Spatial density of catecholaminergic cells in the carp retina

Exp. Eye Res. (]980) 31, 711-719

Spatial Density of Catecholaminergic Cells in the Carp Retina K. NEGISHI, T. NAKAMURA AND W. HAYASHI

Departments of Neurophysiology and Biophysics, Neuroinformation Research Institute, University of Kanazawa School of Medicine, Kanazawa, Ishikawa 920, Japan (Received 19 June 1979 and in revisedform 11 April 1980, New York) Histochemical studies on the cell density of eateeholaminergie (CA-) cells were conducted with flat-mounted preparations of the carp (Cyprinus carpio) retina. Pretreatment of the fish with intramuscular injection of reserpine (R) completely depleted endogenous CA from the retina. CA-cell fluorescence was accentuated and the number of cells in which a positive reaction was obtained was considerably increased by intraperitoneal injection of 5-DOPA ("pDOPA" preparations) or by intravitreal injection of noradrenaline (NA) to reserpinized carps ("R§ preparations). The average CA-cell density was 52• 84• and 104• cells/ram2 in control, "pDOPA" and '~177 preparations, respectively. Differences in the spatial density were found to be statistically insignificant among the quartered areas with the optic disk as center, whereas they were significant between the central or intermediate zone and the peripheral zone. The spatial density in control retinas was 43• 48• and 68• cells/mm2 in the three concentric zones, respectively. The size of CA-cells tended to be smaller where the cell density was higher. Key words: carp retina; catecholaminergic cells; fluorescence microscope; reserpine; n-DOPA; noradrenaline; spatiel cell density; statistical comparison.

1. Introduction Since Falck (1962) established a fluorescence method for the cellular localization of monoamines, this method has been frequently used to study the retina (Ehinger, Falck and Laties, 1969; Boycott, Dowling, Fisher, Kolb and Laties, 1975; Dowling and Ehinger, 1975; Dowling, Ehinger and Hedden, 1976; Ehinger, 1976 ; Dowling and Ehinger, 1978a, b). Boycott et al. (1975) examined the mammalian retina by means of this method in combination with the Golgi-EM technique, and demonstrated the presence of catecholamine (CA)-containing amacrine-like cells in the inner border of the inner nuclear layer (INL) as well as a sparse distribution of such cells in the outer part of the inner plexiform layer (IPL). These cells extend their processes towards both the outer plexiform layer (OPL) and IPL in the fish and monkey retinas (Dowling and Ehinger, 1975). We confirmed the presence of CA-cells in the carp retina using a FGS fluorescence technique (Nakamura, 1979) which represents a modification of the Fa]gu method (Furness, Costa and Wilson, 1977), and pharmacologically manipulated cells in which a positive staining response was obtained (Negishi, Hayashi, Nakamura and Drujan, 1979). In the present study, similar pharmacological treatments of carps were applied, and the cell density of fluorescent cells was calculated in flat-mounted preparations of the whole retina. Differences in the cellular population were compared statistically in different areas of the retina.

2. Methods Kistochemical studies on catecholaminergic cells were conducted with fiat-mounted preparations of the carp (Cyprinus carpio) retina. The retinas were isolated from fish which had been dark-adapted for about 1 hr. A solution for tissue fixation (FGS solution) consisted of distilled water (25 ml at 60~ paraformaldehyde (2 g), 1 N-NaOH (2 drops), 0014-4835/80/120711+09 $01.00/o

9 1980 Academic Press Inc. (London)Limited 711

712

K. NEGISHI, T. NAKAMURA AND T. HAYASHI

0.2 m-phosphate buffer at pI-I 7.4 (24=ml), 20.o/0 glutaraldehyde (1.25 ml), and sucrose (2.5 g). The isolated retina was kept in a gently stirred volume of the fixation solution for 1 hr at 4~ Sucrose (5 g) was then added to the solution three times at 1 hr intervals, maintainiilg gentle stirring at 4=~ Following fixation the tissue was mounted, vitreous side facing upwards, on a deck glassplate, and the vitreous body was removed from the retinal tissue. The flat-mounted preparations were dried over Zeolite in a desiccator for 1-3 days, then covered with Entellan and examined under a fluorescent epi-illumination system (Olympus BR-RFL). The filters used consisted of a BG-3/IF-4=05 for emission and a Y-475 for absorption, Tissue prepared in this manner did not appear to show shrinkage that differed significantly from unfixed preparations. Most of the fish were pretreated with an i.m. injection of reserpine (3 mg/kg body weight) 20 hr prior to enucleation, which was followed by an i.v. injection of noradrenaline (NA) to one eye (20/~g in a 0.01 ml Ringer). These preparations are referred to as "1%" and "1%+NA" (the NA-injected side) preparations, respectively, in the text, table, and figure captions. Some carps were given i.p. injections of L-DOPA (5 mg/kg) 1 hr prior to enueleation; these are labeled by "pDOPA". Since the "R~-NA" preparations were found to show optimal accentuation of cellular fluorescence (Negishi et al., 1979) and the i.p. injection of L-DOPA was assumed to avoid possible damage to the retina caused by intravitreal injection, these two preparations were used in the present series of experiments. The number of fluorescent cells were counted in the flat-mounted preparations. Special consideration was given to the possibility that significant differences in the spatial cell density exist in different areas of the retina. 3. Results

Treatments with reserTine, N A or L-DOPA Figure 1 shows changes in cellular fluorescence and population caused by pharmacological treatments. All the photomicrographs were obtained from the intermediate zone of the retina. Micrograph (a) (control) was taken from the retina of an intact carp. When reserpine (3 mg/kg) was injected intramuscularly 20 bx before eye-removal, the retina of such carps ("R" preparation) did not show any fluorescent cells [Fig. l(b)], indicating that reserpine caused a complete depletion of endogenous CA. On the other hand, cellular fluorescence was accentuated and the number of cells in which a positive staining response was obtained was increased by intravitreal injection of NA (20/xg) to one eye of the reserpinized carps 3 hr prior to enucleation [ " R + N A " preparation; Fig. l(c)], or by intraperitoneal injection of L-DOPA ("pDOPA" preparation; Fig. 2).

Cell density in different retinal areas Fluorescent cells were counted in each photomicrograph covering an area of 0.12 mm2; the counts from 126-155 micrographs per tissue sample obtained from each group of preparations were summed to get the average cell density. It was found to be 51• 84• none and 104• cells/ram~ in the control (n = 6), "pDOPA" (8), " R " (19) and "R +NA" (19) preparations, respectively (Fig. 3). In order to determine whether significant differences exist in the cellular population in different regions of control preparations, the cell densities were calculated in different areas and statistically compared. The results obtained are shown in Fig. 4 and Table I(a). The large deviation ranges obtained indicate that the spatial arrangement of cells is quite irregular over the surface of a single retina and also between different retinas. No significant differences were found when the cell densities were compared

~

o

v

o

~

.~ ~ ~2

.~ o ~3

4~

~o

"

I~

~

~

9

~ee~ ~,~

~

~

~,.~ "~ ~

0

~ .

~

~.~

~'r~

CATECHOLAMINERGIC

CELLS IN THE CARP RETINA

715

between quartered areas of the retina using the students t-test [Fig. 4 and Table I(a)]. However, significant differences were obtained between the cell densities in the peripheral zone (p) and the central (c) or intermediate zone (i) [P < 0.005, respectively; Table I(a)]. In the latter case, the central zone covered a circular area defined by an arbitrarily set 2.5 mm radius with the optic disk as center, the intermediate zone covered an annulus of the inner radius of 2.5 mm and the outer radius of 5.5 mm, and the peripheral zone was the remainder outside the intermediate. The peripheral zone did not always contain all of the outer margin because of technical difficulties in the isolation procedure; this was one of responsible factors for the large standard devii~tion. When the retina was optimally prepared the diameter of the whole area was approximately 16 ram, the same value as before the tissue fixation. 14C

12C

t

I00

E E

I

80

60

40

20

0

Control

)DOPA

R

R+NA

(6)

(8)

(~9)

(~9)

FIo. 3. Histograms illustrating changes in the CA-cell density induced by pharmacological treatments. Average cell numbers (bars) per millimeter square (ram~) were obtained from each group of experiments. Cont, control; "pDOPA", with intraperitoneal injection of L-DOPA (1 hr before enueleation); "R", with intramuscular injection of reserpine (20 hr before); " R + N A " , with reserpine-injection followed by intravitreal injection of NA (3 hr before). Numbers in brackets are the number of retinas examined. 100

80 tif 1 o

to

~) 4 0

2O

ad

av

pv

pd

(28)

(28)

(30l

(30)

(38)

i

p

(4- I)

(47)

:FI~, 4. Histograms of the spatial density of CA-cells in control retinas. Bars represent the average cell densities in different regions, vertical lines ~he standard deviations and numbers in brackelbs are the numbers of photomicrographs examined. At the left, the average densities in the quartered areas (ad, dnterodorsal; av, antroventral; pv, posteroventral; pd, posterdorsal) with the optic disk as center. At the right, those in the concentric zones (v, central; i, intermediate; p, peripheral). See also Table I(a).

716

K. N E G I S H I , T. N A K A M U R A AND T. H A Y A S H I

The same comparisons were made with cell counts in the "pDOPA" and "R +NA" preparations. Differences were again found to be insignificant among the densities in the quartered areas, while they were significant in the concentric three zones [-Figs 5 and 6; Table I(b), (c)]. It was noticed that the size of cells tended to be smaller where the cell density is higher (Fig. 2), that cells with various sizes were considerably increased in number

i80 160 ~

120~ ~00

60 4O 20 C

ad

ov

pv

pj

(38)

(40)

(37)

(40)

(51)

(59)

(45)

Fro. 5. Histograms of the spatial density of CA-cells in the "pDOPA" preparation. See also Fig. 2 and Table I(b). 200

180 160 140 120 ~-~ I00

3

go 60 40 2O 0

od

(36)

,ov (56)

pv (41)

pd (37)

c (46)

i (44)

p (60)

FIG. 6. Histograms of the spatial density of CA-celts in the " R + N A " preparation. See also Fig. 1 and Tsble I(c),

CATECHOLAMINERGIC

CELLS

IN THE

717

CARP RETINA

TABLE I

Statistical comparison (with Student's t-test) of the cell densities in different preparations and areas of retinas (a) Control ad ad av pv pd

<0.50 <0.10 <0.10 v

av

<0.10 <0.10

pv

<0.50

ad av pv pd

<0.25 <0.50 <0.50

i

C

i

(b) " p D O P A " ad

av

pv

<0.50 <0.25

<0.50

c

i

<0'02* <0'001"

<0.001"

C

<0.20 <0.005*

i p

<0.005*

(e) "R+NA" ad av pv pd

ad

av

pv

<0.10 <0.10 <0.25

<0.50 <0.50

<0.50

c

i

<0.025* <0.001"

<0.005*

C

i p

* Significant difference.

by the treatments, and that many pairs of cells with various intercellular distances appeared in the treated preparations [for instance, as seen at the right-top and leftbottom corners of Fig. l(c)]. 4. Discussion

Uptake of L-DOPA was found to be non-specific to CA-cells in the fish retina (Negishi, Drujan and Laufer, 1980). Even after an intramuscular injection of L-DOPA (3 mg/kg), numerous amacrine and ganglion cells often became fluorescent in Certain estuary and marine fishes; this did not occur in the carp retina. On the other hand, uptake of DA or NA was found to be specific to CA-cells in various species of fishes (Negishi et al., 1980), because the number of fluorescent cells, revealed in flat-mounted preparations, was approximately consistent in a range of intravitreally injected DA or NA between 5 and 40/~g. Therefore, the cell density in the "R +NA" preparation in the present study is assumed to represent the actual cell population. The number of fluorescent cells in the "pDOPA" preparation did not surpass that in the "R +NA", indicating that L-DOPA appeared not to be taken up by cells other than CA-cells in the present study. The increase in the cell density of fluorescent cells caused by the pharmacological treatments may suggest that a half population of CA-cells cannot be detected in control retinas with the FGS method used, possibly due to the low content of endogenous DA. The average density of fluorescent cells was 52:k6 and 104• cells/mm2

718

K. N E G I S H I , T. N A K A M U R A

AND

T. H A Y A S H I

in the control and "R-~NA" preparations, respectively. Therefore, among so-called amacrine cells (104 cells/ram2; Tachibana, 1978), approximately 5-10/1000 cells were fluorescence-positive in the carp retina. Significant difference in the spatial density (cells/ram~) was found between the peripheral zone and the remaining (i.e. central and intermediate) regions (Table I). Furthermore, the size of CA-cells tended to be smaller where their numbers were larger, and cells with various sizes became fluorescent after the treatments of the fish (the "pDOPA" and "R +NA" preparations). Unfortunately, data concerning the spatial distribution of other classes of cells in the carp retina are not available at present for comparison with our findings. The spatial distribution of CA-cells was found to be similar in the retina of various species of fresh water and estuary fishes (Negishi et. al., 1980). The particular distribution pattern of CA-cells was opposite to that commonly described for ganglion cells in the retinas of various species of vertebrate, which have a fovea (Van Buren, 1963; Rodieck, 1973). Recen~ electrophysiological studies (Negishi and Drnjan, 1978, 1979a, b, c) carried out with the isolated fish retina revealed that DA applied in the perfusate (0.10.2 raM/1 for about 3 sec) augments horizontal cell response to focal illumination and attenuates the response to the surrounding area. The effects of DA are assumed to mimic the activity of CA-containing interplexiform cells, because these cells send processes towards the outer plexiform layer (OPL) forming dense fiber-networks surrounding horizontal cells, as described by Boycott et al. (1975) and DoMing and Ehinger (1975) and as confirmed in the carp retina by Negishi et al. (1979). Horizontal cells have been shown to possess a characteristic area-summation property of light-induced S-potential (Tomita, 1965; Naka and Rushton, 1967; Werblin and DoMing, 1969; Negishi and Sutija, 1969), and to participate in the lateral inhibition mechanism at the OPL (Svaetichin and Muriel, 1970; Hedden and DoMing, 1978; Naka, Davis and Chan, 1978). Our electrophysiological 6ndings (Negishi and Drujan, 1978, 1979a, b, e) may indicate 'that CA-cells modulate the lateral spread of S-potential along the horizontal cell layer. Therefore, the particular distribution pattern of CA-cells found in the present histofluorescence study might suggest an anatomical substrate for the visual processing of patterns or directional detection in the fish retina. ACKNOWLEDGMENTS

We express our great gratitude to Dr G. Rose (School of Social Sciences, University of California, Irvine) for his correction of English in the text, and to Drs T. Shoin, K. Hashimoto and K. Kawasaki (School of Medicine, University of Kanazawa) for their valuable suggestions in preparation of the manuscript. We also acknowledge Mrs Tami Urano for her secretarial help. Some of the fish used were supplied by the Kanazawa Acquarium (Director; Mr Y. Matsumoto). This work was supported in part by research grants from the Ministry of Education of Japan (Nos 144055 and 387043), the Japan Society for the Promotion of Science, and from the Takaoka-Johnan Hospital (Director; Dr J. Ishiguro). REFERENCES Boycott, B. B., DoMing, J. E., Fisher, S. K., Kolb, H. and Laties, A. M. (1975). Interp|exiform cells of the mammalian retina and their comparison with catecholamine-containing retinal cells. Prec. R. See. Lend. B. 191, 353-68. DoMing, J. E. and Ehinger, ]3. (1975). Synaptie organization of the amine-containing interplexiform cells of the goldfish and cebus monkey retinas. Science 188, 270-3.

CATECHOLAMINERGIC CELLS IN THE CARP R E T I N A

719

Dowling, J. E. and Ehinger, B. (1978a). Synaptic organization of the dopaminergic neurons in the rabbit retina. J. Comp. Neurol. 180, 203-20. Dowling, J. E. and Ehinger, B. (1978b). The interplexiform cell system I. Synapses of the dopaminergic neurons of the goldfish retina. Proc. R. Soc. Lond. B. 201, 7-26. Dowling, J. E., Ehinger, B. and Hedden, W. L. (1976). The interplexiform cell: A new type of retinal neuron. Invest. Ophthalmol. 15, 916-26. Ehinger, B. (1976). ]3iogenic monoamines as transmitters in the retina. In Transmitters in the Visual Process (Ed. Boating, S. L.). Pp. 145-63. Pergamon Press, Oxford, London. Ehinger, ]3., Falck, ]3. and Laties, A. M. 0969). Adrenergic neurons in teleost retina. Zeitschrift fi~r Zellforsehung 97, 285-97. Falck, ]3. (1962). Observations on the possibilities of the cellular localization of monoamines by a fluorescence method. Acta Physiol. Scand. 56 (Suppl. 197), 1-25. Furness, J. ]3., Costa, M. and Wilson, A. L. (1977). Waterstable fluorophores, produced by reaction with aldehyde solutions, for the histochemical localization of catechol and indolethylamines. Histochem. 52, 159-70. Hedden, W. L. and Dowling, J. E. (1978). The interplexiform cell system, i L Effects of dopamine on goldfish retinal cells. Proc. R. Soc. Lond. B 201, 27-55. l~aka, K.-I., Davis, W. and Chan, R. Y. (1978). Receptive field organizations in catfish retina. Sensory Processes 2, 366-74. l~aka, K,-I. and Rushton, W. A. H. (1967). The generation and spread of S-potentials in fish (Cyprinidae). J. Physiol. (London) 192, 437-61. lqakamura, T. (1979). Application of the Faglu method (Furness et al.) for the histochemical demonstration of catecholamines to the cryostat section method. Acta Histochem. Cytoehem. 12, 182. Negishi, K. and Drujan, B. D. (1978). Effects of catecholamines on the horizontal cell membrane potential in the fish retina. Sensory Processes 2, 388-95. Negishi, K. and Drujan, B. D. (1979a). Reciprocal changes in center and surrounding S-potentials of fish retina in response to dopamine. Neurochem. Res. 4, 313-18. Negishi, K. and Drujan, B. D. (1979b). Effects of catecholamines and related compounds on horizontal cells in the fish retina. J. Neurosci. Res. 4, 311-34. ~Ncgishi, K. and Drujan, ]3. D. (1979c). Similarities in effects of acethylcholine and dopamine on horizontal cells in the fish retina. J. Neurosci. Res. 4, 335-49. l~egishi, K., Drujan, ]3. D. and Laufer, M. (1980). Spatial distribution of catecholaminergic cells in the fish retina. J. iVeurosci. Res. 5 (in press). Negishi, K., ttayashi, T., Nakamura, T. and Drujan, B. D. (1979). Histochemical studies on catecholaminergic cells in the carp retina. Neurochem. Res. 4, 473-82. l~egishi, K. and Sutija, V. (1969). Lateral spread of light-induced potentials along different cell layers in the telcost retina. Vision Res. 9, 881-93. Rodieck, R. W. (1973). The Vertebrate Retina. Pp. 35945. W. H. Freeman and Company, San Francisco. Svaetichin, G. and Muriel, C. (1970). Funcion retiniana y control automatico. Rev. Oftal. Ven. 24, 41-70. Tachibana, M. (1978). Displaced ganglion cells in carp retina revealed by the horseradish peroxidase technique. Neurosci. Letters 9, 153-7. Tomita, T. (1965). Electrophysiological study of the mechanisms subserving color coding in the fish retina. Cold Spring Harb. Symp. Quant. Biol. 30, 559-66. Van Buren, J. M. (1963). The Retinal Ganglion Cell Layer. Pp. 60-4. Charles C. Thomas, Springfield, Illinois. Werblin, F. S. and Dowling, J. E. (1969). Organization of retina of the mudpuppy, Necturus maculosus. II. Intraeellular recording. J. 1Veurophysiol. 32, 339-55.