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The three members of the Pou4f family of transcription factors: Pou4f1, Pou4f2, Pou4f3 (Brn3a, Brn3b and Brn3c, respectively) play, during development, essential roles in the differentiation and survival of sensory neurons. The purpose of this work is to study the expression of the three Brn3 factors in the albino and pigmented adult rat. Animals were divided into these groups: i) untouched; ii) fluorogold (FG) tracing from both superior colliculli; iii) FG-tracing from one superior colliculus; iv) intraorbital optic nerve transection or crush. All retinas were dissected as flat-mounts and subjected to single, double or triple immunohistofluorescence The total number of FG-traced, Brn3a, Brn3b, Brn3c or Brn3 expressing RGCs was automatically quantified and their spatial distribution assessed using specific routines. Brn3 factors were studied in the general RGC population, and in the intrinsically photosensitive (ip-RGCs) and ipsilateral RGC sub-populations. Our results show that: i) 70% of RGCs co- express two or three Brn3s and the remaining 30% express only Brn3a (26%) or Brn3b; ii) the most abundant Brn3 member is Brn3a followed by Brn3b and finally Brn3c; iii) Brn3 a-, b- or c- expressing RGCs are similarly distributed in the retina; iv) The vast majority of ip-RGCs do not express Brn3; v) The main difference between both rat strains was found in the population of ipsilateral-RGCs, which accounts for 4.2% and 2.5% of the total RGC population in the pigmented and albino strain, respectively. However, more ipsilateral-RGCs express Brn3 factors in the albino than in the pigmented rat; vi) RGCs that express only Brn3b and RGCs that co-express the three Brn3 members have the biggest nuclei; vii) After axonal injury the level of Brn3a expression in the surviving RGCs decreases compared to control retinas. Finally, this work strengthens the validity of Brn3a as a marker to identify and quantify rat RGCs.

Glaucoma, the second most common cause of blindness, is characterized by a progressive loss of retinal ganglion cells and their axons, with a concomitant loss of the visual field. Although the exact pathogenesis of glaucoma is not completely understood, a critical risk factor is the elevation, above normal values, of the intraocular pressure. Consequently, deciphering the anatomical and functional changes occurring in the rodent retina as a result of ocular hypertension has potential value, as it may help elucidate the pathology of retinal ganglion cell degeneration induced by glaucoma in humans. This paper predominantly reviews the cumulative information from our laboratory's previous, recent and ongoing studies, and discusses the deleterious anatomical and functional effects of ocular hypertension on retinal ganglion cells (RGCs) in adult rodents. In adult rats and mice, perilimbar and episcleral vein photocauterization induces ocular hypertension, which in turn results in devastating damage of the RGC population. In wide triangular sectors, preferentially located in the dorsal retina, RGCs lose their retrograde axonal transport, first by a functional impairment and after by mechanical causes. This axonal damage affects up to 80% of the RGC population, and eventually causes their death, with somal and intra-retinal axonal degeneration that resembles that observed after optic nerve crush. Importantly, while ocular hypertension affects the RGC population, it spares non-RGC neurons located in the ganglion cell layer of the retina. In addition, functional and morphological studies show permanent alterations of the inner and outer retinal layers, indicating that further to a crush-like injury of axon bundles in the optic nerve head there may by additional insults to the retina, perhaps of ischemic nature.

In axotomized mouse retinas, a single intravitreal injection of BDNF protects RGCs throughout the whole retina. There is a strong contralateral response that involves microglial activation and OHSt phagocytosis.

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We have studied in parallel the population of displaced retinal ganglion cells (dRGCs) and normally placed (orthotopic RGCs, oRGCs) in albino and pigmented rats. Using retrograde tracing from the optic nerve, from both superior colliculi (SC) or from the ipsilateral SC in conjunction with Brn3 and melanopsin immunodetection, we report for the first time their total number and topography as well as the number and distribution of those dRGCs and oRGCs that project ipsi- or contralaterally and/or that express any of the three Brn3 isoforms or melanopsin. The total number of RGCs (oRGCs+dRGCs) is 84,706 ± 1249 in albino and 90,440 ± 2236 in pigmented, out of which 2383 and 2428 are melanopsin positive (m-RGCs), respectively. Regarding dRGCs: i/ albino rats have a significantly lower number of dRGCs than pigmented animals (0.5% of the total number of RGCs vs. 2.5%, respectively), ii/ dRGCs project massively to the contralateral SC, iii/ the percentage of ipsilaterality is higher for dRGCs than for oRGCs, iv/ a higher proportion of ipsilateral dRGCs is observed in albino than pigmented animals, v/ dRGC topography is very specific, they predominate in the equatorial temporal retina, being densest where the oRGCs are densest, vi/ Brn3a detects all dRGCs except half of the ipsilateral ones and those that express melanopsin, vii/ the proportion of dRGCs that express Brn3b or Brn3c is slightly lower than in the oRGC population, viii/ a higher percentage of dRGCs (13% albino, 9% pigmented) than oRGCs (2.6%) express melanopsin, ix/ few m-RGCs (displaced and orthotopic) project to the ipsilateral SC, x/ the topography of m-dRGCs does not resemble the general distribution of dRGCs, xi/ The soma size in m-oRGCs ranges from 10 to 21 μm and in m-dRGCs from 8 to 15 μm, xii/ oRGCs and dRGCs have the same susceptibility to axonal injury and ocular hypertension. Although the role of mammalian dRGCs remains to be determined, our data suggest that they are not misplaced by an ontogenic mistake.

In the GCL, only RGCs are lost after axotomy, and there are important differences in the course of loss and rate of survival between melanopsin+RGCs and the rest of RGCs.

We purpose here to analyze and compare the population and topography of cone photoreceptors in two mouse strains using automated routines, and to design a method of retinal sampling for their accurate manual quantification. In whole-mounted retinas from pigmented C57/BL6 and albino Swiss mice, the longwave-sensitive (L) and the shortwave-sensitive (S) opsins were immunodetected to analyze the population of each cone type. In another group of retinas both opsins were detected with the same fluorophore to quantify all cones. In a third set of retinas, L-opsin and Brn3a were immunodetected to determine whether L-opsin+cones and retinal ganglion cells (RGCs) have a parallel distribution. Cones and RGCs were automatically quantified and their topography illustrated with isodensity maps. Our results show that pigmented mice have a significantly higher number of total cones (all-cones) and of L-opsin+cones than albinos which, in turn, have a higher population of S-opsin+cones. In pigmented animals 40% of cones are dual (cones that express both opsins), 34% genuine-L (cones that only express the L-opsin), and 26% genuine-S (cones that only express the S-opsin). In albinos, 23% of cones are genuine-S and the proportion of dual cones increases to 76% at the expense of genuine-L cones. In both strains, L-opsin+cones are denser in the central than peripheral retina, and all-cones density increases dorso-ventrally. In pigmented animals S-opsin+cones are scarce in the dorsal retina and very numerous in the ventral retina, being densest in its nasal aspect. In albinos, S-opsin+cones are abundant in the dorsal retina, although their highest densities are also ventral. Based on the densities of each cone population, we propose a sampling method to manually quantify and infer their total population. In conclusion, these data provide the basis to study cone degeneration and its prevention in pathologic conditions.