Calretinin-Immunoreactive Amacrine Cells and Ganglion Cells in the Greater Horseshoe Bat, Rhinolophus ferrumequinum

Vol. 12, No. 4, pp. 133-139, November 2007

Calretinin-Immunoreactive Amacrine Cells and Ganglion Cells in the Greater Horseshoe Bat, Rhinolophus ferrumequinum

Young-Ki Jeon*

** and Chang-Jin Jeon*

*Department of Biology, College of Natural Sciences, Kyungpook National University, Daegu 702-701, Korea

**Department of Ophthalmic Optics, Kundong University, Andong 760-833, Korea (Received August 25, 2007: Revised manuscript received September 20, 2007)

···

Although the physiological roles of calretinin have not been established, it may simply work as a calcium buffer or may actively work in calcium-mediated signal transduction. Calretinin plays a little role in the transport and physiological buffering of calcium in the adult photoreceptor cells, bipolar cells and horizontal cells of the human retina. We identified the calretinin-immunoreactive neurons in the inner nuclear cell layer and ganglion cell layer and the distribution pattern of the labeled neurons in the retina of a bat, Rhinolophus ferrumequinum , in this study. We observed the existence of calretinin-immunoreactive AII amacrine cell in the inner nuclear layer and ganglion cells in the ganglion cell layer of bat retina through this study. This observation must be significant along with our previous studies as we need to study for more understanding about the unsolved issue of a bat vision and the unique behavioral aspects of bat flight maneuverability.

Key words: Bat, Distribution, Immunocytochemistry, Calretinin, Parvalbumin, Amacrine cell, Ganglion cell

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Introduction

As a kind of bat used in this study, the greater horse- shoe bat, Rhinolophus ferrumequinum , is one of about 65 species of the genus Rhinolophus , which has one of the largest geographic distributions, ranging from Europe and Northern Africa in the west to China, Korea, and Japan in the east. This species is mainly cave-dwelling. It is insectivorous, using its echolocation capability

. It is a common misconception that bats are blind. In general, bats are better known for echolocation rather than for visual abilities. Nevertheless, bats clearly have eyes, and many of them rely on visual clues for sight, both day and night, to find prey and monitor their environment in order to protect themselves from predators

.

Among the many calcium-binding proteins found in the central nervous system calbindin D28K, calretinin, and parvalbumin have been shown to be markers of specific neuronal populations in the central nervous system

.

Calretinin is a 29 kDa calcium-bindind protein that was

first isolated as a cDNA clone from the chick retina. The amino acid sequence of calretinin is also known to have 58% homology to that of calbindin D28K in the chick

. Although the physiological roles of calretinin have not been established, it may simply work as a calcium buffer or may actively work in calcium-mediated signal trans- duction. Unlike calbindin and parvalbumin, calretinin plays little role in the transport and physiological buffer- ing of calcium in the adult photoreceptor cells, bipolar cells and horizontal cells of the human retina. It appears, however, that calretinin is predominantly involved in amacrines in postnatal and adult retinas and ganglion cells' differentiation

. The distribution of calretinin in retina from rabbit, cat, and dog was reported

. We also studied the morphology of calretinin positive cells in ox and pig retina

.

Amacrine cells in the mammalian retina represent approximately 40% of all neurons of the inner nuclear layer

. Amacrine cells are variable in morphology and physiology. Each type of amacrine neuron carries out

1st Author Contact Address: Chang-Jin Jeon, Neuroscience Lab. Department of Biology College of Natural Sciences, Kyungpook National University, 1370

Sankyuk-dong, Daegu 702-701, Korea TEL: 82-53-950-5343, FAX: 82-53-953-3066, E-mail: [email protected]

amacrine cells were identified in a recent study in rabbit retina

. Recently, we reported the distributional pattern and the cell type of parvalbumin-immunoreactive cells in the inner nuclear layer of bat

. Parvalbumin was specif- ically localized in AII amacrine cells in the bat retina. As AII amacrine cells are critically involved in both rod- and cone-driven signals by sending a vertical flow of infor- mation within the On- and Off-layers, the existence of AII amacrine cells suggests that bats have retinas involved in both rod- and cone-driven signals. For a bet- ter understanding of the unsolved issue of the bat vision, the present study is undertaken to identify calretinin-con- taining cells in the inner nuclear and ganglion cell layer of the greater horseshoe bat retina primarily as a series of our studies about the retina of the bat.

Materials and Methods

Animals and tissue preparation

Adult greater horseshoe bats ( Rhinolophus ferrumequi- num ) were used in this study. The bats were anesthetized with a mixture of ketamine hydrochloride (30-40 mg/kg) and xylazine (3-6 mg/kg). A local anesthetic, propara- caine hydrochloride (100-200 μ l), was applied to the cor- nea of the bats to suppress blink reflexes. Their eyes were enucleated, and the animals were euthanized by an overdose of the same anesthetics. The anterior segments of the eyes were removed, and the retinas were dissected from the eyecups. The retinas were fixed in 4% para- formaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) for 2 hr at 4-5°C, and then they were rinsed 3 × 10 min in 0.1 M PB. The guidelines of the National Institute of Health regarding the Care and Use of Laboratory Ani- mals were followed in all experimental procedures.

Immunocytochemistry

Antibodies against parvalbumin and calretinin were obtained from Sigma Chemical (St. Louis, USA) and Chemicon (USA). The immunocytochemical methods were described in detail in our previous reports

.

For immunocytochemistry, the retinas were incubated in 1% sodium borohydride (NaBH

) for 30 min. Subse- quently, these tissues were rinsed for 3 × 10 min in 0.1 M PB, and incubated in 0.1 M PB with 4% normal horse

whole mounts were processed free floating in small vials at 4-5°C with gentle agitation. The retinas were then incubated in the primary antiserum in 0.1 M PB with 4%

normal horse serum for 72 hr with 0.5% Triton X-100 added. The primary antiserum was diluted 1:1000-1:2000.

Following 3 × 10 min rinses in 0.1 M PB, the retinas were incubated in a 1:200 dilution of biotinylated anti-mouse IgG in 0.1 M PB with 4% normal horse serum for 24 hrwith 0.5% Triton X-100 added. The retinas were then rinsed for 3 × 10 min in 0.1 M PB and incubated in a 1:50 dilution of avidin-biotinylated horseradish peroxidase complex (ABC, Vector lab, USA) in 0.1 M PB for 2 hr.

The tissues were again rinsed in 0.25 M Tris buffer for 3 × 10 min. Finally, the staining was visualized by reacting with 3,3'-diaminobenzidine tetrahydrochloride and hydro- gen peroxide in 0.25 M Tris buffer for 3-5 min using a DAB reagent set (Kirkegaard & Perry, USA). The retinas were then rinsed in 0.25 M Tris buffer before mounting.

The whole mounts were cover slipped in glycerol without dehydration procedure. The tissue was examined and pho- tographed on a Zeiss Axioplan microscope using either conventional or differential interference contrast (DIC) optics.

Results and Discussion

These observations show that there are considerable similarities in the calretinin distribution among species.

Generally, calretinin immunoreactivity was found in nerve fibers in the nerve fiber layer, in cells in the ganglion cell layer, in AII amacrine cells and in horizontal cells in the inner nuclear layer, and in fibers in the outer plexiform layer. However, there are some subtle differences between species in calretinin staining.

Fig. 1 shows calretinin-positive cells in the 40 μ m ver- tical sections of bat retina from a mid-peripheral region.

Calretinin-immunoreactive cell bodies are visible in the inner marginal inner nuclear layer and ganglion cell layer.

At the outer part of the inner plexiform layer, these cells show labeled processes though they are stained indis- tinctly.

In Fig. 2A, we can show most AII amacrine cells

labeled faintly and a clear stained cell in the ganglion

cell layer, which is a displaced amacrine cell (Fig. 2A

arrow), of the whole mount of bat retina from the mid- periphery. The results of the lack of axonal staining and this small darkly labeled cell in the ganglion cell layer suggest this cell is a displaced amacrine cell. In a similar previous study, displaced parvalbumin-immunoreactive amacrine cells are clearly distinguishable as small and more darkly labeled neurons in the ganglion cell layer of the rabbit retina

. We have also reported parvalbumin- immunoreactive displaced amacrine cells in previous study

.

In contrast, Fig. 2B shows well labeled cells in the inner nuclear layer of the whole mount of bat retina from the mid-periphery. These cell bodies create a regular mosaic as well. The labeled processes in the inner plexi- form layer and the regular mosaic of their cell bodies in the inner nuclear layer are characteristic of AII amacrine cells

. Calretinin has been used to label AII ama- crine cells in monkey retina

, rabbit retina

and pig retina

. Our results and the previous results in monkey, pig and rabbit suggest that the calretinin-labeled cells in the inner nuclear layer of the bat retina are AII amacrine cells.

Other cells in the inner nuclear layer and photoreceptor cell layer were negative for this antibody.

Fig. 3A shows calretinin-immunoreactive cell bodies in

the inner nuclear layer which are thought to be AII ama- crine cells and several ganglion cells in the ganglion cell layer. In Fig. 3B, parvalbumin immunoreactivity was found in ganglion cells in the ganglion cell layer and in AII amacrine cells in the inner nuclear layer.

Fig. 3C shows colocalization of the calretinin- and par- valbumin-immunoreactive cells in the inner nuclear layer and in the ganglion layer of the retina of the bat. In this figure, we can identify that almost all of the AII amacrire cells in the retina of the bat are colocalized in the inner nuclear layer by calretinin and parvalbumin immunoreac- tivity. However, only some ganglion cells in the ganglion cell layer are colocalized by calretinin and parvalbumin- immunoreactivity partly. That is, Calretinin-immunoreac- tive ganglion cells exist independently (Fig. 3A arrows, 3C).

Fig. 2. Calretinin-immunoreactive cells in the whole mount of bat retina from the mid-peripheral region (A) This micrograph shows calretinin immunoreactivity in the ganglion cell layer. Focus on the soma of the displaced amacrine in the ganglion cell layer (arrow). (B) Calretinin-positive AII amacrine cells reveal a regular mosaic. The focus is on cell bodies in the inner nuclear layer. Bar=20 μ m.

Fig. 1. Calretinin-immunoreactive cells in a 40 μ m vertical section of fluorescence-reacted bat retina. A low-power photomicrograph shows well labeled cells both at the lower inner nuclear layer and at the ganglion cell layer.

AII amacrine cells labeled in the inner nuclear layer and ganglion cells labeled in the ganglion cell layer are observed. opl, outer plexiform layer; inl, inner nuclear layer; ipl, inner plexiform layer; gcl, ganglion cell layer.

Bar=20 μ m.

This study suggests strongly that bat retina contain AII type of amacrine cells in the inner nuclear layer just as our previous study as to parvalbumin-immunoreactive AII amacrine in the retina of a greater horseshoe bat

. Ama- crine cells in the inner nuclear layer are labeled with cal- retinin antibody in all species studied so far

. Also, calretinin antibody labels AII amacrine cells in the mon- key, rabbit

, cat

, dog

and pig

retina. AII ama- crine cells are a distinctive type of narrow-field inter- neuron that have been described in a wide variety of Golgi-impregnated mammalian retinas, including the cat, dog, monkey, rat, rabbit, and wallaby

. They prove the pivotal link between the scotopic (pure rod) and photopic (pure cone) neuronal pathways in that they connect the rod bipolar cells with different types of cone bipolar cells. Recently, it was reported that AII amacrine cells limit scotopic acuity in central macaque retina

. AII amacrine cells are thus the third-order neurons in the rod pathway and are sometimes termed rod amacrine cells

.

In this study, strongly labeled cells in the inner nuclear layer of bat retina were also found. The cell bodies were distributed in regular mosaics and they were located in the inner marginal portion of the inner nuclear layer.

Even though they were not labeled clear, the processes of AII amacrine cells are shown as the bilaminar morpholo- gies of AII amacrine cells, with lobular appendages in sublamina a and arboreal dendrites in sublamina b of the inner plexiform layer. These properties are consistent with previous descriptions of AII amacrine cells

.

immunoreactive cells in the inner nuclear layer of bat ret- ina are AII amacrine cells and that these cells are immu- noreactive for parvalbumin at the same time.

Cells in the ganglion cell layer are also labeled with calretinin antibody in all species studied to date

.

Retinal ganglion cells collect visual information in the eyes and finally send it to the brain for visual perception.

At least 11 different types of retinal ganglion cells have been identified in rabbit retina using a combination of modern anatomical techniques

. More than 10 morpho- logically distinct ganglion cell types have been revealed in mouse retina, by the introduction of particle-mediated gene transfer

and by the expression of the gene encoding an alkaline phosphatase

.

Calretinin has been localized in the retina of chick

, pig, sheep, rat, cat, pigeon, salamander

, monkey

, lamprey

, dog

and rabbit

. Photoreceptors were not labeled with calretinin antibody in any species except in monkey

.

In our previous study, we reported the existence of well-organized parvalbumin-immunoreactive retinal gan- glion cells in the ganglion cell layer of the greater horse- shoe bats

.

Also, we can observe calretinin-immunoreactive gan- glion cells in the ganglion cell layer at the same material this time. But a part of parvalbumin-immunoreactive gan- glion cells colocalize calretinin positive ganglion cells in the ganglion cell layer of the bat retina. This observation demonstrates that non parvalbumin-immunoreactive but calretinin containing ganglion cells exist independently in the bat retina.

Conclusion

In this study, we identified the existence of calretinin- immunoreactive retinal AII amacrine cells in the inner nuclear layer and ganglion cells in the ganglion cell layer of the greater horseshoe bats. Out of previous and present data, these combined results of the existence of AII ama- crine and the subpopulation of calretinin-containing reti- nal ganglion cells strongly suggest that microbats not only rely on echolocation but also have functional eyes to help flight maneuverability.

And the discordance of localization in calretinin-immu- Fig. 3. Calretinin-immunoreactive cells (A), parvalbumin-

immunoreactive cells (B) and calretinin and parv-

albumin-immunoreactive cells (C) in a 40 μ m vertical

section of fluorescence-reacted bat retina. (A) Well

stained ganglion cells in the ganglion cell layer can be

seen but in microphotograph (B), there are unlabeled

cells in parvalbumin-immunoreactivity in the ganglion

cell layer (A, arrows). (C) Ganglion cells which are

labeled by calretinin but are not labeled by parvalbumin

are shown. Bar=20 μ m.

noreactive ganglion cells with parvalbumin-immunoreac- tive ganglion cells in the ganglion cell layer of the retina of greater horseshoe bats implies the existence of differ- ent function among ganglion cells in the retina of the bat and the specification among species as well.

Acknowledgements

This work was supported by the Korea Research Foun- dation Grant funded by the Korean Goverment(KRF- 2006-311-E00370).

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한국관박쥐망막에서 칼레티닌 면역반응성의 무축삭세포 및 신경절 세포에서의 관찰

전영기 *

** ·전창진 *

*경북대학교 자연과학대학 생물학과, **건동대학교 안경광학과 (2007년 8월 25일 받음, 2007년 9월 20일 수정본 받음)

Calretinin ^{의 생리적 역할은 아직 확립되지 않았지만} , ^칼슘 buffer ^{로서의 작용과 칼슘 중재의 신호 변환에 적극적}

으로 작용하고 있다 . Calretinin ^{은 인간의 망막에서 성체 광수용체 세포와 이극세포} , ^{수평세포에서 칼슘의 수송과 칼}

슘의 생리적 buffering 으로서의 역할을 하고 있다 . 본 연구에서 한국관 박쥐의 망막의 내핵층과 신경절세포층에서

calretinin 의 면역세포화학반응 신경세포들과 그 분포 양상을 조사하여 우리는 박쥐 망막에서 calretinin 에 면역세포

화학반응을 일으키는 AII 무축삭세포를 내핵층에서 그리고 신경절세포층에서는 신경절세포들을 확인할 수 있었다 .

이번의 관찰연구는 우리의 이전 연구와 더불어 박쥐 시력의 풀리지 않는 문제와 박쥐비행의 기동성의 독특한 행동

적인 측면들을 더욱 많이 이해하는데 필요하다고 생각한다 .

주제어: 박쥐 , ^분포 , ^{면역세포화학반응} , Calretinin, Parvalbumin, ^{무축삭세포} , ^{신경절세포}