fig. 1 of this paper with Fig. 1 in Rehkamper et al., 1994). It seems that the retinogeniculocortical system of naked mole-rats therefore is
less regressed than that of the blind mole-rat but markedly more so than that of the laboratory rat. These results further support reports that although the naked mole-rat has an atrophied visual cortex, these animals can indeed detect light, albeit in an unstructured manner with poor image formation. They do indeed respond to light detection and use these visual cues to escape potential life-threatening events (such as predation) by moving away from the source of light. Hetling et al. (2005) report pronounced individual differences in response to experimental illumination, such that the dominant breeding female, in keeping with her status and importance as the sole-breeding female in a colony (Jarvis, 1981; O'Riain et al., 2000), was more likely to leave (or avoid) an illuminated chamber than other non-breeding individuals. The “working castes” of these eusocial rodents were less evasive of experimental illumination, and this may reflect that they are indeed more likely to encounter a breached burrow system, which they would need to recognize as such and repair thereby ensuring the continued safety of the entire colony (Brett, 1991). Mole-rat nervous systems provide a unique model for studying neural development, plasticity, and neural regression. While the imaging function of the retina of mole-rats has regressed in response to evolving in a subterranean habitat, the retinohypothalamic pathway in naked mole-rats, like that of the blind mole-rat, S. ehrenbergi (Cooper et al., 1993a; Negroni et al., 1997) is retained and relatively well developed. This may reflect the employment of some photoendocrine
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responses (Buffenstein, 1996); however, the pineal gland of naked mole-rats is the smallest in absolute size of any other known rodent and is generally considered atrophic (Quay, 1981). This may nevertheless contribute to the preserved ability in some but not all individuals to experimentally entrain activity periods, body temperature, and metabolism to light cues (Riccio and Goldman, 2000). This photoperiodic regulation of circadian rhythm may be especially important in “dispersomorph castes” (O'Riain et al., 1996) that may leave burrow systems at night in search of outbreeding opportunities (Braude, 2000). The suprachiasmatic nucleus (SCN) in both the Spalacidae (blind mole-rats) and the Bathyergidae is organized into fundamental subdivisions that are comparable to those of above-ground dwelling rodents (Negroni et al.,
1997; Nemec et al., 2004). However, the density of retinal projections to both the SCN and the bed nucleus of the stria terminalis (BNST) differs substantially. These regions in molerat brains receive about 20% of the total retinal projection, while in other rodents, that are active above ground, these nuclei receive only a small proportion (less than 1%) of the total retinal projection (Cooper et al., 1993a; Levine et al., 1991). These specific features may reflect both phylogenetic traits and evolutionarily conserved adaptations to a subterranean habitat. Although a subterranean insectivore, the European mole (Talpa europaea), also exhibits a regressed visual system, it appears to direct the bulk of the retinal output to the pretectum (Lund and Lund, 1965). In contrast, the pretectum and other subcortical visual structures of bathyergids (such as
Fig. 4 – Comparison of naked mole-rat LG with that of laboratory rat. (A) A series of 40 μm sections was used to locate the LG. (B) Enlarged view of the insets in panel A showing the location and shape of naked mole-rat LG. Panels B1 and B2 are Nissl-stained sections adjacent to insets in panel B. (C) 3D reconstruction of the naked mole-rat LG. (D) A series of 40 μm sections showing the location and shape of laboratory rat LG. Panels D1 and D2 are sections with insets in panel D subsequently stained with cresyl violet. (E) 3D reconstruction of the laboratory rat LG. IGL, intergeniculate leaflet; LGd, dorsal LG; LGvl, lateral division of ventral LG; LGvm, medial division of ventral LG; D, dorsal; V, ventral; R, rostral; C, caudal. Conventions as in Fig. 1.
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Fig. 4 (continued ).
4.
Experimental procedures
225.4 g were euthanized with sodium pentobarbital (100 mg/kg i.p.) and intracardially perfused with a saline rinse solution, followed by 4% paraformaldehyde in 0.02 M phosphate-buffered saline (PBS: pH 7.4). After a 30-min period of initial fixation, brains were removed and sunk in 4% paraformaldehyde containing 30% sucrose. The brains were embedded in Frozen Section Medium (Richard-Allan Scientific) and cut on a cryostat (Microm, HM560) into 10, 40, or 100 μm thick sections. Both coronal and sagittal sections were cut; however, here, we present only data from coronal sections for studies based on both planes of sectioning yielded similar data. Sections were collected directly into 0.02 M phosphate buffer (PB). Some sections were mounted on gelatinsubbed glass slides and were stained with cresyl violet later; others were kept in a free floating environment and stained for cytochrome oxidase (CO) later using a modified method of WongRiley (1979). We mounted these sections with our newly designed mounting chamber (Xiao and Levitt, 2005).
4.1.
Perfusion and tissue sectioning
4.2.
the LG and accessory optic system), as well as the superior colliculus receive sparse retinal input (Cooper et al., 1993a; Nemec et al., 2004). Collectively, these anatomical and morphological characteristics reflect a severely degenerate visual sensory system in subterranean mole-rats. In conclusion, we report that the naked mole-rat LG is markedly reduced and cytoarchitecturally underdeveloped when compared to that of the laboratory rat; it is, however, substantially bigger than that of the blind mole-rat. This implies that while markedly more regressed than that of rodent species that rely on sight, naked mole-rats are nevertheless better able to perceive light than the blind mole-rat.
Naked mole-rats (H. glaber) with an average mass of 32.7 g and common laboratory rats (Sprague–Dawley) with an average mass of
Preparing the sections
0.02 M PB was filtered (Fisher Scientific filter paper, 09-801F. Porosity: Medium. Flow Rate: Slow). 200–300 μl filtered 0.02 M PB
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was used to seal the slide (75 × 38 mm) with a cover glass. Sections were observed after PB had penetrated the section for 3 min. Sections that had previously been stained for cresyl violet had coverslips removed, were rehydrated, rinsed in PB, and coverslipped as above. Sections that had previously been paraffinembedded were deparaffinized by placing in a 55 °C oven for 10 min to melt the paraffin, then rehydrated and coverslipped as above. We used Permount (Fisher, SP-15-500) to coverslip sections stained by other methods. 4.3.
Neural fiber observation using a novel technique
Unlike conventionally stained sections that rely on transmitted light for observation, sections prepared by this method use reflected light. We used an external light source with 2 optic fiber arms to illuminate the section from above (the slide is placed on a black background for best contrast). We compared our simple new method with that previously described by Gallyas (1979). This method involves the use of a pyridine solution to suppress the staining of non-myelinated tissue elements, followed by a diammine silver solution and a silver intensification reaction. In addition, we tested if other stains interfere with this new technique by first staining tissue with cresyl violet or for CO, respectively, and thereafter following our light reflectance method. 4.4.
Calculation of LG dimensions
A series of sections was used to investigate the morphology of the LG. First, we used 100 μm sections to find the approximate relative position of LG to Bregma. Then we used 40 μm sections to study the size, shape, and stereotaxic location of the LG. Every fourth section was used to reconstruct the 3D structure of the naked mole-rat LG; every sixth section for laboratory rat LG. The same series of sections were also used to calculate the average thickness and the volume of naked mole-rat and laboratory rat LG. A grid (50 × 50 μm for naked mole-rat; 100 × 100 μm for laboratory rat) was superimposed on each section in Neurolucida 4.34 (MicroBrightField Inc., Williston, VT) to facilitate the calculation of the average thickness of LG. The points on the medial edge of the LG that were closest to grid intersection points in alternate grid rows were used to sample the thickness of the LG. LG thickness at each point was defined as the distance spanning the LG, orthogonal to a contour running parallel to the long axis of the LG. The average thickness is the average of all the individual sampling measurements. The contours of the LG and the brain in each section were traced in Neurolucida; the area of the LG in each section was therefore calculated in Neurolucida. The LG volume was determined by summing the average area of the LG in each pair of adjacent sections, each averaged area multiplied by the distance (D) between the adjacent sections (160 μm for naked mole-rat; 240 μm for laboratory rat). These volumes were then summed over the entire extent of the nucleus. The total volume was thus defined as volume = ∑i [D × (areai + areai + 1) / 2]). All dimensions were corrected for shrinkage by making fiducial marks in the tissue prior to tissue processing. Cresyl violetstained sections were also used to study the morphology and lamination of the LG.
Acknowledgments We thank Dr. Gina Cantone, Ms. Lin Yang, and Ms. Geping Zhang for technical assistance and the Animal Care Staff at City College of New York for their help in maintaining our naked mole-rat colony. This work was supported by NIH grants S06GM08168, AG022891, EY12781, and G12RR-03060.
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Larsen, M., Bjarkam, C.R., Stoltenberg, M., Sorensen, J.C., Danscher, G., 2003. An autometallographic technique for myelin staining in formaldehyde-fixed tissue. Histol. Histopathol. 18, 1125–1130. Levine, J.D., Weiss, M.L., Rosenwasser, A.M., Miselis, R.R., 1991. Retinohypothalamic tract in the female albino rat: a study using horseradish peroxidase conjugated to cholera toxin. J. Comp. Neurol. 306, 344–360. Lund, R.D., Lund, J.S., 1965. The visual system of the mole. Talpa europaea. Exp. Neurol. 13, 302–316. Mahon, G.S., 1937. Stain for myelin sheaths in tissues embedded in paraffin. Arch. Neurol. Psych. 38, 103–107. McNally, K.J., Peters, A., 1998. A new method for intense staining of myelin. J. Histochem. Cytochem. 46, 541–545. Mills, L.S., Catania, K.C., 2004. Identification of retinal neurons in a regressive rodent eye (the naked mole rat). Vis. Neurosci. 21, 107–117. Namba, T., Nakamura, T., Grob, D., 1967. Staining for nerve fiber and cholinesterase activity in fresh frozen sections. Am. J. Clin. Pathol. 47, 74–77. Necker, R., Rehkamper, G., Nevo, E., 1992. Electrophysiological mapping of body representation in the cortex of the blind mole rat. NeuroReport 3, 505–508. Negroni, J., Nevo, E., Cooper, H.M., 1997. Neuropeptidergic organization of the suprachiasmatic nucleus in the blind mole rat (Spalax ehrenbergi). Brain Res. Bull. 44, 633–639. Nemec, P., Burda, H., Peichl, L., 2004. Subcortical visual system of the African mole-rat Cryptomys anselli: to see or not to see? Eur. J. Neurosci. 20, 757–768. Nevo, E., 1979. Adaptive convergence and divergence of subterranean mammals. Ann. Rev. Ecolog. Syst. 10, 269–308. O'Riain, M.J., Jarvis, J.U.M., Faulkes, C.G., 1996. A dispersomorph in the naked mole-rat. Nature 380, 619–621. O'Riain, M.J., Jarvis, J.U.M., Alexander, R., Buffenstein, R., Peeters, C., 2000. Morphological castes in a vertebrate. Proc. Natl. Acad. Sci. U. S. A. 97, 13194–13197.
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Nikitina, N.V., Maughan-Brown, B., O'Riain, M.J., Kidson, S.H., 2004. Postnatal development of the eye in the naked mole rat (Heterocephalus glaber). Anat. Rec. A Discov. Mol. Cell. Evol. Biol. 277, 317–337. Quay, W.B., 1981. Pineal atrophy and other neuroendocrine and circumventricular features of the naked mole-rat, Heterocephalus glaber (Ruppell), a fossorial, equatorial rodent. J. Neural Transm. 52, 107–115. Rehkamper, G., Necker, R., Nevo, E., 1994. Functional anatomy of the thalamus in the blind mole rat Spalax ehrenbergi: an architectonic and electrophysiologically controlled tracing study. J. Comp. Neurol. 347, 570–584. Riccio, A.P., Goldman, B.D., 2000. Circadian rhythms of locomotor activity in naked mole-rats (Heterocephalus glaber). Physiol. Behav. 71, 1–13. Schmidt-Nielsen, K., 1984. Scaling; Why is Animal Size So Important? Cambridge Univ. Press, Cambridge, UK. Schmued, L.C., 1990. A rapid, sensitive histochemical stain for myelin in frozen brain sections. J. Histochem. Cytochem. 38, 717–720. Smith, B., Dunbar, R.A., 1966. Mode of action of certain myelin stains. J. Pathol. Bacteriol. 91, 117–121. Waiss, C.B., Curtis, R.L., Anderson, F.D., 1971. A sensitive myelin-sheath stain particularly useful for small mammals and neonatal cats. Stain Technol. 46, 289–295. Weigert, C., 1884. Ausfuhrliche Beschreibung der in no. 2 dieser zeitschrift erwahnten neuen farbungs methode für das centralnervensystem. Fortschr. Deutsch Med. 2, 190–192. Wong-Riley, M., 1979. Changes in the visual system of monocularly sutured or enucleated cats demonstrable with cytochrome oxidase histochemistry. Brain Res. 171, 11–28. Xiao, J., Levitt, J., 2005. A new chamber method for mounting tissue sections. J. Neurosci. Methods 144, 235–240.
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
The use of a novel and simple method of revealing neural fibers to show the regression of the lateral geniculate nucleus in the naked mole-rat (Heterocephalus glaber ) Jun Xiao ⁎, Jonathan B. Levitt, Rochelle Buffenstein Biology Department, The City College of New York, 138th Street and Convent Avenue, New York, NY 10031, USA
A R T I C LE I N FO
AB S T R A C T
Article history:
The lateral geniculate nucleus (LG) is an important subcortical nucleus in the visual system.
Accepted 6 January 2006
It receives primary projections from the retina and relays these to central visual structures.
Available online 14 February 2006
Although there are studies on the retina and visual cortex of animals with regressed vision, little is known about the LG in such animals. The strictly subterranean naked mole-rat
Keywords:
(Heterocephalus glaber) has markedly reduced visual acuity with concomitant pronounced
Thalamus
changes in the visual cortex. We used a novel method to reveal myelinated neural fibers in a
Neuroanatomy
histological study assessing if the LG shows regressive changes commensurate with the
Histology
level of reliance on vision by this rodent. Myelin detection here relies on significant
Visual system
differences in visible light reflection between neural fibers and the gray matter. Moreover,
Myelin staining
this simple method does not interfere with further staining for additional analyses. This method reveals that the contribution of the LG to brain volume in the naked mole-rat is less than a third of that of the rat. This shows that the retinogeniculocortical system in the naked mole-rat is considerably smaller than that of rodents that rely heavily on their visual system, but is nevertheless less regressed than that of the extensively studied blind molerat; this may facilitate limited responses to visual stimuli. © 2005 Elsevier B.V. All rights reserved.
1.
Introduction
Naked mole-rats (Heterocephalus glaber) are strictly subterranean rodents that spend almost their entire lives below ground. Occasional forays onto the surface, although uncommon, may occur when animals excavate tunnels and kick loose soil onto the surface, or when a burrow system is breached (Braude, 2000). Living in this dark and sandy milieu, it is not surprising that these mammals rely heavily upon tactile stimuli and mechanosensory hairs scattered over the body (Crish et al., 2003). Naked mole-rats' eyes are markedly reduced in size, and generally these rodents move about with
their eyes closed, suggesting that these animals do not rely on vision. Nevertheless, they have retained most of the wellcharacterized mammalian cell types found in the eye, although their eye structural organization is considerably less regular than that in more sighted mammals (Mills and Catania, 2004; Nikitina et al., 2004). The naked mole-rat reportedly cannot perceive images but can detect amorphous light (Hetling et al., 2005). Similarly, although the blind molerat (Spalax ehrenbergi) has a subcutaneous eye and atrophied structures involved with image formation, non-image forming visual pathways involved in photoperiod perception are well developed (Cooper et al., 1993a,b). In contrast to the exten-
⁎ Corresponding author. Fax: +1 212 650 8585. E-mail address: [email protected] (J. Xiao). 0006-8993/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.01.021
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sively studied visual system of the Israeli blind mole-rat, the visual system of Bathyergid mole-rats has received much less attention (Bronchti et al., 1991; Catania and Remple, 2002; Cooper et al., 1993a,b; Nikitina et al., 2004; Rehkamper et al., 1994), even though from a comparative evolutionary perspective, it would be both interesting and informative to study this regressed visual system as a viable model for the study of evolutionary remodeling. Most of the studies to date assessing the visual system of subterranean rodents have focused on eye morphology (Nevo, 1979; Nikitina et al., 2004), retinal structure (Cernuda-Cernuda et al., 2003; Mills and Catania, 2004), and the visual cortex (Bronchti et al., 2002; Heil et al., 1991; Necker et al., 1992). Although the lateral geniculate nucleus (LG) is an important sensory nucleus governing the flow of visual information from retina to visual cortex, little is known about this region in subterranean rodents. The primary purpose of this paper is to assess if this structure, like the eye and visual cortex, differs from that of above ground dwelling animals. The LG in rodents can be easily identified using myelin staining to identify the superior thalamic radiation (str: a bundle of myelinated neural fibers) and the dorsolateral edge of the thalamus as landmarks. Although there are a variety of myelin staining methods, most of these are labor intensive, involve many reagents, and each method requires specific and/or different modes of section preparation. To facilitate experiments in this study, we introduce a simple and reliable method that can reveal neural fibers using a novel mechanism. This new method reveals myelinated axons using differences in visible light reflection of the myelinated axons and the gray matter. The main advantages of this method are that it is simple and reliable, and does not interfere with other staining techniques.
2.
Results
We first demonstrate the validity of our new method, and then use it to document and compare the extent of the LG in naked mole-rats and laboratory rats.
2.1.
Assessment of our method for revealing neural fibers
The new method works well on brain sections from both naked mole-rats and common laboratory rats (Fig. 1) as well as carnivorous species like ferrets (data not shown). The black background plate gives excellent contrast of the neural fibers that are in white. The sections in Figs. 1A and B show myelinated fibers revealed by the new method from naked mole-rat forebrain 1.90 mm posterior to Bregma. This section had previously been reacted to reveal cytochrome oxidase (CO). The sections in Figs. 1D and E show myelinated fibers of the laboratory rat forebrain 4.16 mm posterior to Bregma. This method reveals both fiber bundles and single axons in either frozen or paraffin sections. Figs. 2A–C shows that our method of revealing myelinated fibers in the naked mole-rat forebrain gives comparable results to that obtained from the widely used Gallyas (1979) method (Fig. 2D), confirming that our novel method does reveal myelinated neural fibers.
A significant advantage of this method is that sections processed by this method first (e.g. Figs. 1D and E) can be stained later by any other method (Fig. 1F, see Discussion). The section in Fig. 1C was stained by cresyl violet with no other processing. There are no differences between the staining quality of the section in Fig. 1F and that in Fig. 1C. Alternatively, sections may be stained first by other methods, dried, coverslipped, and then processed some time later to reveal myelin by our method. CO staining did not essentially affect myelinated fibers observation (Figs. 1A, B). However, when cresyl violet was used first on a section and then subsequently used for our method, we noted lower resolution. This was most likely due to the dehydration and hydration process, for although the fibers could be seen clearly with the new method (Fig. 3B), the resolution was lower than that of CO-stained or unstained sections. Myelin may have been disrupted by the dehydration process. Thinner sections provided better resolution. Fig. 3C shows a 10 μm thick section. Single fibers can be identified under high amplification. Because the neural fibers are not confined to a strict 10 μm thick plane, the fiber bundles seem to be cut off periodically. In contrast, the fiber bundles in thicker sections were less cut off (Fig. 3D, 100 μm thick). Therefore, while thinner sections provide better single fiber observations, thicker sections support better global observations.
2.2. Regression of the naked mole-rat lateral geniculate nucleus The size of naked mole-rat LG was reduced significantly compared with that of the laboratory rat (Fig. 1). The naked mole-rat LG also appeared to be cytoarchitecturally underdeveloped for there is no obvious division into laminae or divisions (Figs. 1C vs. F, 4B1 and B2 vs. D1 and D2). Nevertheless, typical LG subdivisions of the naked mole-rat dorsal lateral geniculate nucleus (LGd), ventral lateral geniculate nucleus (LGv), and intergeniculate leaflet (IGL) were architectonically distinct entities. However, the boundary between the medial and lateral divisions of the LGv (LGvm and LGvl) was not clear in naked mole-rat brain sections (Figs. 1C, 4B1 and B2). In contrast, the rat LGv could be divided into LGvm (parvicellular part) and LGvl (magnocellular part) (Figs. 1F, 4D1 and D2). The cells in LGvm were smaller than cells in LGvl (Fig. 1F). The naked mole-rat LG was found to typically extend from 0.90 mm to 2.36 mm posterior to Bregma (Fig. 4A). The overall shape of the naked mole-rat LG was like a bending sheet with an average thickness of 81 μm, occupying roughly 3.4% of the width of the thalamus (from the midline to the lateral edge of the thalamus). The thickness reached its maximum (198 μm) at roughly 1.23 mm posterior to Bregma (Fig. 4B). The length dimension along the curved band in the lateroventral– mediodorsal direction reached its maximum (864 μm) at roughly 1.23 mm posterior to Bregma. The volume of naked mole-rat LG was roughly 0.075 mm3, occupying roughly 0.019% of the entire brain volume. In contrast, the average thickness of the rat LG was 311 μm, occupying roughly 7.8% of the width of the thalamus. The thickness reached its maximum (537 μm) at roughly 4.32 mm posterior to Bregma (Fig. 4D). The dimension along the curved band in the lateroventral–mediodorsal
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Fig. 1 – Neural fibers revealed by the new method in naked mole-rat (A and B) and laboratory rat (D and E). Panel B is the inset in panel A; panel E is the inset in panel D. Panel C illustrates a section adjacent to that in panels A and B, showing a region corresponding to the inset in panel B, and was stained with cresyl violet without any other processing. Panel F is the same section as in panel E, showing the region indicated by the inset in panel E. It was processed by the new method first and then was stained with cresyl violet. ml, medial lemniscus; opt, optic tract; str, superior thalamic radiation; IGL, intergeniculate leaflet; LGd, dorsal lateral geniculate nucleus; LGv(m,l), ventral lateral geniculate nucleus (medial/lateral divisions). All sections are 40 μm thick frozen sections. The number at the upper right corner in each panel indicates the A–P distance from Bregma.
direction reached its maximum (1677 μm) at roughly 4.08 mm posterior to Bregma. The volume of rat LG was roughly 0.83 mm3, occupying roughly 0.059% of the entire brain volume. Thus, the relative thickness (percent of the thalamus width) of the naked mole-rat LG was less than half that of the laboratory rat, while the relative volume (percent of the entire brain) of the naked mole-rat LG was roughly one-third that of the laboratory rat. Indeed, if the relative volumes of the LG were scaled to body mass0.67, as is commonly done in allometric based biological studies (Schmidt-Nielsen, 1984), the naked molerat LG would be predicted to be over three times larger. The diminished relative size of this nucleus in naked mole-rats
suggests that the LG in this subterranean species is indeed markedly regressed.
3.
Discussion
This paper set out to describe both a novel histological method for assessing myelinated regions in neural tissue, and the use of this method to compare among naked mole-rats and laboratory rats the histology and size of the LG. This easy myelin-specific method relies upon physical properties of the myelinated axon and yields similar results to those of
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Fig. 2 – Neural fibers revealed by the new method. (A) Frozen section (40 μm) 0.65 mm posterior to Bregma. cc, corpus callosum; dhc, dorsal hippocampal commissure; ec, external capsule; fi, fimbria of the hippocampus; ic, internal capsule. (B) High amplification view of the inset in panel A. (C) A paraffin-embedded section (10 μm) from another brain of approximately the same position as the inset in panel A. (D) A frozen section (40 μm) of similar location as the inset in panel A from another brain stained by the silver method (Gallyas, 1979).
conventional methods (Gallyas, 1979). Using this method, we have noted that differences in the LG between rats and naked mole-rats are commensurate with reported differences in eye morphology and visual cortex (Bronchti et al., 2002; CernudaCernuda et al., 2003; Francescoli, 2001; Heil et al., 1991; Mills and Catania, 2004; Necker et al., 1992; Nevo, 1979; Nikitina et al., 2004). Our data confirm that the entire visual system of naked mole-rats has regressed in a similar manner, suggesting a reduced ability to perceive images. Myelin staining is a fundamental technique employed in many areas of neuroscience. Although myelin staining has been employed for over a century (Cohnheim, 1866; Weigert, 1884), new less complex and reliable methods are continuously being sought after (Ciaccio, 1909; Fuentes, 1960; Gallyas, 1979; Kaatz et al., 1992; Kutscher et al., 1987; Larsen et al., 2003; McNally and Peters, 1998; Schmued, 1990). A common feature of the many previous complex myelin-staining procedures is that they exploit chemical or biological properties of the histological section: either using oil-soluble dyes to stain lipophilic components of the fibers (including the lipid phase of the myelin, complex phospholipids, oligodendrocytes), or binding metal particles to some components of myelin (for review, see Schmued, 1990). Each method has its drawbacks or limitations: some are suitable for only one type of section (e.g. frozen or paraffin sections), others work best on sections cut at a specific thickness (Gallyas, 1979; Mahon, 1937), and still others require that tissues are postfixed for prolonged periods
(Larsen et al., 2003) and/or kept at high temperatures (e.g. 50 °C in Waiss et al., 1971). These latter harsh conditions not only result in the loss of stainable phospholipid (for review, see Smith and Dunbar, 1966) but also inactivate enzymes (Namba et al., 1967), thereby preventing the use of myelin-stained sections in multiple co-staining procedures, especially those based upon enzymatic procedures. These shortcomings thus curb the amount of information obtained in neurohistological studies. A key advantage of our method is that it does not change chemical and biological properties of the section. This allows additional section staining with both enzyme and dye based techniques (e.g. cytochrome oxidase and cresyl violet). Furthermore, our method can be used with both fresh sections and dehydrated sections (even those that have been dehydrated for prolonged, ∼N1 month, periods) that cannot be stained for myelin using conventional protocols (Schmued, 1990). Our simple reliable technique is quick and does not require postfixing. In contrast, conventional methods not only involve numerous steps and chemicals but also typically require meticulous care to maintain a metal-free environment. Unlike traditional methods that work well only on one type of section (for example, Klüver and Barrera (1953) technique only on paraffin sections), this method can be used on different kinds of sections (frozen, paraffin, and celloidin) and on sections that vary in thickness (10–100 μm and up). We used this method to examine the cyto- and
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Fig. 3 – The new method reveals neural fibers in sections that had been stained by other methods (A and B) and in sections of different thickness (C and D). The section in panel A had been stained for CO first; that in panel B had been stained by cresyl violet first. Sections in panels A and B are 40 μm thick. (C and D) Effect of section thickness on resolution. Thin section (C: 10 μm) provides better resolution than thick section (D: 100 μm). Conventions as in Fig. 1.
myeloarchitecture of the LG so as to assess if the visual thalamic relay nuclei are regressed in a highly eusocial, strictly subterranean rodent, the naked mole-rat. Subterranean animals have received considerable attention as a viable model for the study of mosaic evolutionary remodeling. Like other subterranean mammals, the Bathyergid family of mole-rats (which include naked mole-rats) shows a suite of morphological and physiological adaptations to the underground habitat they have inhabited since the early Miocene (Buffenstein, 2001). Although the naked mole-rat eye retains a typical mammalian organization, eyes are markedly reduced in size, and the optic nerve cross sectional area is extremely small with a very low fiber density (Hetling et al., 2005). Furthermore Catania and Remple (2002) have reported that the somatosensory cortex in these animals encompasses large regions of the neocortex normally devoted to vision in above-ground inhabitant. The LG is a key nucleus in the central nervous system to receive projections from retina and to relay the information received to the cerebral cortex. In the blind mole-rat S. ehrenbergi, the LG has regressed resulting in a very small superficial thalamic area (Cooper et al., 1993a,b; Rehkamper et al., 1994). Similarly, the LG of naked mole-rats is also markedly attenuated and cytoarchitecturally underdeveloped when compared to laboratory rats, but nevertheless substantially bigger than that of the blind mole-rat (compare Fig. 1 of this paper with Fig. 1 in Rehkamper et al., 1994). It seems that the retinogeniculocortical system of naked mole-rats therefore is
less regressed than that of the blind mole-rat but markedly more so than that of the laboratory rat. These results further support reports that although the naked mole-rat has an atrophied visual cortex, these animals can indeed detect light, albeit in an unstructured manner with poor image formation. They do indeed respond to light detection and use these visual cues to escape potential life-threatening events (such as predation) by moving away from the source of light. Hetling et al. (2005) report pronounced individual differences in response to experimental illumination, such that the dominant breeding female, in keeping with her status and importance as the sole-breeding female in a colony (Jarvis, 1981; O'Riain et al., 2000), was more likely to leave (or avoid) an illuminated chamber than other non-breeding individuals. The “working castes” of these eusocial rodents were less evasive of experimental illumination, and this may reflect that they are indeed more likely to encounter a breached burrow system, which they would need to recognize as such and repair thereby ensuring the continued safety of the entire colony (Brett, 1991). Mole-rat nervous systems provide a unique model for studying neural development, plasticity, and neural regression. While the imaging function of the retina of mole-rats has regressed in response to evolving in a subterranean habitat, the retinohypothalamic pathway in naked mole-rats, like that of the blind mole-rat, S. ehrenbergi (Cooper et al., 1993a; Negroni et al., 1997) is retained and relatively well developed. This may reflect the employment of some photoendocrine
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responses (Buffenstein, 1996); however, the pineal gland of naked mole-rats is the smallest in absolute size of any other known rodent and is generally considered atrophic (Quay, 1981). This may nevertheless contribute to the preserved ability in some but not all individuals to experimentally entrain activity periods, body temperature, and metabolism to light cues (Riccio and Goldman, 2000). This photoperiodic regulation of circadian rhythm may be especially important in “dispersomorph castes” (O'Riain et al., 1996) that may leave burrow systems at night in search of outbreeding opportunities (Braude, 2000). The suprachiasmatic nucleus (SCN) in both the Spalacidae (blind mole-rats) and the Bathyergidae is organized into fundamental subdivisions that are comparable to those of above-ground dwelling rodents (Negroni et al.,
1997; Nemec et al., 2004). However, the density of retinal projections to both the SCN and the bed nucleus of the stria terminalis (BNST) differs substantially. These regions in molerat brains receive about 20% of the total retinal projection, while in other rodents, that are active above ground, these nuclei receive only a small proportion (less than 1%) of the total retinal projection (Cooper et al., 1993a; Levine et al., 1991). These specific features may reflect both phylogenetic traits and evolutionarily conserved adaptations to a subterranean habitat. Although a subterranean insectivore, the European mole (Talpa europaea), also exhibits a regressed visual system, it appears to direct the bulk of the retinal output to the pretectum (Lund and Lund, 1965). In contrast, the pretectum and other subcortical visual structures of bathyergids (such as
Fig. 4 – Comparison of naked mole-rat LG with that of laboratory rat. (A) A series of 40 μm sections was used to locate the LG. (B) Enlarged view of the insets in panel A showing the location and shape of naked mole-rat LG. Panels B1 and B2 are Nissl-stained sections adjacent to insets in panel B. (C) 3D reconstruction of the naked mole-rat LG. (D) A series of 40 μm sections showing the location and shape of laboratory rat LG. Panels D1 and D2 are sections with insets in panel D subsequently stained with cresyl violet. (E) 3D reconstruction of the laboratory rat LG. IGL, intergeniculate leaflet; LGd, dorsal LG; LGvl, lateral division of ventral LG; LGvm, medial division of ventral LG; D, dorsal; V, ventral; R, rostral; C, caudal. Conventions as in Fig. 1.
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Fig. 4 (continued ).
4.
Experimental procedures
225.4 g were euthanized with sodium pentobarbital (100 mg/kg i.p.) and intracardially perfused with a saline rinse solution, followed by 4% paraformaldehyde in 0.02 M phosphate-buffered saline (PBS: pH 7.4). After a 30-min period of initial fixation, brains were removed and sunk in 4% paraformaldehyde containing 30% sucrose. The brains were embedded in Frozen Section Medium (Richard-Allan Scientific) and cut on a cryostat (Microm, HM560) into 10, 40, or 100 μm thick sections. Both coronal and sagittal sections were cut; however, here, we present only data from coronal sections for studies based on both planes of sectioning yielded similar data. Sections were collected directly into 0.02 M phosphate buffer (PB). Some sections were mounted on gelatinsubbed glass slides and were stained with cresyl violet later; others were kept in a free floating environment and stained for cytochrome oxidase (CO) later using a modified method of WongRiley (1979). We mounted these sections with our newly designed mounting chamber (Xiao and Levitt, 2005).
4.1.
Perfusion and tissue sectioning
4.2.
the LG and accessory optic system), as well as the superior colliculus receive sparse retinal input (Cooper et al., 1993a; Nemec et al., 2004). Collectively, these anatomical and morphological characteristics reflect a severely degenerate visual sensory system in subterranean mole-rats. In conclusion, we report that the naked mole-rat LG is markedly reduced and cytoarchitecturally underdeveloped when compared to that of the laboratory rat; it is, however, substantially bigger than that of the blind mole-rat. This implies that while markedly more regressed than that of rodent species that rely on sight, naked mole-rats are nevertheless better able to perceive light than the blind mole-rat.
Naked mole-rats (H. glaber) with an average mass of 32.7 g and common laboratory rats (Sprague–Dawley) with an average mass of
Preparing the sections
0.02 M PB was filtered (Fisher Scientific filter paper, 09-801F. Porosity: Medium. Flow Rate: Slow). 200–300 μl filtered 0.02 M PB
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was used to seal the slide (75 × 38 mm) with a cover glass. Sections were observed after PB had penetrated the section for 3 min. Sections that had previously been stained for cresyl violet had coverslips removed, were rehydrated, rinsed in PB, and coverslipped as above. Sections that had previously been paraffinembedded were deparaffinized by placing in a 55 °C oven for 10 min to melt the paraffin, then rehydrated and coverslipped as above. We used Permount (Fisher, SP-15-500) to coverslip sections stained by other methods. 4.3.
Neural fiber observation using a novel technique
Unlike conventionally stained sections that rely on transmitted light for observation, sections prepared by this method use reflected light. We used an external light source with 2 optic fiber arms to illuminate the section from above (the slide is placed on a black background for best contrast). We compared our simple new method with that previously described by Gallyas (1979). This method involves the use of a pyridine solution to suppress the staining of non-myelinated tissue elements, followed by a diammine silver solution and a silver intensification reaction. In addition, we tested if other stains interfere with this new technique by first staining tissue with cresyl violet or for CO, respectively, and thereafter following our light reflectance method. 4.4.
Calculation of LG dimensions
A series of sections was used to investigate the morphology of the LG. First, we used 100 μm sections to find the approximate relative position of LG to Bregma. Then we used 40 μm sections to study the size, shape, and stereotaxic location of the LG. Every fourth section was used to reconstruct the 3D structure of the naked mole-rat LG; every sixth section for laboratory rat LG. The same series of sections were also used to calculate the average thickness and the volume of naked mole-rat and laboratory rat LG. A grid (50 × 50 μm for naked mole-rat; 100 × 100 μm for laboratory rat) was superimposed on each section in Neurolucida 4.34 (MicroBrightField Inc., Williston, VT) to facilitate the calculation of the average thickness of LG. The points on the medial edge of the LG that were closest to grid intersection points in alternate grid rows were used to sample the thickness of the LG. LG thickness at each point was defined as the distance spanning the LG, orthogonal to a contour running parallel to the long axis of the LG. The average thickness is the average of all the individual sampling measurements. The contours of the LG and the brain in each section were traced in Neurolucida; the area of the LG in each section was therefore calculated in Neurolucida. The LG volume was determined by summing the average area of the LG in each pair of adjacent sections, each averaged area multiplied by the distance (D) between the adjacent sections (160 μm for naked mole-rat; 240 μm for laboratory rat). These volumes were then summed over the entire extent of the nucleus. The total volume was thus defined as volume = ∑i [D × (areai + areai + 1) / 2]). All dimensions were corrected for shrinkage by making fiducial marks in the tissue prior to tissue processing. Cresyl violetstained sections were also used to study the morphology and lamination of the LG.
Acknowledgments We thank Dr. Gina Cantone, Ms. Lin Yang, and Ms. Geping Zhang for technical assistance and the Animal Care Staff at City College of New York for their help in maintaining our naked mole-rat colony. This work was supported by NIH grants S06GM08168, AG022891, EY12781, and G12RR-03060.
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