Proc. Nail. Acad. Sci. USA Vol. 87, pp. 449-453, January 1990 Neurobiology Innervation patterns of single physiologically identified geniculocortical axons in the striate cortex of the tree shrew (geniculocortical afferents//on- and off-center pathways/binocular convergence) DAVID FITZPATRICK* AND DENiS RACZKOWSKI Department of Neurobiology, Duke University, Durham, NC 27710 Communicated by Irving T. Diamond, October 10, 1989

ABSTRACT We examined the termination patterns of MATERIALS AND METHODS single geniculocortical axons in the striate cortex of the tree shrew by using intracellular recording and horseradish per- We obtained the data for this report from adult tree shrews oxidase staining methods. Axons were classified by whether raised in our breeding colony. At the outset ofan experiment, they responded to light onset (ON center) or light offset (OFF we anesthetized the tree shrew with ketamine and xylazine, center) and whether they were driven by the ipsi- or contra- cannulated a vein for infusion of a dextrose/saline solution lateral eye. Afferents with ON-center responses end in the containing sodium thiopental (delivered at a rate of 3 upper part oflayer IV (IVa) whereas afferents with OFF-center mgkg-1hr-1 for the remainder of the experiment), per- responses end in the lower part of layer IV (IVb). Within each formed a tracheotomy, and placed a catheter in the bladder. tier, axons driven by the ipsilateral and contralateral eye The animal was placed in a stereotaxic apparatus, paralyzed overlap. These results suggest that binocular convergence with pancuronium bromide to prevent eye movements and occurs within layer IV without mixing the information from the ventilated with a respirator to maintain expired CO2 between ON- and OFF-center pathways and we consider the significance 2 and 3%. Body temperature was maintained at 37.50C by of this arrangement for visual cortical function. using a thermostatically controlled heating blanket. The pupils were dilated with atropine and contact lenses were The tree shrew is a good species in which to study the used to protect the corneas and to focus the eyes on visual organization of the because it shows a remark- stimuli presented on a tangent screen. able degree of segregation of visual pathways in the lateral We encountered geniculocortical axons in the deep layers geniculate nucleus and in the striate cortex. In most mam- of the cortex or in the adjacent white matter by using mals, the inputs from the two eyes are segregated into fine-tipped glass micropipettes filled with HRP. Axons were different layers of the lateral geniculate nucleus, and the tree characterized by their response to the onset or offset ofa light shrew is no exception. But, in addition to ocular segregation, presented to the ipsilateral or contralateral eye. lamination in the tree shrew lateral geniculate nucleus seg- Then, we attempted to impale the axon with the micropipette. regates the projections of ganglion cells that respond to light If successful, we rechecked its properties to onset (ON-center) from those that respond to light offset ensure that it was the same fiber and injected HRP by passing (OFF-center). Conway and Schiller (1) showed that the two current. All injected axons had response properties entirely most medial layers, 1 and 2, contain mostly ON-center consistent with those recording from in the lateral neurons and layers 4 and 5 contain mostly OFF-center geniculate nucleus (1, 7): they were monocular, nonoriented, neurons. and had receptive field centers that responded to stationary The segregation of the ON and OFF pathways appears to flashing stimuli. be maintained by the projections of the lateral geniculate Several hours after the last intracellular injection, the nucleus to layer IV of striate cortex. Layer IV in the tree animal was deeply anesthetized with barbiturate and per- shrew consists of two horizontal tiers separated by a - fused with aldehyde fixatives. We removed the brain, sparse cleft and the tract tracing studies of Harting et al. (2) trimmed it to a block containing the visual cortex, and and Conley et al. (3) showed that the ON-center geniculate refrigerated this block overnight in a solution of 5.0% (wt/ layers project to the upper tier, IVa, whereas the OFF-center vol) sucrose in 0.1 M sodium phosphate, pH 7.4. The next layers project to the lower tier, IVb. The studies of Norton day, 80-,.m-thick sections were cut in the coronal plane using and colleagues (4, 5) provided support for these anatomical a vibratome and these were reacted with 3,3'-diaminoben- experiments by showing that neurons in IVa have ON zidine tetrahydrochloride as the chromogen with nickel and responses whereas neurons in IVb have OFF responses. cobalt intensification (8). Here we tested the idea that the ON and OFF pathways Sixteen axons that were labeled by intraaxonal injection of terminate in separate tiers by combining intracellular record- HRP and recovered for morphological analysis form the basis ing and horseradish peroxidase (HRP) staining methods to for our conclusions. Twelve of these axons (6 ON center and visualize the termination patterns of individual physiologi- 6 OFF center) were reconstructed using a microscope cally characterized geniculocortical axons. The results ob- equipped with 63x and 100x oil-immersion objectives (NA tained confirm that the axons of individual ON- and OFF- 1.40 and 1.32, respectively) and a drawing tube. The relation center geniculate neurons end in distinct subtiers of layer IV of axon terminal arbors to the layers of the visual cortex was and we consider the significance of this segregation for the assessed by counterstaining selected sections with cresyl function of visual cortex. Some of these results have ap- violet. peared in abstract form (6). Abbreviations: ON, light onset; OFF, light offset; HRP, horseradish peroxidase. The publication costs of this article were defrayed in part by page charge *To whom reprint requests should be addressed at: Department of payment. This article must therefore be hereby marked "advertisement" Neurobiology, Box 3209 Medical Center, Duke University, in accordance with 18 U.S.C. §1734 solely to indicate this fact. Durham, NC 27710. 449 Downloaded by guest on October 3, 2021 450 Neurobiology: Fitzpatrick and Raczkowski Proc. Natl. Acad. Sci. USA 87 (1990) RESULTS cesses arborized in layer IlIb and in the cleft that separates layer IVa and IVb. A few processes also ended in layer IVb. Fig. 1 shows a cross section of the striate cortex in the tree Neither this nor any other geniculocortical axon in our sample shrew stained for Nissl substance. Layer IV stands out as a had processes that arborized in layer VI. prominent cell-rich band that is divided into two tiers by a The laminar projections of a representative OFF-center cell-sparse cleft. The upper tier is designated IVa and the geniculocortical axon driven by the ipsilateral eye is illus- lower tier is IVb. trated in Fig. 4. The first bifurcation point occurred just Fig. 2 illustrates the morphological features seen in the below where the axon arborized densely in the lower tier of terminal arbors of several geniculocortical axons in layer IV; layer IV (layer IVb). In addition to the cell's main arbor, a few the two classes, ON and OFF, are virtually indistinguishable boutons ended in layer V just below its border with layer IV. from one another. For both, the arbors consist mainly of very Only a few labeled terminal boutons occupied the cleft fine axon collaterals, less than 0.5 ,um in diameter, that are between layers IVa and IVb. studded with many boutons en passant and that end with a In both tiers, axons driven by the contralateral eye and single bouton or terminal. Only occasionally do these bou- those driven by the ipsilateral eye were distributed across the tons occur in clusters. The boutons vary in diameter from 0.5 depth of the tier. Since there is no evidence for columnar to 3.0 ,um. segregation of the inputs from the two eyes in layer IV of the The main point of this report is that all of the ON-center tree shrew's striate cortex (2, 9-11), axons driven by the geniculocortical axons, whether driven by the left or right eye, contralateral and ipsilateral eyes must overlap extensively. had terminal fields largely confined to cortical layer IVa, while There are, however, differences in the spatial distribution of all of the OFF-center axons had terminal fields mainly re- ipsilateral and contralateral axons. For example, axons stricted to IVb. The terminal arborization pattern of a repre- driven by the ipsilateral eye have a larger lateral extent than sentative ON-center geniculocortical axon driven by the con- those driven by the contralateral eye. Also, ipsilateral axons tralateral eye is illustrated in Fig. 3. Traced from the site of display more boutons near the borders of layer IV (i.e., the injection in the white matter (data not illustrated), the parent upper part of IVa and the lower part of IVb) than in the middle axon coursed unbranched until it neared its termination in the of the layer. These differences will be considered in more striate cortex. There, the axon turned sharply to enter into the detail elsewhere. gray matter where it bifurcated several times to form a dense collection of preterminal axon branches and terminal boutons DISCUSSION in layer IV. For this ON-center axon, most ofthe bouton-laden What might the segregation of the ON and OFF pathways in processes arborized densely in the upper tier oflayer IV (layer tree shrew layer IV tell us about the functional organization IVa). Besides the main arborization in layer IVa, some pro- of striate cortex? To address this question it is first necessary

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FIG. 1. Cross section of the striate cortex in the tree shrew, stainedX by the Nissl method, which reveals the cytoarchitecture. The dense granular layer IV is especially clear and it is divided in half by a cell-sparse cleft. WM, white matter. Downloaded by guest on October 3, 2021 Neurobiology: Fitzpatrick and Raczkowski Proc. Natl. Acad. Sci. USA 87 (1990) 451

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.~~~~~~~~ .i 5~~~~ I~A A PY2?L FIG. 2. Individual HRP-labeled ,, geniculocortical axons in the tree shrew striate cortex. (A) Lower power view of ON-center axon termi- nating in layer IV. The dashed lines bound layer IV. Note that the termi- - * nal arbor is limited to the upper halfof layer IV. (B) Higher power view of I* .14 Iftl same terminal arbor depicted in A. Most of the axonal collaterals contain boutons that are spaced rather evenly I and occur en passant. These collat- 0 1 4 erals end as a single bouton (the ar- .i ilt. 4* rows in A and B point to the same I 20, terminal swelling). (C) Higher power ./ view ofa portion of the terminal arbor of an OFF-center geniculocortical 1010PMMm axon terminating in the lower half of B layer IV. to consider what happens to the ON and OFF pathways overlap of ipsilateral and contralateral axons within layer IV beyond layer IV: do they remain separate or are they brought is not a sure sign that convergence occurs, the existence of together? In all other species that have been examined, the binocular convergence within layer IV is supported by the ON and OFF pathways ultimately converge in the striate physiological studies ofHumphrey et al. (11). Thus, matching cortex (e.g., refs. 12-15): individual cortical neurons have pathways from each eye are brought together to generate both ON and OFF responses and these are acquired by binocular ON neurons in layer IVa and binocular OFF combining the excitatory responses of ON-center and OFF- neurons in layer IVb; only then do the axons from the center geniculate neurons (12, 16, 17). The tree shrew seems neurons in IVa and IVb converge onto neurons in the to be no exception: most neurons outside of layer IV show superficial layers to generate binocular neurons that respond excitatory responses to both the onset and the offset ofa light to both the onset and offset of a light stimulus. stimulus (unpublished results). Moreover, experiments using Is there any significance to having binocular convergence microinjections of HRP show that the projections of neurons occur prior to the convergence of the ON and OFF path- in the ON and OFF tiers of layer IV overlap extensively ways? We argue that the answer is yes. To understand why within the superficial cortical layers (18). this is so requires reviewing two features of the spatial We view the segregation of the ON and OFF pathways as organization of cortical receptive fields that are relevant to an opportunity to gain insights into the cortical circuitry our argument. (i) Cortical neurons that receive inputs from responsible for bringing these two pathways together. The both the ON and OFF pathways combine these inputs in key issue is the relation between the convergence of the ON many different ways (e.g., refs. 12, 14, and 19). In one class and OFF pathways and the convergence of the pathways (simple cells), ON and OFF responses can be elicited from from the two eyes. Because individual geniculocortical fibers separate regions within the receptive field, and the number carry both traits, ON/OFF convergence and binocular con- and position of these regions vary widely. For example, in vergence could occur simultaneously or in separate stages some simple cells, there are two separate ON and OFF and the organization of layer IV in the tree shrew allows us regions; in others there are three: a central ON flank sur- to distinguish between these alternatives. rounded by two OFF flanks or a central OFF region sur- In the tree shrew at least, the two forms of convergence rounded by two ON flanks. In another class (complex cells), occur separately with binocular convergence preceding the ON and OFF responses are not spatially separate and both convergence of the ON and OFF pathways. Although the responses can be elicited at almost every point in the recep- Downloaded by guest on October 3, 2021 452 Neurobiology: Fitzpatrick and Raczkowski Proc. Natl. Acad. Sci. USA 87 (1990) tive field. (ii) Despite this variation, binocular ON/OFF Our view of the neural circuitry in the tree shrew that neurons always show identical responses to stimulation of generates receptive fields with these features is as follows: We either eye: whatever the spatial arrangement of ON and OFF assume that neurons in layer IVa have ON receptive fields that responses seen in the receptive field of the left eye, the same are identical in size, shape, and position for the two eyes and arrangement is found in the right (12, 20). that similar features hold for the OFF neurons in layer IVb

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FIG. 4. Reconstruction of an OFF-center geniculocortical axon excited by the ipsilateral eye. Here, the arbor is wide and limited to the lower half of layer IV (see text for additional details). Downloaded by guest on October 3, 2021 Neurobiology: Fitzpatrick and Raczkowski Proc. Natl. Acad. Sci. USA 87 (1990) 453 (Fig. 5). These single-sign binocular neurons are then used as we argue that monocular ON/OFF neurons are not the "building blocks" to generate a variety of receptive field building blocks for binocular ones and that monocular and arrangements in layer III. Since the inputs from the two eyes binocular ON/OFF neurons may be generated in parallel. already have been matched in layer IV, any subsequent The final issue for discussion is whether the organization combination of the ON and OFF pathways achieved by the we have described in the tree shrew has applications to convergence of the axons of IVa and IVb neurons will result ON/OFF and binocular convergence in other species. There in neurons with matching receptive fields in the two eyes. is no question that the contribution of geniculocortical ter- Of course, a similar result could be achieved by mixing the minations to ON/OFF and binocular convergence varies But considerably across species. For example, in Old World ON and OFF pathways prior to binocular convergence. monkeys, neither binocular convergence nor ON/OFF con- in this case, all varieties of ON and OFF convergence would vergence occurs in layer IV (13, 20, 22), whereas in carni- have to be established independently for each eye and vores both seem to occur (e.g., ref. 15). Still, regardless ofthe considerable specificity in synaptic connections would be sites where ON/OFF and binocular convergence occur, the required to ensure that binocular neurons receive inputs from advantage of the sequence we have described in the tree only those monocular neurons that have the same spatial shrew, binocular convergence preceding ON/OFF conver- arrangement of ON and OFF zones within their receptive gence, remains the same. The facts that binocular neurons fields. with single ON or OFF zones are found in the visual cortex Our model is not meant to imply that binocular conver- of both cat and monkey and that most simple and complex gence is a prerequisite for ON/OFF convergence; indeed, cells in these species are binocular leave open the possibility monocular neurons are present within IVa and IVb ofthe tree that this sequence may apply to other species as well. shrew's striate cortex (11) and monocular ON/OFF neurons are present outside of layer IV (unpublished results). Rather, We thank Martha Foster and Patricia Hicks for their superb technical assistance. We also thank Dr. Irving T. Diamond for his Connections c IDof Receptive Field generous support and his helpffil comments on earlier versions ofthis Layer IV Organization paper. This work was supported by a National Institutes of Health grant (EY06821) and a National Institute of Mental Health grant (MH04849). 686886

Binocular ON/OFF A 1. Conway, J. L. & Schiller, P. H. (1983) J. Neurophysiol. 50, cm 1330-1342. A&& A A± A8 A A--A A 2. Harting, J. K., Diamond, I. T. & Hall, W. C. (1973) J. Comp. Striate w Neurol. 150, 393-440. A AA Cortex * ^-A 6\6 "A AA±A C,) 3. Conley, M., Fitzpatrick, D. & Diamond, 1. T. (1984) J. Neu- Ill ,A Layer A 66-& A±AA± AA A&A rosci. 4, 171-197. A8&AAA A±86± 4. Norton, T. T., Rager, G. & Kretz, G. (1985) Brain Res. 327, A6 '8A± A A 66A A6±68±8 319-323. A 5. Kretz, G., Rager, G. & Norton, T. T. (1986) J. Comp. Neurol. 251, 135-145. 6. Raczkowski, D. & Fitzpatrick, D. (1985) Soc. Neurosci. Abstr. 11, 227. 7. Sherman, S. M., Norton, T. T. & Casagrande, V. A. (1975) Brain Res. 93, 152-157. IVa Binocular ON lo± 8. Adams, J. C. (1981) J. Histochem. Cytochem. 29, 77 (abstr.). 9. Hubel, D. H. (1975) Brain Res. 96, 41-50. Striate I- 10. Casagrande, V. A. & Harting, J. K. (1975) Brain Res. 96, Cortex -- Layer IV 367-372. 11. Humphrey, A. L., Albano, J. E. & Norton, T. T. (1977) Brain Binocular OFF A±± AAA Res. 134, 225-236. lVb 12. Hubel, D. H. & Wiesel, T. N. (1%2) J. Physiol. (London) 160, Ipsi Contra 106-154. 13. Hubel, D. H. & Wiesel, T. N. (1968) J. Physiol. (London) 195, LGN 215-243. 14. Schiller, P. H., Finlay, B. L. & Volman, S. F. (1976) J. Neu- rophysiol. 39, 1288-1319. FIG. 5. Summary of the proposed stages involved in the conver- 15. LeVay, S., McConnell, S. K. & Luskin, M. B. (1987) J. Comp. gence of the ON and OFF pathways from the two eyes in tree shrew Neurol. 257, 422-441. striate cortex. Stage 1: ON-center axons driven by the ipsilateral 16. Schiller, P. H. (1982) Nature (London) 297, 580-583. (Ipsi) and contralateral (Contra) eye converge onto individual neu- 17. Sherk, H. & Horton, J. C. (1984) J. Neurosci. 4, 381-393. rons in layer lVa to generate binocular ON receptive fields (open 18. Muly, E. C., Fitzpatrick, D. & Raczkowski, D. (1989) Soc. triangles) that are matched in size, shape, and position for the two Neurosci. Abstr. 15, 1398. eyes. The same process leads to the formation of binocular OFF 19. Palmer, L. A. & Davis, T. L. (1981) J. Neurophysiol. 46, receptive fields in layer IVb (solid triangles). Stage 2: axons of 260-276. neurons in layers IVa and IVb converge upon neurons in layer III to 20. Hubel, D. H. & Wiesel, T. N. (1977) Proc. R. Soc. London Ser. generate a variety of different types of binocular ON/OFF receptive B. 198, 1-59. fields. Our depiction of the receptive fields of neurons in layers III 21. Humphrey, A. L. & Norton, T. T. (1980) J. Comp. Neurol. and IV as orientation selective is consistent with the results of 192, 531-547. Humphrey et al. (21). LGN, lateral geniculate nucleus. 22. Blasdel, G. G. & Fitzpatrick, D. (1984)J. Neurosci. 4,880-895. Downloaded by guest on October 3, 2021