Role of target tissue in regulating the development of retinal ganglion cells in the albino rat: Effects of kainate lesions in the superior colliculus

THE JOURNAL OF COMPARATIVE NEUROLOGY 251:240-259 (1986)

Role of Target Tissue in Regulating the Development of Retinal Ganglion Cells in

the Albino Rat: Effects of Kainate Lesions in the Superior Colliculus

PAUL CARPENTER, ANN JERVIE SEFTON, BOGDAN DREHER,

Departments of Physiology, (P.C., A.J.S., W.-L.L.) and Anatomy (B.D.), University of Sydney, N.S.W. 2006, Australia

AND WEI-LENG LIM

ABSTRACT Kainic acid or ibotenic acid was injected unilaterally into the major

target regions of the axons of retinal ganglion cells-the superior colliculus (SC) or dorsal lateral geniculate nucleus (DLG)-of rat pups ranging in age from postnatal day 0 to postnatal day 10 (PO - P10). While the collicular or geniculate neurons within the injection site died within 48 hours of the injection, damage to axons and terminals of extrinsic origin within the injected region was not apparent. The neuronal degeneration induced by the neurotoxins, observed at both the light and electron microscopic levels, resembled the neuronal degeneration that occurs in the colliculus during normal development. Macrophages were identified in the regions containing degenerating cells.

Two to three weeks after the injections of neurotoxin, massive injections of the enzyme, horseradish peroxidase (HRP), were made into the retinorecipient nuclei. After about 24-hour survival time the numbers of retinal ganglion cells were estimated by counting the number of neurons containing HRP reaction products in sample areas distributed in a regular rectangular array across the entire retinal surface.

In the animals in which the neurotoxin was injected into the SC during the first 4 postnatal days, there was a substantial reduction (on average 41.5%; the range: 27.5-65.5%) in the normal number (mean value of 113,000- Potts et al.: Deu. Brain Res.3:481-486, ‘82) of retinal ganglion cells surviving the period of “naturally occurring ganglion cell death” in the retinae contralateral to the injected SC. By contrast, injections of neurotoxins into the DLG and/or the optic tract of newborn rats did not result in a significant reduction in the numbers of retinal ganglion cells surviving the period of naturally occurring ganglion cell death. The period of sensitivity of retinal ganglion cells to the injection of neurotoxin into the colliculi extends from birth to about the end of the first postnatal week; the greatest sensitivity seems to be restricted t o the first 3-4 postnatal days.

In the retinae in which the total number (and density) of ganglion cells was substantially reduced by the selective destruction of their target cells, the centroperipheral difference in the somal diameters of the ganglion cells (apparent in normal animals) was abolished, both amongst the whole population of ganglion cells and amongst the ganglion cells with the largest somata, relatively thick axons, and large-gauge primary dendrites (Class I cells). The number and distribution of the Class I cells in the depleted retinae were, however, unaltered. The abolition of the centro-peripheral difference in somal sizes results mainly from an increase in the somal size of the centrally located ganglion cells. Key words: kainic acid, cell death, neuronal degeneration, period of sensitivity

0 1986 ALAN R. LISS, INC. Accepted April 1,1986.

KAINATE LESIONS IN THE SUPERIOR COLLICULUS 241

During the development of the visual system of birds and out damaging axons of passage or axonal terminals of cells both marsupial and placental mammals, there is a substan- extrinsic to the injected region. Since it is crucial for the tial overproduction of retinal ganglion cells, many of which interpretation of these experiments to establish if indeed are subsequently lost during a period of naturally occurring the neurotoxins eliminate neurons located within the in- cell death (chick-Hughes and McLoon, '79; Rager and Ra- jected area but spare axons of cells extrinsic to the injected ger, '78; Rager, '80; Nurcombe and Bennett, '81; marsupial region, several control experiments were performed. Sefonix bruchyurus-Beazley and Dunlop, '83; Braekevelt Since our evidence indicated that indeed the neurotoxins et al., '86; rat-Lam et al., '82; Potts et al., '82; Dreher et specifically destroy cells but not axons of extrinsic origin or al., '83; Perry et al., '83 Jeffery, '84; Sefton and Lam, '84; terminals in the SC of the neonatal rat, we injected the Crespo et al., '85; cat-Ng and Stone, '82; Williams et al., neurotoxins into the SC of rats varying in age from new- '83; Chalupa and Williams, '84; monkey-Rakic and Riley, born to 10 days. At about 20 days of age, that is, well after '83; human-Provis et al., '85b). Several observations in the ganglion cell numbers normally stabilize (Potts et al., '82; rat suggest that the number of ganglion cells that survive Perry et al., '83), we made massive injections of the enzyme the period of "naturally occurring cell death" is critically horseradish peroxidase (HRP) into virtually all retinoreci- dependent on the amount of specific target tissue available pient nuclei and counted the number of HRP-labeled retinal during that period. First, retinal ganglion cells from neo- ganglion cells present. We were thus able to determine the natal rats dispersed in tissue culture survive longer in the effects on ganglion cells of the selective removal of neurons presence of the superior colliculus (SC) (to which over 90% in their major terminal site and to study the period of of retinal ganglion cells project-Linden and Perry, '83; sensitivity to target removal. Some of our findings have Dreher et al., '85) whereas non-retinorecipient nuclei (e.g., already been published in a short communication (Carpen- cerebellum) do not provide the same level of support (Mc- ter et al., '84).

METHODS CatTery et al., '82). Second, when transplanted to the SC of newborn rats, fetal ganglion cells innervate the retinorecipient laminae in the host animals and survive (McLoon and Injections of neurotoxins McLoon, '84) to develop the morphological characteristics Sprague-Dawley albino rats aged from birth (Po) to of all the classes of ganglion cells normally Seen (Perry et postnatal day 10 810) were injected with kainic acid or al., '85). Third, cells in fetal retinal explants 01: from disso- ibotenic acid. The rat pups were anesthetized with 0.5- ciated fetal retinae transplanted to visual cortex (Matthews 1.0% halothane in a nitrous oxide/oxygen mixture (65%/ and West, '82; Matthews et al., '82; McLoon and Lurid, '8% 35%) and then placed in a plaster mold (Lithgow and Barr, cerebellum (McLoon and Lund, '83, or dorsal column nuclei '82). The head was immobilized with dental impression in neonatal rats (McLoon et al., '83) fail to make Connec- compound and the skin overlying the skull was incised. tions with host nuclei and die. Collicular injections. In animals aged from PO to P6, a

The dependence of rat retinal ganglion cells on their piece of cartilage overlying the SC caudal to the transverse COlliCUlar t is also Suggested bY the demonstration sinus (readily visible through the translucent cartilagenous that the number of cells surviving the naturally occurring skull) was reflected. In elder animals, a piece of bone just period of ganglion cell death is substantially reduced if the rostra1 to the transverse sinus was reflected. A glass micro- SC is surgically removed within the first postnatal week pipette (tip diameter 30-50 pm) filled with a solution of (Perry and Cowey, '79a, %la, '82; Dreher et al., '83). How- either kainic acid (5 mg) or ibotenic acid (20 mg) dissolved ever, the interpretation of the loss of ganglion cells follow- in 1 m1 of physiological saline was inserted 400-500 pm ing the surgical removal of the sc postnatally is rather below the surface of the SC. The neurotoxin (250-500 nl, complex. Thus, since some axons Of retinal ganglion cells that is, 1.2-2.5 pg) was injected by pressure over 1-2 min- reach the sc by embryonic day 16 and the majority by the utes and the pipette was left in place for a further 5 minutes time of birth (Bunt et al., '83), the surgical procedure per- before withbawd. formed even in the newborn rat not only removes the target Gniculate and/or optic tract injections. In several ani- (collicular) cells but also severs retinocollicular axons. Be- mals aged from Po to p2 a piece of cartilage in front of the cause young neurons are particularly sensitive to damage transverse sinus was removed and the dura was reflected. of their axons (see Lieberman, '74; Allcutt et al., '84a,b), For optic tract (OT) injections a micropipette containing the reduction in the numbers of retinal ganglion cells after kainic acid (2 mg per ml) was inserted at a position 2.1-2.3 surgical ablation of the SC in the newborn rat may reflect rostra1 to the transverse suture and 1.8-1.9 mm lateral no more than their rapid retrograde degeneration following to the sagittal suture while for the dorsal lateral geniculate axonal damage. nucleus (DLG) injections the micropipettes were inserted in

In the present study we have attempted to elucidate the 1.8-1.9 mm rostra1 to the transverse suture and 1.9 mm mechanism(s) underlying the death of retinal ganglion ceIls lateral to the sagittal suture. OT injections were made at following the removal of their major target during the early 2.5-3.5 mm below the cortical surface. The DLG injections postnatal period. We therefore aimed to remove the collicu- were made 2.0-3.0 mm below the cortical surface. About lar target cells in neonatal rats without damaging retino- 200-300 nl was injected at a given depth over a period of collicular axons. 1-2 minutes. The micropipette was then moved up 0.2-0.3

In order to destroy the target selectively, we used either mm and again 200-300 nl was injected over 1-2 minutes. kainic acid or ibotenic acid, which have been shown specif- After several such injections interspaced by 0.2-0.3 mm the ically to destroy neurons in the brain of adult rodents (Coyle micropipette was left in place for about 5 minutes in order et al., '78; McGeer and McGeer '81; Coyle, '83; Coyle and to prevent any backflow of kainic acid. Altogether, about Kohler, '83; Kohler and Schwarcz, '83; Contestabile et al., 600-800 nl(l.2-1.6 pg) kainate was injected. The defect in '84), including those in the visual system of adult rats (sc- the skull was covered with a piece of paraan film and the Merker, '78; dorsal lateral geniculate nucleus-Woodward scalp was sutured. Animals were placed in an incubator (at and Coull, '82) and embryonic chicks (retina-Catsicas and 27°C) for about 1 hour to recover before they were returned Clarke, '84; Gibson and Rief-Lehrer, '84), apparently with- to their mothers.

242 P. CARPENTER ET AL.

Iniection of HRP into the eye and the DLG on both sides of the brain (Sherwood and 1

Timiras, '70). A 10-pl microsyringe filled with a 30-40% solution of HRP (grade I, lyophilized, Boehringer-Mann-

after a delay lasting from a few hours to 16 days, ~ o m e PUPS heim) in nis.cl buffer (o.l M, pH 7.6) containing 5% were re-anesthetized and placed again into the plaster mold. dimethyl sulphoxide was inserted into the target nuclei at

the appropriate depth. A large volume (1.25-1.5 pl) was An incision was made along the palpebral fissure to sepa- rate the eyelids and expose the cornea* A micropipette injected slowly Over a 20-minute period into each nucleus containing a 40-50% solution of HRP (grade I, lyophilized, (potts et al., ,82). Boehringer-Mannheim) in Tris/HCl buffer (0.1 M, pH 7.6)

Either immediately after the injection of neurotoxin or

containiig 5% dimethyl sulphoxide was carefully inserted into the vitreous humor bv uenetrating the sclera near the Tissue preparation for microscopy

" _ I

ora serrata just posterior to the iris. After an injection of 1- For light microscopy, the rats were anesthetized and per- 2 pl, the pipette was left in place for 5-10 minutes before fused through the heart with warm (37°C) Hartmann's withdrawal, in order to prevent leakage of HRP. solution, followed bv a fixative solution containing 0.5% -

paraformaldehyde, -1.25% glutaraldehyde in phoiphate buffer (0.1 M. DH 7.4): finallv the fixative was washed out Injection of HRP into retinorecipient nuclei

On P19 or later (Table l), rats that had been injected with with a' solutio> of 10% sucrose in phosphate buffer. For the neurotoxin were anesthetized and immobilized in the HRP histochemistry, tissue perfusion was performed about plaster mold as described above. By using a dental drill, 24 hours after the injection of HRP. The eyes and brains four small holes were made in the skull overlying the SC were dissected out and the brains were sectioned frozen at

TABLE 1. Numbers of Retinal Ganglion Cells Following Kainate Injections Into the SC, DLG, or Optic Tract

Day of Ipsi Contra % Difference Volume injection Day of count count % Difference std'd ipsihontra (postnatal) perfusion ( x 1,000) ( x 1,000) (contrdipsi) (1113,000) (%I

SC injections 0 21 97.5 82.5 -16 -27.5 90 0 19 94 64.5 -31.5 -43 87 0 23 114.5 68 -40.5 -40 59

Mean

Mean

Mean

Mean

Mean

Mean

0 C 0

2 2 2

3 3 3 3

5 5 5

7 7 7

10 10 10

24 23 21

19 23 20

20 22 22 22

21 20 20

21 21 23

33 21 20

DLG injections 0 44 0 22

Mean Optic tract injections

0 22 0 24

Mean

92 116 101.5 102.5

91 88 90 89.5

109

98 98

101.5

113 94

111 106

101.5 103 102 102

105 113 108 108.5

-

60 79 40.5 66

78 74 39 63.5

66 74.5 65.5 70 69

88 64.5 74 75.5

94 88.5 93.5 92

99.5 106.5 104.5 103.5

102.5 99 99 102

101 100.5

111 111 - 116

113.5

-34.5 - 32 -60 -35.5

- 14 -15.5 -56.5 -29

-39.5

-33 -28.5 - 32

-22 -31.5 -33.5 - 29

- 7.5 - 14 - 8 - 10

-5 -6 -3 -4.5

-3.5 +3 -0.5

0

-

-

-47 -30 - 64 -41.5

-31 - 34 -65.5 -44

-41.5 - 34 - 42 - 38 - 39

-22 -43 -34.5 -33

-17 -22 -17.5 -18.5

- 12 -6 -7.5 -9

-12.5 -9.5

-11

-2 +3 +0.5

35 33 0

51

80 67

74

90 74 65 41 68

69 63

66

77 63 40 60

74

-

-

- -

67 50 59

'SC, superior colliculus; DLG, dorsal lateral geniculate nucleus.


selected from the high (above 5,000 cells per mm2), medium (3,000-5,000 cells per mm2), and low (fewer than 3,000 cells per mm2) ganglion cell density regions of the control retinae, and every cell in each area was measured. Topograph- ically corresponding areas were selected in the retinae contralateral to the kainate injection or the surgical ablation, and again the soma1 diameter of ever cell in each area was measured.

Class I cells. Although, after massive injections of HRP into the optic tract and retinorecipient nuclei of both sides of the brain, virtually all the retinal ganglion cells are retrogradely labelled, the fine details of the dendritic morphology upon which the classification of retinal ganglion cells is largely based (see Dreher et al., '85) are not apparent for the majority of ganglion cells in such retinae. How- ever, Class I cells (Dreher et al., '85 cf. Type I cells of Perry, '791, even when their dendritic trees are not completely filled, can be easily distinguished from ganglion cells of other classes on the basis of their large somata (18-29 pm), characteristic three to seven large-gauge primary dendrites, and relatively large-gauge axons with a fairly con- stant intraretinal diameter of 0.6-1.2 pm (Dreher et al., '85). We mapped the location of all Class I cells in four pairs of retinae: in two animals in which kainate was injected into the SC and in two animals in which the SC had been ablated surgically. In the two animals in which kainate was injected into the SC, the perikaryal diameters of Class I cells selected from the three density regions were measured. Retinal maps and histograms of perikaryal diameter plotted by using an X-Y plotter interfaced with the microcomputer.

RESULTS Effective dose of neurotoxin

In adult central nervous tissue ibotenic acid has been reported to be less toxic than kainic acid and to produce more discrete lesions (Schwarcz et al., '79; Kohler and Schwarcz, '83). On the other hand, in immature (P7) rats, ibotenic acid injected into the hippocampus or striatum produces a complete loss of neuronal perikarya within the injected region, while injections of kainic acid do not seem to produce neuronal lesions (Steiner et al., '84). However, we were unable to discern any difference in the appearance or size of the lesion made by either toxin in the neonatal SC. Because of the need to use a larger dose and the much greater cost of ibotenate, we chose routinely to use kainic acid and found that it caused damage largely restricted to one SC when injected at a dose of 1.2-2.5 pg. Similarly, 1.2- 1.6 pg of kainic acid injected into the DLG caused substantial damage to the DLG (see further). However, as has been previously reported in studies on adult rats, the dose-response curve in the SC is very steep (Coyle, '78); in neo- nates, 4-5 pg usually proved fatal, while injections of amounts less than 1.0 pg did not seem to produce significant damage to the neonatal tissue. However, the dose of 3-5 pg was not fatal in rats 5 days (or more) old (G. Hors- burgh, personal communication).

Pathology of the lesion in SC Within 2 days of the kainate injections made in the period

PO-P2, the line of damage caused by the pipette can be readily identified; it is often associated with a considerable amount of bleeding (6. Kohler and Schwarcz, '83). As seen in Figures 1B and 3B, in the region surrounding the electrode track there is substantial oedema and disruption of

50 pm in the coronal plane. In animals that did not undergo HRP injections, perfusion was performed at various time intervals after the injections of neurotoxin; the brains were dissected out, sectioned coronally at 4-6 pm, and stained with either haematoxylin and eosin or cresyl violet. For electron microscopy, the animals were anesthetized and perfused with a weak and strong fixative solution (Peters, '70). The brain was dissected out, sectioned coronally at 100 pm with a Vibratome, osmicated, and embedded in Spurr's resin. Ultrathin sections were cut and, after staining with uranyl acetate and lead citrate, the grids were viewed in an electron microscope.

Retrograde labeling with HRP Alternate sections were either processed to demonstrate

the presence of HRP in the injection site by using the method of Hanker et al. ('77) or stained either with haema- toxylideosin or cresyl violet. The retinae of these animals were dissected out (Stone, '81) and then treated by the procedure of Leventhal('82) before being wholemounted on to glass slides.

Anterograde labeling with HRP Serial sections were treated with tetramethyl benzidine

as a chromogen (Mesulam, '82), mounted, and counterstained with neutral red.

Identification of macrophages Some rats were reanesthetized 48-72 hours after the

kainate lesion and perfused transcardially with Hart- mann's solution, followed by 10% formalin in physiological saline and then by 10% sucrose in phosphate buffer (pH 7.4). The spleen and parts of the brain including the injection site were dissected out, frozen in isopentane, and sectioned at 4-6 pm with a cryostat. These tissues were treated together by using an immunoperoxidase method (Lojda et al., '79) that identifies macrophages by the presence of endogenous myeloperoxidases or by using a modified esterase method @. Cutts, personal communication) that de- pends on the presence of endogenous nonspecific esterases.

Estimates of the amount of damage In order to estimate the amount of tissue in the SC or

DLG destroyed by the injection of the neurotoxin, cresyl- violet-stained sections from injected animals were inspected for signs of neuronal loss. Outlines of the SC and DLG were traced by using a drawing tube attached to the microscope; areas of adjacent sections were measured with a graphics tablet interfaced with a computer; and the volumes of the two sides were compared both in injected and in normal animals.

Wholemount analysis lbtal numbers. An outline drawing of each retina was

made on a graphics tablet interfaced with an Apple microcomputer at a magnification of 10 x with a microscope fitted with a camera lucida attachment. The total number of HRP-labeled cells was estimated by counting, with a 100 x oil-immersion objective, the number of cells in 200-550 areas (each 6,400 pm2) distributed across the entire retinal surface in a regular rectangular array; 6-11% of the retinal surface was sampled in this way.

Ganglion cell sizes. In selected retinae, the diameters of lucida system and graphics "mouse" interfaced with the microcomputer (Halasz and Martin, '84). Sample areas were


Fig. 1. Coronal sections through the superior colliculus (SC) following an injection of kainic acid. Sections in A,B, and E stained with cresyl violet. A. Forty-eight hours after a kainate injection; note the pale region (arrowed) from which neurons have disappeared. Scale bar, 0.2mm. B. Twenty-four hours after injection; note that many condensed and fragmented nuclei are present in the region of the injection site. Scale bar, 10 pm. Sections in C and D treated to demonstrate esterases and counterstained with haematoxylin, 72 hours after injection of kainate. C. A number of macrophages can be identified (double arrows). Scale bar, 30 pm. D. A higher-power view of

two macrophages (double arrows) adjacent to two pyknotic cells (single arrows). Scale bar, 10pm. E. Coronal section through the midbrain of a 3- week-old rat at the level a t which the injected SC (here shown on the left) reaches its maximum width. Note that the kainate-injected SC is substantially narrower than the noninjected SC on the other side. Arrows indicate the medial and lateral extent of the colliculi. Scale bar, 1 mm. F. Photograph of the base of the brain of a 3-week-old rat in which kainate was injected into the right SC on PO. Note the smaller right optic tract and left optic nerve (arrowed). Scale bar, 5 mm.

the cytoarchitecture. Cells in various stages of degenera- kainate injection (Fig. lA), the region surrounding the in- tion are prominent, exhibiting condensation of the nucleus, jection site is characteristically pale in sections stained an increase in basophilia and clumping of chromatin, and with cresyl violet, presumably due to a loss of cells; those pyknosis of the nucleus with or without fragmentation; cells remaining are in the later stages of degeneration and examples of phagocytosis are also apparent although there phagocytosis is prominent pig. 1B). Within 2448 hours of is no infiltration of neutrophils. About 48 hours after a the kainate injection, the SC contralateral to the injection

KAINATE LESIONS IN THE SUPERIOR COLLICULUS

appears to be very similar to normal, with only a few scattered degenerating cells present (6. the SC of newborn rats-Giordano et al., '80; hamsters-Finlay et al., '82).

Three days after the injection of kainate, macrophages identified by the presence of their characteristic esterases Fig. lC,D) and similar in appearance to those seen in the control tissue (spleen) are conspicuous in the lesion site. At that stage, many macrophages lie in close proximity to darkly pyknotic cells that they appear to be engulfing. The esterase technique also demonstrates the presence of macrophages in the ganglion cell layer of the retina during the first 5 postnatal days. On the other hand, with the technique that identifies macrophages by the presence of myeloperoxidase enzymes we failed to stain the macrophages in either colliculus or retina, although abundant examples were found in the control tissue (spleen). This negative result suggests that the myeloperoxidase might be insuffi- ciently reactive or present in too low a concentration in brain macrophages for its detection.

245

Pathology of the lesions in DLG andfor OT As in the case of collicular injections, during the first 2

days after kainate injections into the DLG and/or OT a considerable amount of disruption and tissue damage as well as signs of neuronal degeneration in the form of massive numbers of pyknotic cells were apparent (see Fig. 2A,B). Similarly, in the case of the injections aimed at parts of the OT located ventrally and rostrally to the DLG, the bleeding, oedema, and unequivocal signs of neuronal degeneration (in the form of massive numbers of pyknotic cells in the structures within the injected areas) were also apparent.

Injection sites 3 weeks after kainate injections By the time of the HRP injections at P19 or later, the

lesions of the SC, DLG, and the structures around the more ventrorostral injections aimed at the OT have been re- solved. However, at least some disruption of the normal cytoarchitecture was apparent. At the same time, rostrally to the injected SC, both the pretectum and the DLG appeared normal. Similarly, in both cases of the DLG lesions, the lateral posterior nucleus (LP) and the ventral geniculate nucleus (VLG) were apparently not affected Fig. 2C). Since in every case the injected SC was clearly smaller both rostrocaudally and mediolaterally than its uninjected counterpart (see Figs. IE, 4A), we have estimated the extent of the lesion in each animal by comparing the volume of the injected SC or DLG to the volume of its uninjected counterpart. In normal, uninjected animals, we have found the voIumes of the SC and DLG on opposite sides of the brain are virtually identical, with differences not exceeding 5%. However, our estimates of the volumes of the injected SC and DLG do not necessarily reflect the total amount of tissue damaged, because patches of tissue depleted of neurons are sometimes present (see Figs. 1A,2A,C). The volumes of the kainate-injected DLGs were reduced to 50-65% of its contralateral counterparts that had not been injected with kainate (see Table 1, Fig. 4C). The assessment of the amount of damage tissue around the parts of the OT located rostra1 to and below the DLG was not attempted but the disruption of the cytoarchitecture of these structures located within the injected sites was unequivocal in both cases presented in the study (Fig. 4B).

Fig. 2. Coronal sections through dorsal lateral geniculate nucleus (DLG) following injections of kainate on PO, stained with cresyl violet. A. After 30 hours, the injection site within DLG is paler than the surrounding normal tissue. Scale bar, 100 pm. B. Higher-power view of the area outlined in A showing pyknotic nuclei and degenerating cells; unaffected cells can be seen at bottom left. Scale bar, 15 pm. C. Patch depleted of neurons in the DLG of a rat perfused on P37 after an injection of kainic acid on PO. Scale bar, 0.2 mm. VLG, ventral lateral geniculate nucleus.

Survival of axons Several control experiments demonstrated the presence

in the injected colliculus of axons and terminals normal in appearance and capable of transporting HRP in an anterograde direction. Thus, in animals in which HRP was in-


jected into the eye 1-16 days after the kainate injection, direction are present adjacent to the needle track (Fig. 3B) abundant labeled terminals of retinocollicular axons are as well as rostrally and caudally throughout the SC (Fig. apparent in the upper layers of the contralateral, kainate- 2C), resembling those seen in normal animals of the same injected SC (Fig. 3B-D). In particular, 1 day after the kain- age (Fig. 3A). Similarly, abundant labeled terminals are ate injection, axons and terminals labeled in anterograde present 18 days after the kainate injection (Fig. 3D). Sec-

Fig. 3. A-E. Coronal sections through the SC, 24 hours after an injection of HRP into the contralateral eye; treated with tetramethyl benzidine and counterstained with neutral red. Note the presence of dense HRP-filled terminals in the retinorecipient layers demonstrating the anterograde transport of HRP. Scale bars, 400 pm. A. Normal 3-day-old rat. B. Three- day-old rat, 24 hours after an injection of kainate into the SC. Note that although oedema and bleeding are apparent, many filled terminals are present in the region of, and adjacent to, the lesion. C. More caudal section through the SC of the animal illustrated in B. Note that terminals are

abundant. D. Densely labeled retinocollicular terminals are present 18 days after the injection of kainate into SC on P2. E. Two-day-old rat; section through the SC 24 hours after an injection of kainate into the optic tract on PO (see Fig. 4C). Note that labeled terminals are abundant. F, G. Electron micrographs through the region of the optic stratum of the superior colliculus 24 hours after a kainate injection made on P2. Axons, normal in appearance, are present in large numbers. F. Many axons can be seen, close to erythrocytes (dark profiles, arrowedf and profiles containing condensed cytoplasm. Scale bar, 1 Fm. G. At higher magnification. Scale bar, 0.5 pm.


Contralateral A SC

lpsilateral

b Rostral Bregma -5.6

G V

Caudal -u Bregma -7.9

1.0 mm

6 OPTICTRACT Rostral

u 1.0 mm

c DLG Rostral

lpsilateral Contralateral

ondly, after kainate injections into the ipsilateral optic tract (see Fig. 4C) and DLG, labeled retinal terminals are present in the upper (retinorecipient) layers of the SC following injections of HRP into the contralateral eye (Fig. 3E). Thirdly, the electron micrographs of material prepared from the injection site 24 hours after the kainate injection illus- trate the presence of many typically nonmyelinated axons, similar in appearance to axons in the optic nerve of normal animals of the same age (Lam et al., '82; Perry et al., '83; Sefton and Lam, 84; Crespo et al., '85; see Fig. 3F,G). Con- sistent with the light microscopic observations, the electron micrographs reveal neurons with conspicuous degenerative changes, including condensation of their cytoplasm, close t o the apparently intact axons. Erythrocytes can also be identified (Fig. 3F). Although it cannot be firmly concluded that the intact axons seen in electron micrographs are indeed of retinal origin, there is evidence to suggest that they are: (1) the axons are located in the optic stratum in which the retinocollicular axons are normally found; (2) the other major projection to the retinorecipient layers of the SC, that from the ipsilateral visual cortex, has not at this stage reached the retinorecipient collicular laminae (Thong and Dreher, '86).

'""1 Q

"1 --*

1 3 5 7 9 11 19 2 1 23 Adult Age (Postnatal D a y s )

Fig. 5. Plot of the estimated number of ganglion cells in the retinae of albino and hooded rats of different ages. Open circles: estimated numbers of ganglion cells in the retinae of normal albino rats at different ages (from Potts et al., '82). Open triangle: the estimated numbers in normal Manch- ester hooded rats (unpublished data of R.A. Potts and B. Dreher). Black circles: estimated numbers of ganglion cells in retinae of albino rats contralateral to the SC injected with kainic acid during the first 5 postnatal days (the period of naturally occurring cell death). Black triangles: estimated numbers of retinal ganglion cells in albino animals in which the contralat- era1 DLG or optic tract has been injected with kainic acid on PO. Note that in the animals in which the SC was injected with kainate the numbers of retinal ganglion cells are substantially below those. estimated in normal albino and hooded animals of the same age. Note also the normal numbers of ganglion cells in the retinae contralateral to the kainate injected DLG and/or optic tract.

Fig. 4. Reconstructions from camera lucida tracings of coronal sections of tissue damaged by kainate injections. The sections were stained with cresyl violet. The locations of the sections are indicated with reference to the coordinates of the atlas of Paxinos and Watson, '82). A. The SC illustrated on the left was injected with kainate on P3. The animal was perfused on P22. The injected colliculus is 41% of the volume of the noninjected SC illlustrated on the right. B. Tracings of two sections of the brain of a P1 rat, treated with tetramethyl benzidine and counterstained with neutral red. On PO, kainate was injected into the vicinity of the optic tract and HRP was injected into the contralateral eye. The kainate injection site (cross-hatched) lies in the trajectory of the optic tract fibres that are heavily labeled with HRP reaction product. Abundant labeled terminals are present in the DLG (stippled) and SC (see Fig. 2E). C. The DLG, injected with kainate on PO. The animal was perfused on P22. The injected nucleus (left) is 50% of the volume of its noninjected counterpart (right).

Caudal


labeled retinal ganglion cells in the retinae ipsilateral to the kainate injection were also 9-12.5% lower than the mean control value (Table 1).

Timing of the lesion. Figure 6 illustrates the reduction in the number of gan-

glion cells, relative to the normal stabilized value of about 113,000, following an injection of kainate at different postnatal ages. Considering first the retinae with the maximal loss at each age, the kainate injection was clearly most effective in reducing the number of ganglion cells surviving the period of naturally occurring cell death if performed within the first 2 postnatal days (maximal loss -65.5%). By contrast, an injection at P10 caused only a very small loss of ganglion cells (6-12%, Table 1). However, even if the maximum loss at a given age is not regarded as the most significant feature, Figure 6 demonstrates that between P3 and P10 there is a clear-cut decline in the sensitivity of the ganglion cells to a reduction in size of their collicular targets. Thus, the mean losses of ganglion cells (relative to the control value of 113,000) in retinae contralateral to the SC injected with kainate on PO, P2, and P3 were, respectively, 41.9% (SD f 12%, n = 61, 43.5% (SD f 15.6%, n = 31, and 38.9% (SD +_ 3.2%, n = 41, whereas the mean losses of ganglion cells in animals injected on P5, P7, and P10 were, respectively, 33.2% (SD f 8.6%, n = 31, 18.9% (SD k 2.3%, n = 31, and 8.5% (SD k 2.6%, n = 3).

Ganglion cell numbers AtZer kainate injections into superior colliculus.

Reference to Table 1 indicates that there is only a weak correlation between the reduction in volume of the injected SC relative t o its uninjected counterpart and the final reduction in the numbers of ganglion cells in the contralateral retina. The correlation might, however, be partially obscured by the fact that some injections probably spread across the midline, reducing the size of the opposite SC (see below). It should also be noted that although there were substantial reductions in the volume of the SC in animals injected on P7 or P10, the numbers of ganglion cells in those animals were close to normal. However, in the animal with a complete collicular lesion, with no trace of the superficial layers of the injected SC, only 40,500 ganglion cells remained in the contralateral retina.

Since over 97% of ganglion cells in the albino rat project contralaterally (Jeffery, ’84; Dreher et al. ’851, the bulk of the loss of ganglion cells would be expected to occur in the retina contralateral to the kainate lesion. Due to the small size of the ipsilateral projection, in cases in which the kainate has not spread across the midline, the retina ipsilateral to the injection should serve as a control. In all 21 animals in which both retinae were available for study, fewer HRP- labeled cells were present in the retina contralateral to the kainate-injected SC than in the ipsilateral (control) retina (see Table 1, Fig. 5). Since in 12 of the 21 animals the number of ganglion cells in the retinae ipsilateral to the kainate injections lay within 10% of the normal control value (113,000-Potts et al., ’82; Perry et al., ’83; see Fig. 51, we conclude that in these 12 rats the kainate injections were essentially restricted to one side. In the remaining nine animals in which the number of ganglion cells in the ipsilateral retina was counted, the number was 13-22% of the control value. The reduction in numbers in the “control” retinae could be a result of some spread of kainate across the midline. When the counts in both retinae are compared in each animal, it is evident that the greatest reductions in the number of cells in the retina contralateral to the kainate-injected SC are 55-60%. The loss when compared with the mean number of retinal ganglion cells present in normal adult animals is even higher-about 65%; indeed, a reduction of that magnitude is reflected by an obvious thinning of the optic nerve contralateral to, and optic tract ipsilateral to, the injected SC (see Fig. 1F).

In those animals in which the numbers of ganglion cells were most dramatically reduced, the density of ganglion cells was reduced right across the retina. In the remaining animals, the density of ganglion cells was normal in some parts of the retina, while in other retinal areas, presumably corresponding retinotopically to the region of spread of the kainate injected into the SC, the density of surviving ganglion cells was reduced.

M e r kainate injections into DLG and/or optic tract. In the retinae of two animals in which kainic acid had been injected into optic tract (see Fig. 4B,C), the numbers of labeled retinal ganglion cells following massive injections of HRP into virtually all retinorecipient nuclei were within 3% of those in normal adult animals (Table 1, Fig. 5). By contrast, the numbers of ganglion cells in retinae contralateral to the DLG injected with kainate in two animals were 9.5% and 12.5% lower than the mean value for normal

Soma1 sizes of retinal ganglion cells following the kainate lesion of SC

We measured the sizes of ganglion cells located in closely corresponding areas of the retinae of the two animals in which there was the greatest reduction in ganglion cell numbers, with only 39,000 and 40,500 ganglion cells remaining in the retinae contralateral to the kainate-injected SC. The results are illustrated in Figure 7. As has been reported for normal retinae (Fukuda, ’77; Schober and Gruschka, ’77; Dreher et al., ’84), in the retinae ipsilateral to the kainate injection, the somata of ganglion cells located in the high-density region (the “area centralis”) are smaller than those located in the low-density (peripheral) regions. By contrast, in the retinae contralateral to the kainate injection, as in the retina contralateral to a surgically ablated SC (Dreher et al., ’84), the centro-peripheral gradient is not apparent: that is, the mean somal size in the high- and medium-density regions is equal to, or is even slightly greater than, the value normally found in the low- density regions. In all but one case, the mean size of the ganglion cell somata in the retinae contralateral to the kainate injection is significantly greater than that of ganglion cells from the corresponding region in the ipsilateral (control) retina (P < .01, Kruskal-Wallis test). In the case in which no significant difference was found (comparing somal sizes in the lower temporal regions), the kainate injection was probably not completely restricted to one side, since the number of cells in the ipsilateral retina was reduced to 90,000-that is, about 20% below mean control values. It is possible that cells in the lower temporal region of the ipsilateral retina that project to the SC on both sides of the brain (Cowey and Franzini, ’79) had been particularly affected by the spead of kainate across the midline. Support for this suggestion comes from the fact that the somal

animals. However, in both those animals the numbers of diameters i f t he cells in the lower temporal region of the


7 0- 0

6 0-

5 0-

0 40-

8

* O 1

0

0 r)

O ! I

0 1 1 3 4 !i B -). Q Q 1 ' 0 1 ' 1 I

Postnatal Day of Injection

N

N

CONTRALATERAL IPSILATERAL

Fig. 6. The effect of the age at which the kainate was injected into the superior colliculus on the loss of ganglion cells from the contralateral retina. Above: Graph illustrating the reduction in the number of retinal ganglion cells as a percentage of the normal adult number following injections at different ages. Note that the greatest losses occurred after injections made during the PO-P3 period and that injections made after the first postnatal week had little or no effect. Note also that the curve would be similar if graphed as the percentage of the mean of all ipsilateral (control) counts made in these experiments (102,000). Below: Two pairs of retinae illustrat-

ing the distribution and density of retinal ganglion cells following a kainate injection on P2 (above) and P10 below). Cells were counted in a regular array across the retina in an area of 6,400 Fm2. The diameter of each spot corresponds t o the number of cells in each area. Note that following an injection on P2 the number of cells in the contralateral retina is considerably reduced, and that (unlike in the control ipsilateral retina) they are fairly evenly distributed Following an injection at P10, there is little difference evident between the retinae. L, lower; N, nasal; T, temporal; U, upper.

A HIGH DENSITY n v - IPS1

V - CONTRA I -. . .k. -. c 10 0

CI, C

.- - m a 0

- . . . . . . - . - - . - . - . - -~-- . - - - 5 10 15 20 25 Soma diameter ( pm Soma diameter ( p m 1

B CONTRALATERAL IPSILATERAL c ' -CENTRAL (HIGH) n

Q) 0

5 10 15 20 25 Soma diameter ( p m )

C CONTRALATERAL

Q

5 10 15 20 25 Soma diameter ( p m 1

IPSILATERAL

Figure 7


Fig. 8. Photomicrographs of HRP-labeled cells in the corresponding regions of retinae ipsilateral (A,C) and contralateral (B,D) to the SC injected with kainate at PO. The largest cell in each photograph is a typical Class I cell; note the large samata and thick primary dendrites. Scale bar, 20 pm. A. Central region of the retina, ipsilateral to the injection site (control). Note the high density of the ganglion cells, and the relatively small soma size of the Class I cell when compared with the other samples illustrated. B. Retina contralateral to the kainate-injected SC, central region, the optic disk lies in the upper left-hand corner. Note the reduced density of the cells and their larger diameter. C. Retina, ipsilateral to the kainate injected SC, peripheral (low density) region. D. Retina, contralateral to the kainate injected SC, peripheral region.

Fig. 7. Diameters of the somata of ganglion cells from different retinal locations. A. Histograms of the distributions of the somal diameters in the retinae ipsilateral (control, thin line) and contralateral (thick line) to the SC injected with kainate on P2. Cells from the higli-density region (above 5,000 cells per mm'), medium-density region (3,000-5,000 cells per mm'), together with cells from nasal and temporal areas of low-density regions (below 3,000 cells per mm2) from the control retina and from topographically corresponding regions of the retina contralateral to the kainate injection are shown separately. The mean somal sizes for cells in the contralateral and ipsilateral retinae are indicated, respectively, by black and white arrowheads. Note that the ganglion cells in the ipsilateral (control) retina (with the exception of those in the temporal region-see text) are smaller than those in the contralateral retina. B. Histograms of the distribution of the somal diameters in the high and low ganglion cell density regions (nasal and temporal combined). Note that in the retina contralateral to the kainate injection the centro-peripheral difference in ganglion cell somal diameters seen in the ipsilateral retina is no longer apparent. C. Drawings of wholemounted retinae of a rat injected with kainate on P2. HRP was injected into the retinorecipient nuclei on P19 and the rat was perfused on P20. The total numbers of labeled retinal ganglion cells are 39,000 in the retina contralat- era1 to the kainate injected SC and 90,000 in the ipsilateral retina. Isodens- ity lines (5,000, 3,000, and 1,000 cells per mm2) have been indicated on the control retina (ipsilateral, shown on the right). The squares indicate the regions sampled in both retinae; the diameter of every cell in each square

ipsilateral retina are apparently significantly larger than those for similarly located cells in the other control retina in which the injection appeared to be confined to one side.

Class I retinal ganglion after a kainate or surgical lesion of SC

Following removal of their major target, the cells likely to survive would be those with sustaining collaterals projecting to other retinorecipient nuclei. Prominent amongst the retinal ganglion cells that branch to innervate SC and DLG are the Class I cells (Type I in the terminology of Perry, '79). These cells, with their large somata, three to seven thick primary dendrites, and relatively thick axon, can readily be identified in well-labeled HRP material (Dreher et al., '85). Accordingly, the locations of all clearly identifiable Class I cells (illustrated in Fig. 8) were mapped in the two pairs of retinae which, after kainate lesions of the SC, revealed the greatest reduction in total numbers of contralaterally projecting ganglion cells (40,500 and 39,000); the maps of Class I cells in both retinae of one of these rats are illustrated in Figure 9. As seen in Table 2, the values for the number of Class I cells lie within the range reported for the population of Class I cells in normal adult albino rats, and as in normal rats, Class I cells are relatively evenly distributed (Dreher et al., '85). Also shown in Table 2 are the numbers of Class I cells found in two pairs of retinae from rats that had undergone a surgical lesion of the SC at birth; these totals also lie within the normal range (Dreher et al., '85).

The somal diameters of Class I cells were measured in the two Dairs of retinae from the kainate-lesioned animals.

was measured. N, nasal; T, temporal; U, upper. Cells from the high-, medium-, and low-density regions of


CONTRALATERAL IPSILATERAL

A

6 HIGH DENSITY MEDIUM DENSITY

LOW DENSITY (TEMPORAL) L O W DENSITY (NASAL) - c c

8 F

-20 - 9

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al u - -

I - = 10 0 - I (3r

d - W

15 20 25 30 Soma diameter ( u m )

15 20 25 30 Soma diameter ( p m 1

n CONTRALATERAL IPSlL A T ER A L r c

E -CENTRAL (HIGH) v 'PERIPHERAL 7

I

I

15 20 25 30 15 20 25 30 Soma diameter ( p m ) Soma diameter ( p m )

Fig. 9. Distribution and somal sizes of Class I cells in the retinae of a rat in which kainate was injected into one superior colliculus on P2. A. Map of morphologically identified Class I cells across the flat-mounted retina; each cell is represented by a dot. Temporal edges of the retinae lie niedially. Total cell numbers were 39,000 in the contralateral retina and 90,000 in the retina ipsilateral to the kainate-injected colliculus (Fig. 7 0 . Note the similarity in the distribution and numbers of Class I cells (653 in the contralat- era1 retina which had 15% of the retinal surface obscured to the extent that Class I cells could not be identified and counted; 806 in the ipsilateral retina). B. Histograms of the somal diameters of Class I cells sampled from

the high, medium, and low ganglion cell density regions (see Fig. 7). Note that in each case the mean somal diameter of Class I cells (indicated by white and hlack arrowheads) is smaller in the ipsilateral (control) retina, and that the greatest difference is seen between the high-density regions. C. Histograms of the distribution of somal diameters of Class I cells in the central regions (normally high density, thin line) and peripheral regions, both nasal and temporal (normally low density, thick line). Note that the centro-peripheral difference apparent in the ipsilateral retina (on the right) is not apparent in the contralateral retina (left). N, nasal; T, temporal; U, upper.


TABLE 2. Numbers of Class I Retinal Ganglion Cells After Kainate Injection and Surgical Ablation of the SC at Birth

253

brain to the toxic effects of ibotenic acid have also been reported (Steiner et al., '84). By contrast, our data indicate that neurons in the SC of the rat, although morphologically immature (Warton and Jones, '851, have already developed

of cells Class I cells of cells Class I cells their sensitivity to kainic acid (and presumably the appro- in contra Ipsi in contra Ipsi priate binding sites) by the time of birth. Thus, it appears

retina kontrol) Contra retina (control) Contra that the neurons in the sc and DLG of the rat become 40,500 793 865 83,000 753 810 sensitive to the neurotoxic effects of kainic acid earlier than 39,000 806 653l 73,000 770 759 those in the striatum, hippocampus, or cerebellum. How-

not be identified and therefore counted. was required to produce an effective lesion of the SC and DLG in neonatal rats than the doses required to destroy

the retinae ipsilateral to the kainate injection a& in COT- the sc in adult rodents (0.5-2 pg, Merker, '78). The lowest responding in the contralateral retinae were aria- effective doses reported for adult striatum are even lower lyzed separately, and the results are presented in Figure 9. (0.5 CLg, %kmlhde et al.. '78; 0.4 pg, Divac et al., '78). It Thus, in the two retinae ipsilateral to the kainate lesion would be interesting in this context to determine the time (controls), as has been previously reported (Dreher et al., COurse of development of kainic acid binding sites and the '85), the somal diameters of class I ganglion cells in the sensitivity to the neurotoxic effects of kainic acid in differ- high-density region are smaller (16.5k2.6 pm and 19.8k2.2 ent Parts ofthe brain SO as to relate them to the morpholog- pm, respectively, for each retina) than those located within ical stages Of

the medium-density regions (respectively, 19.9k1.8 pm and The finding of labeled retinocollicular axons and termi- 22.3k1.6 pm), with those located in the low-density regions nals many days after an injection of kainate into the SC, having the largest somata (respectively, 21.4k2.5 pm and DLG, or optic tract, taken in conjunction with the electron 23.6k1.7 pm). These differences are significant (p< .001 in microscopic observations of the presence of intact axons, each case, Kruskal-Wallis test). By contrast, in the retinae suggests that in irf-mature rats ( a ~ previously claimed for contalateral to the kainate injection, the soma1 diameters the Of whose somata lie outside the of Class I cells (21.3k2.3 pm and 21.1k2.0 pm respectively, injected retinorecipient region are indeed spared while the for each retina) located in tbe central region of the retina Cells located within the injected region degenerate. Simi- (corresponding to the high&nsity region in normal reti- larly, ibotenic acid injected into the brain of adult rats or nae) are very similar to those (22.6k2.1 pm and 21.5k1.8 into the hiPPocmPus ofP7 rats appears to destroy Only the pm, respectively, for each retina) of cells located in the somata Of neurons located within the injected region. Fur- peripheral regions (corresponding to the low-density region thermore, in the present study, kainic acid injections made in normal retinae); thus, the differences in somal size of into the optic tract on Po that did not involve either the sc Class I cells normally present between the regions have O r DLG did not cause any reduction in the m d ~ r of been abolished (Fig. 9). ganglion cells surviving the period of naturally occurring

ganglion cell death. Surprisingly, ibotenate injected into the striatum of P7 rats appears to destroy not only the neuronal somata lying within the injection site but also

DISCUSSION axons and axonal terminals originating from regions ex-

Neurotoxic effects of kainic acid in neonatal trinsic to the injected region (Steiner et al., '84). It is possible, however, that the death of axons and axonal terminals originating from the regions extrinsic to the injected re- animals

Clear-cut effects of kainic acid injections have been pre- gions is related to the death of cells whose targets have viously described for two retinorecipient nuclei of adult been destroyed by the ibotenate injections (see further). rodents. the SC Merker, '78) and DLG (Woodward and Since the remaining retinocollicular axons and terminals Coull, '82). The results reported here indicate that kainic are abundant despite a considerable reduction in the num- (and ibotenic) acids, when injected into the SC or DLG of ber of ganglion cells present in the retina, it appears that neonatal animals, are similarly neurotoxic, destroying cells they may represent branches of axom that have secured in the vicinity of the injection site without apparently dam- synaptic connections with other nuclei of the visual system aging afferent axonal terminals or axon6 passing nearby. (see below). Furthermore, in P18 animals in which kainate While in the striatum of adult rats larger neurons appear was injected into the SC at PO, the plexus of retinal termi- to be more resistant to kainate (Kohler and Schwarcz, '831, nals in the SC appears to be denser than that seen in we found no evidence of any differential effect on the rather younger animals. This observation suggests that either the uniformly sized population of immature neurons present in anterograde transport of HRP is more effective in older the SC early in postnatal life (6. Warton and Jones, '85). animals, andor, as has been previously reported (Goodman

It is interesting to note in this context that sensitivity to et al., '73; Schneider, '73, '81), terminals of surviving reti- the neurotoxic effects of kainate in the retina of the chick nal ganglion cells that lose their normal targets in the SC varies with age (Catsicas and Clarke, '841, becoming mani- sprout, perhaps to innervate intact parts of the SC includ- fest only from embryonic day 8 onward (Gibson and Rief- ing deeper laminae (Perry and Cowey, '79b). Indeed, intra- Lehrer, '84)-that is, about the time at which kainic acid cerebroventricular or intravenous injections of kainic acid binding sites become functional. Similarly, in rats younger in rats induce sprouting of mossy fibers in the hippocampal than P7, the striatum, hippocampus, and cerebellum are formation (Nadler et al., '80). Similarly, in the retinae of virtually immune to the toxic effects of kainate (Coyle, '78; adult cats and rabbits, intravitreal injections of small doses Campochiaro and Coyle, '781, and differences in the onset of kainic acid induced sprouting of the processes of the A of sensitivity of the different regions of the immature rat type horizontal cells (Peichl and Bolz, '84).

Kainate injection of SC Surgical of sc Total No. Total No.

'15% of the retinal surface obscured to the extent that Class I cells in this region could ever, we found that a larger amount Of kainate (1.2-2.5 pg)

Of the


The dependence of retinal ganglion cells on their collicular target

Several observations suggest that the apparent loss of ganglion cells observed by us following the injection of kainic acid into the SC is not due to the blocking by kainic acid of the retrograde axonal transport of HRP (cf. Gomez- Ramos and Reinoso-Suarez, '83). First, HRP injections were made 2-3 weeks after injections of kainic acid. Second, a significant reduction in numbers of ganglion cells was observed only in those animals in which kainic acid was injected into the SC before postnatal day 10. Third, a substantial reduction in the number of labeled ganglion cells is always accompanied by a dramatic reduction in the size of the optic nerve contralateral to the kainate-injected SC (Fig. 1F). Indeed, Horsburgh and Sefton ('85) observed that 12-24 hours (but not 48 hours) after the injection of kainic acid into the SC of rats at P5, the number of pyknotic (degenerating) profiles in the ganglion cell layer of the contralateral retinae was almost twice that in the ganglion cell layer of the ipsilateral retinae or in the retinae of normal animals of the same age. We therefore conclude that the reduction in the number of labeled ganglion cells following selective lesions in the SC reflects a genuine loss of retinal ganglion cells.

At least two lines of reasoning argue against the idea that the death of the retinal ganglion cells is due to the direct lethal effect of the kainic acid transported retrogradely from the injection site. First, the injections of kainic acid into the optic tract and more importantly the DLG did not produce a significant reduction in the numbers of retinal ganglion cells surviving the period of naturally occurring ganglion cell death. Second, despite substantial damage to the SC after kainic acid injection into the SC of P7 animals, the number of retinal ganglion cells is reduced slightly. While in animals injected at P10 the effect is negligible (Fig. 6, Table 2). Thus, our results demonstrate that if the number of neurons in the SC of the rat is selectively reduced within the first few postnatal days, fewer ganglion cells than in normal retinae survive the period of naturally occurring ganglion cell death. Similarly, when the retinal targets of isthmo-optic cells (amacrine cells) in the chick are selectively removed by using kainate, there is a reduction in the number of cells in the isthmo-optic nucleus that survive the normally occurring wave of cell death (Catsicas and Clarke, '84). Further evidence of the dependence of retinal ganglion cells on their specific target tissues is provided by those experiments in which fetal retinae have been transplanted to the brain of newborn rats. Indeed, only when placed close to an appropriate target region (the SC) do the fetal retinal ganglion cells survive, sending axons into the host tissue and differentiating into the morphological classes normally present (McLoon and Lund, '84; McLoon and McLoon, '84; Perry et al., '85). It appears that retinal ganglion cells depend on their collicular target, perhaps for the provision of some trophic sub- stance necessary for survival during the critical period CMcCafYery et al., '82).

Although the number of ganglion cells surviving the period of naturally occurring cell death is also reduced following an early postnatal surgical lesion of the SC (Perry and Cowey, '79a, '81a, '82; Dreher et al., '83), we do not feel that mechanical damage of the SC has contributed significantly to the results reported in the present study. First, traumatic disruption around the injection site appears to be always confined to the region of the electrode track; the remainder

of the SC shows no obvious histological signs of mechanical damage. Second, while in the present study each kainate injection was made with micropipette, in previous experiments conducted in our laboratories when a much larger microsyringe needle was used for the injection of HRP at birth there was no apparent reduction in the number of ganglion cells surviving through the period of normally occurring cell death (Dreher et al., '83). Third, neonatal injections of kainic acid into the DLG and adjacent optic tract (present study) produce only a very small (about 10%) reduction in the number of retinal ganglion cells surviving through the period of naturally occurring cell death. In view of the fact that the reduction of about 10% was also apparent in the retinae ipsilateral to the injected DLG, the reduction observed by us probably does not indicate a real loss of significant number of retinal ganglion cells. Further- more, the injections centered on the optic tract without involving the retinorecipient nuclei (present study) did not cause any reduction in the number of retinal ganglion cells surviving the period of ganglion cell death.

Pathology of the lesion and phagocytosis At the cellular level, the sequence of events following an

injection of kainic acid into the neonatal SC or DLG (present data) is similar to that reported in the adult rat striatum (Coyle et al., '78). It is of particular interest that the histological appearance of cells affected by neonatal kainate injections into the SC or DLG, with little evidence of idammatory responses, resembles that of cells dying during normal development in the SC of the rat (Giordano et al., '80) and hamster (Finlay et al., '82) as well as in the retina of the rat (Cunningham et al., '82; Horsburgh and Sefton, '85), hamster (Sengelaub and Finlay, '821, mouse (Young, '84), and chick (Hughes and McLoon '79). Thus, kainate-induced neuronal death may serve as a model for the further study of the characteristics of naturally occurring cell death.

Since macrophages appear to be closely associated with dying cells following a kainate lesion of retinorecipient nuclei (present study) and hippocampus (Murabe et al., '811, macrophages are also likely to phagocytose cells damaged by neurotoxic lesions elsewhere in the mammalian brain. Indeed, following section of the axom of retinal ganglion cells in rats, both macrophages and Muller cells (glia) con- tain degenerating material (Miller and Oberdorfer, '81). However, the contribution of macrophages to neuronal phagocytosis during normal development has been a contro- versial issue. On the one hand, Miiller cells rather than macrophages were considered to be the principal scaven- gers of degenerating material during the normal development of the retina in the rat (Kuwabara and Wiedman, '74) and chick (Hughes and LaVelle, '75; Hughes and McLoon, '79). On the other hand, it has been claimed that retinal debris is phagocytosed by ventricular cells (mouse-Theiler et al., '76; chick-Garcia-Porrero and Ojeda, '79), by microglia (rat-Cunningham et al., '82; chick-Rager, '80), or by macrophages that later become microglia (mouse-Hume et al., '83). The observation that macrophages are present in substantial numbers in the ganglion layer of the rat's retina during the period of ganglion cell death lends support to the view that they contribute to the phagocytosis of dying retinal ganglion cells. Furthermore, the similarity in the histological appearances of cells dying during the period of naturally occurring cell death and cells killed by an injec-


tion of kainate suggests that macrophages may be at least partially responsible for the phagocytosis of the dying neurons found in the SC during normal development in rodents (rat-Giordano et al., '80; hamster-Finlay et al., '82).

Branching of axons of retinal ganglion cells In the rat, there is clear-cut electrophysiological evidence

indicating that many ganglion cells innervating the SC branch to supply the DLG (Sefton,'68). Additional evidence of the branching of the axons of retinal ganglion cells to the SC and DLG comes from the observations that while over 90% of retinal ganglion cells project to the SC (Linden and Perry, '83; Dreher et al., '85), representing about 100,000 of a total population of 110,000-115,000 ganglion cells (Potts et al., '82; Perry et al., '83), about 20,000-35,000 project to the DLG (Dreher et al., '85; Martin, '86). Furthermore, only about two-thirds of the total radioactivity found after an injection of the radioactively labeled amino acids into one eye is located in the contralateral SC (Toga and Collins, '81). Finally, combined neonatal ablations of visual cortex (which cause a complete degeneration of the relay cells of the DLG) and SC cause no greater a reduction in ganglion cell numbers in the retinae contralateral to the lesions than does removal of the SC alone (Perry and Cowey, '79a). The last argument has to be treated with caution since the effects of the ablations of the visual cortex might not mimic precisely the effects of the direct damage to the DLG. Thus, before making firm conclusions, one would have to test the effects of combined injections of kainic acid into both the DLG and SC. With this limitation in mind, it appears, nevertheless, that in those animals of the present study in which only about 40,000 ganglion cells were present (those with the most complete kainate lesions), the majority are likely to be those with sustaining collaterals to retinorecipient nuclei other than the SC (DLG, ventral geniculate, pretectal, accessory optic, and suprachiasmatic nuclei). The surviving retinal ganglion cells would presumably include a small number that do not appear to project to the SC. The degree of branching within these noncollicular projections has not been estimated. It is nevertheless very likely that a certain proportion of ganglion cells projecting to the DLG do not send branching collaterals into the SC. Thus, after injections of one fluorescent dye into the DLG and another into a retinotopically corresponding part of the SC, some retinal ganglion cells contained only the dye injected into the SC (P. R. Martin, unpublished observations).

The hypothesis that the large majority of retinal ganglion cells that survive the removal of the SC are those with sustaining collaterals is further supported by the observation that the number and distribution of the morphologically identified Class I cells are unaffected by the kainate lesion or surgical lesion of the SC. Thus, in the present study, about 800 Class I cells were present in each retina (ipsilateral and contralateral to the kainate-injected SC); this number is very similar to that found in normal albino rats (Dreher et al., '85). It has previously been demonstrated that virtually all fast-conducting axons (presumably those of the morphologically identified Class I retinal ganglion cells) branch and project to both the SC and DLG (Sefton, '68). Since similar numbers of Class I cells are found in both retinae following a surgical lesion of one SC, it appears that Class I cells can survive section of their tectal branch as well as the removal of their tectal target cells.

The selective removal of principal retinal target nuclei in at least two other mammalian orders-carnivores and pri-

mates-also causes a substantial loss of retinal ganglion cells. Thus, in cats, in which about 75% of all retinal ganglion cells project to the DLG (Illing and Wassle, '81; Was- sle, '82; Leventhal et al., '851, the unilateral removal of the neonatal visual cortex (areas 17, 18, and parts of area 19) produces a rapid degeneration of the principal cells in the DLG ipsilateral to the cortical lesion and a loss of nearly 80% of the retinal ganglion cells belonging to one specific functional class-the X-cells-in the nasal retina contralat- era1 to the cortical lesion (Tong et al., '82). Furthermore, in a recent study, Payne and his co-workers ('84) found that 18 months (or more) after unilateral neonatal ablation of areas 17 and 18 (with only partial involvement of area 19) of the visual cortex of cat, normal numbers of alpha and gamma ganglion cells (positively identified on the basis of retrograde labeling with KRP) were present in the nasal retinae contralateral to the ablated cortex. By contrast, the population of positively identified beta cells-that is, the pre- sumed morphological counterparts of X-cells (Boycott and Wassle, '74bin the nasal retinae contralateral to the ablated visual cortex was reduced by 90% when compared with normal animals. Payne et al. concluded that the majority of beta cells degenerate following visual cortical ablation because of an almost complete degeneration of their principal target tissue-laminae A and A1 of the DLG ipsilateral to the cortical lesion. Those authors attribute the survival of alpha, gamma, and 10% of the beta cells to their collateral projections to targets other than cells in laminae A and A1 of the DLG.

Relay cells in the DLG of the macaque monkey constitute the principal (or the only) target for 90% of retinal ganglion cells (Perry et al., '84); thus it is not surprising that the neonatal ablation of cortical area 17 (which causes a rapid degeneration of relay cells in DLG) leads to the massive loss of ganglion cells. Indeed, in macaques in which the striate cortices were removed at about 3 weeks of age, about 65% of the total ganglion cell population was lost within 2 months of the operation (Ogren et al., '83). Such a massive loss of ganglion cells is consistent with the fact that the P /3 (Perry and Cowey, '81b) or B (Leventhal et al., '81) cells, which compose about 80% of all ganglion cells, seem to project exclusively to the parvocellular laminae of the DLG (Perry et al., '84). Furthermore, only a small proportion of P Q (Perry and Cowey, '81b) or A cells (Leventhal et al., '811, which constitute about 10% of all ganglion cells in the macaque, project to retinorecipient nuclei other than the magnocelluar laminae of the DLG (Perry and Cowey, '84; Perry et al., '84).

Soma1 sizes of ganglion cells in the retinae with reduced ganglion cell numbers

In retinae in which the density of ganglion cells is considerably reduced following a kainate injection into the SC (present study) or a surgical ablation of SC (Dreher et al., '841, the centro-peripheral gradient in somal sizes is no longer apparent. Indeed, in all but one case reported here (see Results), the increase in somal size was apparent for ganglion cells located in all density regions of the retina (high, medium, and low), with the greatest increase occurring in the region of the retina in which the density is normally highest. There is evidence that the orientation of the dendritic trees of retinal ganglion cells is at least partially determined by interactions with neighboring cells (Linden and Perry, '82; Perry and Linden, '82). The results reported here and those of Dreher et al. ('84) suggest that

P. CARPENTER ET AL. 256

in the retina of the rat, the size of the somata of ganglion cells can be altered by reducing the density of ganglion cells. Similarly, in the cat, a reduction in the density of ganglion cells at the area centralis following optic tract section on P11 is associated with an increase in the somal sizes of retinal ganglion cells (Rapaport and Stone, '83).

An increase in mean somal diameter, as reported here, could be achieved by an increase in the diameter of the majority of retinal ganglion cells and/or by the disappear- ance of a class of small cells. We suggest that both mecha- nisms contribute to the observed increase in the mean somal sizes of ganglion cells and the absence of any cells with small somata from the retinae contralateral to the kainate injected SC. In the rat, the mean somal diameters of ganglion cells projecting to SC are smaller than those projecting to the DLG (Schober and Gruschka, '78; Dreher et al., '84). Those small retinal ganglion cells (Class IIb) that appear to project to the SC but not to the DLG (Dreher et al., '85) would be expected to be most vulnerable to the kainate lesion and many may indeed have died. On the other hand, the apparent absence of cells with small somata could be due to an increase in the somal size of those remaining, as a result of the reduction in overall ganglion cell density. Amongst the morphologically identified Class I cells, it is clear that the normal centro-peripheral difference in size has been abolished, and an overall increase in size has been demonstrated.

Time course of sensitivity of ganglion cells to a reduction in the size of their target tissue

Following a kainate lesion of the SC, the maximum loss of retinal ganglion cells, relative to the normal adult number, was about 65%. A similar maximal loss (67%) followed surgical ablation of the SC but was attributed to retrograde degeneration following axonal section rather than to the reduction in the amount of available target tissue (Perry and Cowey, '79a, %la, '82). Indeed, damage to the axons of retinal ganglion cells in neonatal rodents results in the rapid and complete retrograde degeneration of the cell body, within 48 hours (Miller and Oberdorfer, '81; Perry and Linden , '82; Perry et al., '83; Sefton and Lam, '841, whilst in adults retrograde degeneration is slower (Allcutt et al., '84a,b) and can take up to several months (Grafstein and Ingoglia, '82; Richardson et al., '82; Misantone et al., '84).

In the present study, the maximal sensitivity of retinal ganglion cells to the reduction in the amount of available target tissue occurred within the first 3 days of birth (see Fig. 6). On the other hand, Perry and Cowey (%la, '82) and Linden and Perry ('82) reported that the greatest sensitivity to a surgical ablation of the superior colliculus occurred on P5. However, it is apparent from the present study that the loss of retinal ganglion cells in the retina contralateral to the kainate injection into the SC at birth or on P2 was as great as the maximum loss incurred in animals in which the SC was ablated surgically on P5 (Perry and Cowey, %la, '82).

Since in the rat the retinocollicular axons have already reached the SC by the time of birth (Bunt et al., '83), we suggest that a surgical lesion of the SC performed within the first few days of life induces a loss of retinal ganglion cells due to the combined effects of the removal of their target cells and to axotomy. Beyond 3 days of age, the dependence of retinal ganglion cells on their collicular targets seems t o be progressively reduced until about P10

when the adult pattern of innervation of both the superior colliculus and DLG is established (Land and Lund, '79; Laemle and Labriola, '82; Martin et al., '83; Jeffery, '84; Manford et al., '84).

The sensitivity of retinal ganglion cells to section of their axons, maximal in the neonatal period, is also reduced but more slowly from P5 when the first myelinated axom appear in the optic nerve (Sefton and Lam, '84). In the adult (when myelination of the axons in the optic nerve is complete-Lam et al., '82; Perry et al,, '83; Sefton and Lam, '841, retinal ganglion cells can survive for very long periods following section (Richardson et al., '82) or crushing of their axons (Misantone et al., '82).

As described above, in carnivores (cats) there is a substantial reduction in the numbers of retinal Xcells following ablation of the visual cortex within 2 weeks of birth. By contrast, ablation of the visual cortex in older kittens or in adult cats produces only a minor loss of retinal X-cells (Tong et al., '82; Callahan et al., '84). Although the precise time course of the sensitivity of cat retinal ganglion cells to the removal of their target neurons in the DLG is not known at present, it seem that, as in the rat, the end of the sensitive period coincides with the end of the naturally occurring wave of ganglion cell death (the first 2 postnatal weeks or so-Ng and Stone, '82; Pearson et al., '83; Chalupa and Williams, '84).

In different groups of primates, the time course of the dependence of retinal ganglion cells on their target tissue varies considerably. Neither ablations of the striate cortex in adult owl or squirrel monkeys, nor ablations of visual cortex in 3-week-old squirrel monkeys cause appreciable reductions in the numbers of retinal ganglion cells or any alterations in retinogeniculate projections, despite the massive retrograde degeneration of cells in the DLG (Weller and Kaas, '84). Similarly, in the adult prosimian Galago, ablation of the striate cortex does not result in any obvious loss of retinal ganglion cells (Weller et al., '81). This apparent lack of dependence of retinal ganglion cells on their target cells in prosimians and New World monkeys is consistent with the idea that the visual cortical ablations in those species were performed after the period of naturally occurring ganglion cell death. In Old World monkeys (macaque), the sensitivity of the retinal ganglion cells to the removal of neurons in their principal target tissue @LG) is much greater if the ablation of visual cortex is performed during the period of naturally occurring cell death (Ogren et al., '83; Weller and Kaas, '84). However, lesions of the striate cortex made in juvenile macaques, presumably after the period of naturally occurring ganglion cell death (Rakic and Riley, '83), also result in a substantial transneuronal degeneration of retinal ganglion cells (Weller et al., '79 Dineen and Hendrickson, '81; Dineen et al., '82). It appears that the sensitivity of human retinal ganglion cells to the removal of neurons from their principal target tissue-the DLG-extends well beyond the period of naturally occurring ganglion cell death (Provis et al., '85a,b). Thus, following lesions of the striate cortex in adult humans, there is a substantial transneuronal degeneration of retinal ganglion cells some time after lesions of the striate cortex (Walsh, '47; Haddock and Berlin, '50; Van Buren, '63).

Conclusions Injections of kainate into the SC or DLG in neonatal (or

older) rats appear to destroy selectively neurons located within the injection site while leaving afferent axons and


their terminals intact. During the first postnatal week, the amount of available target tissue (SC) appears to regulate the number of ganglion cells that survive the period of naturally occurring ganglion cell death in the retina and indirectly influences the size of the Somata ofthe surviving ganglion cells. The reduced numbers of cells that survive are likely to be those with collaterals to retinorecipient nuclei other than the SC.

257

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Dineen, J.T., and A.E. Hendrickson (1981) Agecorrelated differences in the amount of retinal degeneration after striate cortex lesions in monkeys. Invest. Ophthal. Vis. Sci. 21 5'49-752.

Dineen, J.T., A.E. Hendrickson, and E.G. Keating (1982) Alterations of ACKNOWLEDGMENTS retinal inputs following striate cortex removalin adult monkey. Exp.

We are pleased to acknowledge the support of the Na- Brain Res, 47:446456, tional and Research Of Divac, I., H.J. Markowitsch, and M. Pritzel(1978) Behavioral and anatomi- We thank Cathy Parmagiani and Ian Thong, as well as the cal consequences of small intrastriatal injections of kainic acid in the staff of the Electron Microscope Unit of the University of rat. Brain Res. 151:523-532. Sydney for technical assistance, Gordon Williams and Clive Dreher, B., R.A. Potts, and M.R. Bennett (1983) Evidence that the early jefferY for photography, and M~~~ J. ~ ~ ~ ~ 1 1 for assis- postnatal reduction in the number of rat retinal ganglion cells is due to

a wave of ganglion cell death. Neurosci. Lett. 36255-260. last but not least, we Dreher, B., R.A. Potts, S.Y.K. Ni, and M.R. Bennett (1984) The development

thank Jonathan Stone and Steve Robinson for helpful corn- of heterogeneities in distribution and soma sizes of rat retinal ganglion ments on the manuscript and Paul Martin for help with the cells. In J. Stone, B. Dreher, and D.H. Rapaport (eds): Development of

with the diagrams.

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Role of target tissue in regulating the development of retinal ganglion cells in the albino rat: Effects of kainate lesions in the superior colliculus