Characterization, distribution, and ontogenesis of adenosine binding sites in cat visual cortex

In vitro autoradiographic techniques were used to characterize binding sites for 3H-cyclohexyladenosine (CHA) and 3H-5′-N- ethylcarboxamidoadenosine (NECA) in cat and kitten visual cortex. 3H- CHA binding sites in adult cat have a Bmax of 1,363 fmol/mg protein and a Kd of 6.8 nM. Displacement experiments indicate that 3H-CHA binds to an adenosine receptor similar to the A1-adenosine receptor described by other investigators. 3H-NECA binding sites in adult cat have a Bmax of 518 fmol/mg protein and a Kd of 15.4 nM. Displacement experiments do not allow us to identify this binding site unambiguously. Bmax values increase during postnatal development for both binding sites, peaking in adulthood for 3H-CHA and at 30 d for 3H-NECA. Kd values show neither consistent nor significant differences during postnatal development for either binding site. 3H-CHA and 3H-NECA binding sites are concentrated in layers 1–3 and upper layer 5 in the visual cortex of adult cats. These laminar patterns, however, change during postnatal development, showing the densest binding in the deep cortical layers (5 and 6) in kittens younger than 30 d of age and a fairly homogeneous binding in older kittens before achieving the adult distribution.

In vitro autoradiographic techniques were used to characterize binding sites for 3H-cyclohexyladenosine (CHA) and  (NECA) in cat and kitten visual cortex. 3H-CHA binding sites in adult cat have a B,, of 1363 fmol/mg protein and a Kd of 6.8 nM. Displacement experiments indicate that 3H-CHA binds to an adenosine receptor similar to the A,-adenosine receptor described by other investigators. 3H-NECA binding sites in adult cat have a B,,,, of 518 fmol/ mg protein and a K,, of 15.4 nM. Displacement experiments do not allow us to identify this binding site unambiguously. B,,,, values increase during postnatal development for both binding sites, peaking in adulthood for 3H-CHA and at 30 d for 3H-NECA. Kd values show neither consistent nor significant differences during postnatal development for either binding site. 3H-CHA and 3H-NECA binding sites are concentrated in layers l-3 and upper layer 5 in the visual cortex of adult cats. These laminar patterns, however, change during postnatal development, showing the densest binding in the deep cortical layers (5 and 6) in kittens younger than 30 d of age and a fairly homogeneous binding in older kittens before achieving the adult distribution.
The visual cortex plays a critical role in the analysis of visual signals concerned with form, motion, and depth. The mechanisms by which the cortex processes visual information have been the subject of numerous investigations at the physiological (Hubel and Wiesel, 1962) anatomical (Lund, 1973) and neurochemical (Emson and Hunt, 1979) levels. In particular, the last few years have witnessed a dramatic increase in our understanding of the chemistry of the cortex. Accumulating evidence gathered with immunocytochemical, iontophoretic, and receptor binding techniques has greatly expanded the list of neurotransmitters/neuromodulators that may be active in cerebral cortex, in particular, visual cortex (Emson and Hunt, 1979;Parnavelas and McDonald, 1983). These findings point toward a complex chemical circuitry of neocortex in which various neurotransmitters can modulate each other's actions in a variety of different ways.
Our own studies have been directed to the examination of the receptors for various neurotransmitters and neuromodulators thought to be active in the cat visual cortex (for a summary, see Shaw et al., 1984~). These data, gathered primarily with the methods of receptor autoradiography (Snyder, 1984;Young and Kuhar, 1979) have provided information on the characteristics and laminar distributions of the various receptor populations. Special attention has been directed towards the study of receptor alterations during normal postnatal development, since several receptor populations show differing characteristics and/or distributions in infant and adult animals (Shaw et al., 1984a. In these cases, receptor alterations take place during the physiologically defined critical period (Cynader et al., 1980;Hubel and Wiesel, 1970;Olson and Freeman, 1980) the period in the first few months of postnatal life of greatest neuronal plasticity. Our working hypothesis is that alterations of receptor distribution, number, and/or affinity may play a role in the mechanism by which visual exposure modifies cortical function during the critical period.
In the present study, we have examined adenosine binding sites in visual cortex using 3H-CHA and 3H-NECA as ligands (Bruns et al., 1980;Goodman et al., 1983). The adenosine binding sites are characterized and their postnatal development charted in terms of number, affinity, and laminar distribution.
While this paper was being revised, a report by Aoki (1985) appeared describing adenosine binding site distribution in normal and dark-reared cats.

Materials and Methods
Fourteen male colony cats were maintained on a 14 hr/l 0 hr light/dark cycle and were sacrificed at approximately the same time of day (1600-1830). Following an overdose-of sodium-pentobarbital, they were perfused through the heart with cold (4°C) phosphate buffer solution (PBS; 0.1 M, pH 7.4). In some animals, the PBS was followed by a O.l-O.2% formaldehyde/PBS solution in order to lightly fix the tissue. This procedure had no discernible effect on the characteristics of adenosine binding sites. The brains were quickly removed and frozen in liquid Freon. Sixteen micron coronal sections were cut on a cryostat and thawmounted onto subbed glass slides, then stored at -20 to -25°C until used. In experiments designed to compare animals of different ages, sections were cut within the same 24 hr period and stored for the same length of time before being used.
3H-CHA (New England Nuclear; specific activity, 25 Ci/mmol), an adenosine agonist, was used to study binding at A,-adenosine binding sites, and 3H-NECA (New England Nuclear; specific activity, 30 Ci/ mmol) was employed in an attempt to label both A, and A, binding sites.
The slide-mounted sections were allowed to warm to 4°C. The slides were then preincubated for 10 min in dishes containing 4°C Tris-HCl buffer (50 mM, pH 7.7). A small amount of formaldehyde (0.2%) was added to lightly fix the tissue. Two additional 5 min washes in buffer alone removed the formaldehyde. Reduction of endogenous adenosine was accomplished through the addition of adenosine deaminase, Sigma type VIII (Goodman and Snyder, 1982), to all 3 preincubation rinses at a concentration of 0.06 IU/ml. In preliminary experiments we determined that raising the preincubation concentration of adenosine deaminase to 1 IU/ml gave, at most, 20% higher binding. For this reason, preincubation rinses contained the lower concentration. All incubation media, however, contained adenosine deaminase at a concentration of 1 III/ml (Goodman and Snyder, 1982). After the preincubation washes, the slides were laid out on a black plastic tray to aid visualization of the sections. The incubation medium containing the tritiated ligand (0.4-52 nM for both ligands) was dripped onto the sections. Nonspecific binding was determined by including 1 x 1O-4 M CHA or L-PIA for 'H-CHA or )H-NECA, respectively, to the incubation medium. Incubations for both ligands were for 120 min at 23°C. The incubations were terminated with two 5 min washes in 4°C buffer. In characterization studies, areas 17-I 9 identified from the cortical maps of Tusa et al. ( 198 1) were scraped off the slides onto Whatman GB/F filter paper, suspended in Aquasol II and the bound ligand was counted in a scintillation counter. For autoradiography, sections were incubated at optimal conditions as determined in the characterization experiments. The sections were dried under a stream of cool air, allowed to dry further for at least 12 hr over desiccant at 4°C and then apposed to LKB tritium-sensitive Ultrofilm for l-4 weeks. The film was conventionally developed and fixed.
Protein content was determined in alternate sections from each animal using the methods of Lowry et al. (195 1) and used for computing B,, values. Saturation binding data were analyzed by Eadie-Hofstee plots as described by Zivin and Waud (1982). Cortical lamination was determined for comparison with the autoradiograms by staining alternate sections with cresyl violet or processing for cytochrome C oxidase activity (Wong-Riley, 1979).

Results
Characterization of adenosine binding sites using 3H-CHA and 'H-NECA Figures 1 and 2 illustrate biochemical experiments in which we characterized the binding sites for 3H-CHA and 3H-NECA, respectively. For each experiment, each point represents at least 3 separate determinations for total binding. Determination of nonspecific binding was always performed in duplicate. All experiments for these characterizations were performed at least twice. All time course and displacement curves thus represent average values for 2 or more experiments.  Table  1. Incubation in each case was in 20 nM 3H-CHA for 120 min at 23°C with the appropriate displacers present. Vol. 6, No. 11, Nov. 1986   The time course of association and dissociation was examined for 3H-CHA binding (Fig. 1, top panel). At 23°C equilibrium binding was achieved by 120 min, remaining stable for at least another hour. The association rate constant (K, ,) was cal-culated to be 2.06 x 1O-3 min-' m-l. Rinsing the sections in an "infinite" dilution led to a rapid decline in specific binding over 10 min, followed by a slower decline. The dissociation rate constant (K-J was calculated to be 1.93 x 1O-2 min-*. The In both cases. incubation was for 120 min at ratio of these rate constants gave a K,, (= K-, -l/K+, + 1) of 9.34 nM. A representative saturation binding experiment for 'H-CHA is illustrated in Figure 1 (middle panel). Increasing the concentration of 'H-CHA resulted in saturable binding. Nonspecific binding was less than 7% of total binding at the highest ligand concentration used. Total binding is represented by filled circles, while specific and nonspecific binding are indicated by open circles and triangles, respectively. Eadie-Hofstee analysis of these data (insert) gave a B,,, of 1363.17 + 89.04 fmol/mg protein and a Kd of 6.83 k 0.7 nM. A Hill plot of these data had a slope of 1.02, suggesting the existence of a single population of binding sites. Figure 1 (bottom panel) illustrates a series of displacement experiments in which we determined the IC,, (concentration required to displace 50% of the bound 3H-CHA) for various agonists and antagonists of adenosine. CHA had the lowest value of the compounds tested, with au IC,, of 5 x 1O-9 M. This value is in good agreement with the previously determined Kd's derived from the rate constant measurements and with other reports in the literature (Aoki, 1985;Bruns et al., 1980). The IC,, values for all of the compounds tested are shown in Table  1. The order of potency of the various compounds suggests that 3H-CHA labels a binding site similar to the A,-adenosine receptor described by other investigators in other preparations (Bruns et al., 1980). Figure 2 illustrates the association and dissociation time courses for binding sites labeled with 3H-NECA (top panel). At 23°C 3H-NECA binding reached equilibrium by 120 min, remaining relatively stable for at least another hour. The association rate constant was calculated to be 7.66 x 1O-4 min-I m-l, almost 3 times slower than the K, , for 3H-CHA. Postincubation rinses produced a rapid early decline in specific binding over 10 min, followed by a slower decline that was similar to that described above for 3H-CHA. The dissociation rate constant was calculated to be 1.5 1 x 1O-2 min-I, a value close to that for 3H-CHA. The Kd calculated from these rate constants was 23°C: Ligand concentrations were 26 nM (A) and 30 tn.4 (B); exposure period for LKB Ultrofilm was 14 d (A) and 2 1 d (B). Adjacent sections in each instance were processed for cytochrome c oxidase activity (Wong-Riley, 1979). Photographs of the autoradiograms and the cytochrome c-stained sections were made to the same magnification. A portion of a photograph of a cytochrome c-stained section (medial bank of area 17) has been superimposed on the photograph of the autoradiogram. Dark arrows indicate layer 4 which stains most densely for cytochrome c oxidase; white arrow in A points to the labeled sublayer in layer 5. Calibration bar, 1 mm. Dorsal (D) and medial (M) directions are as indicated.
19.71 nM. In Figure 2 (middle panel) the results of a representative saturation binding experiment using 3H-NECA are illustrated. Specific binding was saturable. Eadie-Hofstee analysis (insert) gave a B,,,, of 5 17.77 + 7.05 fmol/mg protein and a Kd of 15.41 St 1.84 nM. Nonspecific binding was about 25% of total binding at the highest concentration. A Hill plot of these data gave a slope of 1.02, suggesting the presence of a single population of binding sites.
In Figure 2 (bottom panel) a series of displacement experiments is shown (see also Table 1)   Photographs are from LKES Ultrofilm autoradiograms photographed for actual contrast; tilm exposure, 14 d. Note the changes in laminar distribution during development, from deep layers in the young kittens to superficial layers in adults. For each cat the different laminae were identified in alternate sections stained with cresyl violet or cytochrome c ox&se. Calibration (all panels), 1 mm. Dorsal is up, medial is left for all panels. Shaw et al. Vol. 6, No. 11, Nov. 1986 impervious to additions of up to 1 x 1O-6 M dipyridamole, a blocker of facilitated diffusion of adenosine into cells (Wu et al.. Distributions of 3H-CHA and 3H-NECA binding sites in cat 198 1). Neither the number of binding sites, nor their laminar visual cortex distributions, was altered for either ligand. These data suggest The laminar binding patterns for 3H-CHA and 3H-NECA are that neither 3H-CHA nor 3H-NECA is labeling high-affinity illustrated in Figure 3. These photographs of the autoradiograms adenosine transport sites in cat visual cortex.
have been combined with inserted slices of photographs from alternate sections processed for cytochrome c oxidase. Cytochrome c oxidase labels layer 4 most densely in cat visual cortex (Wong-Riley, 1979), offering a convenient laminar landmark with which to identify laminar patterns of receptor binding. In the adult cat, 3H-CHA (Fig. 3A) showed the densest binding in the supragranular layers (layers l-3). Moderate binding was also seen in the upper portion of layer 5. 3H-NECA labeled binding sites with essentially the same laminar distribution as that of 3H-CHA in the adult cat cortex (Fig. 3B).
Postnatal development of 'H-CHA and 3H-NECA binding patterns Figure 4 illustrates the laminar distribution of 3H-CHA binding sites in kittens of various postnatal ages. The illustrations shown here have been photographed from the original autoradiograms at the same contrast level in order to allow direct comparisons among the different panels of Figure 4. At 3, 15, or 30 d after birth (panels A-C), layers 5-6 were most densely labeled, while the superficial cortical layers were relatively lightly labeled. By 60 d of age, the deep cortical layers were still labeled, but moderate binding had appeared in the superficial layers as well, resulting in a relatively laminarly homogeneous, albeit slightly patchy, binding pattern. By 95 d postnatal (panel E), the adult binding pattern became more apparent: Layers l-3 exhibited dense binding, with layers 4-6 much lighter in comparison. In the adult, 3H-CHA strongly labeled layers 1-3, with an additional sublayer in layer 5 (Fig. 40, a pattern very different from that of the young kittens (Fig. 4, A-C).
3H-NECA binding shows a developmental pattern (Fig. 5) similar to that of 3H-CHA. Again, the deep cortical layers were heavily labeled early in postnatal life. A more homogeneous binding pattern then developed, with the superficial layers becoming heavily labeled only late in postnatal development. Figure 6 illustrates the changes in receptor number (B,J during postnatal development for 3H-CHA and 3H-NECA binding sites. The number of 3H-CHA binding sites increased more than 3-fold from low early values to adulthood. 3H-NECA binding increased until 30 d postnatally, remaining relatively constant thereafter. 3H-CHA and 3H-NECA binding in other cortical and subcortical areas Studies of other receptors in cat and raccoon cortex reveal that receptor binding in primary sensory cortices is often denser and can show different lamination patterns than that in adjacent association cortices (Sampson et al., 1984;Shaw et al., 1984a). 3H-CHA and 3H-NECA binding in cat visual cortex likewise follows this pattern: Areas 17 and 18 have denser binding and more distinct lamination than nearby association cortex. Similarly, auditory cortex shows a highly laminar-specific binding pattern, with the highest binding densities in the supragranular laminae (data not shown). Binding also becomes less dense and more homogeneous across cortical layers in nonsensory cortical areas. Figure 8 illustrates a clear transition between the binding pattern in the ventral-most part of the striate cortex and that of the cingulate cortex. 3H-CHA and 3H-NECA have similar distributions in subcortical structures. Stratum oriens and stratum radiatum of the CA1 to CA3 fields of the hippocampus label densely. The subiculum and the inner molecular layers of the dentate gyrus also label densely for both ligands. The lateral geniculate nucleus (LGN) shows moderate and homogeneous binding in the cell layers, with the interlaminar zones relatively free of label. The medial geniculate nucleus labels moderately with some inhomogeneities. Both inferior and superior colliculi show low-moderate binding in the superficial layers only. Figure 8 shows 'H-CHA binding sites in the cat diencephalon midbrain to illustrate adenosine receptor distribution in some of the regions mentioned above.

Discussion
The identification of the 3H-CHA binding site in the cat visual cortex as an A,-adenosine receptor seems relatively straightfor- Figure 8. Montage of 2 coronal sections of cat diencephalon midbrain and cortex are used to illustrate 'H-CHA binding in the visual cortex and other cortical and subcortical areas. The sections were incubated with 26 nM "H-CHA for 120 min at 23°C. Note in general the dense binding in cortex compared with most of the chencephalon. A, The most densely labeled diencephalic areas are the hippocampus @pp.; see text for further description) and the lateral and medial geniculate bodies (Zgn, mgn). A clear laminar difference is observable between the binding in visual cortex (vc) and cingulate cortex (chg.). B, Low to moderate binding is observed in the superficial layers of the superior colliculus (SC). Film exposure, 14 d. Calibration bar, 1 mm. Dorsal is up, medial is right in A and left in B.
ward. The binding pattern is similar to that reported for A,-adenosine receptors in the cat (Aoki, 1985); in rat cortex the binding pattern differs (Goodman and Snyder, 1982;Goodman et al., 1983). Additionally, the Kd and displacement characteristics are generally similar to those reported for A,-adenosine receptors previously described (Aoki, 1985;Bruns et al., 1980;Goodman and Snyder, 1982), although Aoki (1985) reports 2 distinct binding sites in cat visual cortex using 3H-CHA.
The identification of the cortical 3H-NECA binding site is more problematic. 3H-NECA binds with lower affinity than 'H-CHA, in agreement with reports of A,-adenosine receptor binding in other preparations (Bruns et al., 1980). A Hill plot of the saturation binding data suggests the existence of a single population of binding sites, although 3H-NECA in other preparations is reported to bind to both A, and A, adenosine sites (Fredholm, 1982;Snyder, 1985). The higher Kd for 3H-NECA is attributable to an almost 3-fold slower association rate constant. The dissociation rate constants for the 2 ligands are approximately the same, suggesting that the differences in B,, ('H-CHA nearly 3 times that of 3H-NECA) are due to different numbers of distinct binding sites. If 3H-NECA were binding to both A, and A, sites, we would expect a higher B, than for 3H-CHA, unless the K-, were appreciably faster, which is not the case. In addition, the postnatal time course of variation in B,,, is quite different for the 2 ligands. Taken together, these data argue for a cortical 3H-NECA binding site distinct from the A, site labeled by 'H-CHA. The difficulty with this interpretation is that the order of effectiveness for the various displacer substances tested is very similar, although not identical, for the 2 ligands (Figs. 1; 2; Table 1). Previous reports in other systems have indicated major differences in displacer potencies at the 2 sites. Another difficulty is the nearly identical laminar distribution of 3H-CHA and 3H-NECA binding patterns. These 2 reservations, especially the first, raise questions concerning the identification of the 3H-NECA binding site in the cat cortex as the A,-adenosine receptor described by other investigators. Further experiments are required to establish definitively whether 3H-NECA binds to the same sites as does 3H-CHA or to a distinct subclass of adenosine receptors in cat visual cortex.
Both 'H-CHA and 3H-NECA binding sites show alterations in their laminar binding patterns during postnatal development. Similar changes, or even reversals, of the original neonatal laminar binding pattern have been found in the majority of cortical receptor populations we have studied to date Shaw et al., 1984aShaw et al., , b, 1985. These data are summarized in schematic form for young kittens and adult cats in Figure 9.
Most of the previously studied receptors that have exhibited changes in developmental laminar binding patterns (muscarinic/ cholinergic, cholecystokinin, and /3-adrenergic binding sites) have shown dense concentrations in layer 4 initially, and subsequent alterations to favor the superficial and/or deep layers. Other receptors (GABA, benzodiazepine) are concentrated in layer 4 at all postnatal ages. Adenosine binding sites differ in that layers 5 and 6 show the densest binding initially, layers l-3 are most heavily labeled in the adult, and layer 4 is not a zone of highest concentration at any stage of development. The pattern of alteration of binding sites (from deep to superficial cortical layers) parallels that previously reported for the ontogeny of neurons in the different cortical layers (Rakic, 1974). The relative absence of adenosine receptors in layer 4 at all ages suggests that adenosine does not exert its effects at the first stage of cortical information processing, but rather that it acts on higher-order cortical functions. This reduced presence in layer 4 also characterizes muscarinic acetylcholinergic binding sites of the adult cat (Shaw et al., 1984a), and it is interesting to note that these 2 "modulatory" substances seem to have similar effects on cortical EEG and behavioral arousal. It has long been known that administration of the muscarinic antagonist scopolamine results in cortical EEG synchronization in the absence of behavioral sleep (Steriade and Hobson, 1976). Administration of L-PIA, an adenosine agonist, results in behavioral manifestations of sleep, but with a desynchronized cortical EEG (Meltzer et al., 1984).
As was the case for several other receptors studied in cat visual cortex (Jonsson and Kasamatsu, 1983;Shaw and Cynader, 1985, unpublished observations;Shaw et al., 1984aa;Wilkinson et al., 1983) and the cortex of other species (Candy and Martin, 1979;Pittman et al., 1980), adenosine receptor density (B,d was initially low but increased during postnatal development. 3H-CHA binding density peaked in adulthood, while 3H-NECA binding density peaked at 30 d postnatally. Geiger et al. (1984) have reported an increase in the number of A,-adenosine receptors during postnatal development in rat cerebral cortex.
Kd values for the 2 binding sites showed some developmental variations, but no clear or significant trends were seen, unlike those observed for rat adenosine receptors (Geiger et al., 1984) and for other cortical receptor populations (Shaw et al., 1984c. Of the other receptors studied to date in cat visual cortex, only the P-adrenergic receptors, also purported to be modulatory in action, show no consistent Kd changes during postnatal development . The issue of the cellular location of adenosine receptors is a subject of some controversy. Goodman et al. (1983) have sug-gested a presynaptic locus for A,-adenosine receptors in rat superior colliculus but not for the LGN or visual cortex. In other preparations, adenosine receptors are probably postsynaptic (Geiger et al., 1984). We have examined these issues in a preliminary way in adult cats subjected to unilateral enucleation (3 month survival), unilateral decortication (2-3 week survival), or unilateral LGN lesion (2 week survival). In no case have we noted a significant change in adenosine receptor density or pattern of binding in either the LGN (following enucleation), superior colliculus, or visual cortex. Results were the same whether 3H-CHA or 3H-NECA was employed as a ligand. These results suggest that if adenosine receptors are associated with the retinogeniculate, retinocollicular, geniculocortical, or callosal pathways, then they are not presynaptic in location, at least not in the cat. Any presynaptic loci for adenosine receptors in cat visual cortex are thus likely to arise from intracortical circuits.
The functional role of adenosine receptors within the cortex remains unclear. The evidence that adenosine modulates the synaptic release of a wide variety of different substances (Harms et al., 1979;Hollins and Stone, 1980;Michaelis et al., 1979;Snyder, 1985) allows for a number of different roles. It is interesting, in view of recent findings, that adenosine blocks release of glutamate from cortical slices (Dolphin and Archer, 1983), and that adenosine receptors and binding sites labeled by L-glutamate (Monaghan et al., 1985) show somewhat similar laminar distributions within the visual cortex (C. Shaw and M. Cynader, unpublished observations). We note, however, that the distributions of adenosine receptors and glutamate-related binding sites both alter their laminar distribution during development and that these patterns of changes are clearly different. The developmental patterns, which are specific and different for these, and other, receptors may allow for maximum modulation of the functions of any particular neurotransmitter system by adenosine at a specific time during postnatal development.
The identification of adenosine receptors in cat visual cortex and the description of their laminar binding pattern and ontogenesis add important information to the broadening picture of the chemical circuitry of this region of the brain. In conjunction with the description of the other receptor populations thus far studied, these data reveal a complex system with the possibility of multiple interactions among different neurotransmitters. Unraveling these interactions promises to be a major task for the future.