Feed-forward inhibition as a buffer of the neuronal input-output relation

Neuronal processing depends on the input-output (I/O) relation between the frequency of synaptic stimulation and the resultant axonal firing rate. The all-or-none properties of spike generation and active membrane mechanisms can make the neuronal I/O relation very steep. The ensuing nearly bimodal behavior may severely limit information coding, as minimal input fluctuations within the expected natural variability could cause neuronal output to jump between quiescence and maximum firing rate. Here, using biophysically and anatomically realistic computational models of individual neurons, we demonstrate that feed-forward inhibition, a ubiquitous mechanism in which inhibitory interneurons and their target cells are activated by the same excitatory input, can change a steeply sigmoid I/O curve into a double-sigmoid typical of buffer systems. The addition of an intermediate plateau stabilizes the spiking response over a broad dynamic range of input frequency, ensuring robust integration of noisy synaptic signals. Both the buffered firing rate and its input firing range can be independently and extensively modulated by biologically plausible changes in the weight and number of excitatory synapses on the feed-forward interneuron. By providing a soft switch between essentially digital and analog rate-code, this continuous control of the circuit I/O could dramatically increase the computational power of neuronal integration.     

Functional role of spines in the retinal horizontal cell network.

Compartmental models derived from serial electron-microscopic reconstructions of horizontal cell processes entering cone pedicles and rod spherules are used to show that these processes have the morphological and electrical characteristics of dendritic spines. Properties of these spines are incorporated into a distributed model of the horizontal cell network. Expressions relating the magnitude of conductance changes applied at the spine heads to hyperpolarization of cells within the network are derived. Model analyses show that spine properties play a critical role in determining network responses. Specifically, increasing spine stem resistance increases the network input resistance and space constant, hyperpolarizes the resting potential, decreases response to full-field light stimuli, and increases response to small light spots. Increasing spine-stem resistance also decouples potential at the spine head from potential at the cell body. This result suggests that the location of feedback neurotransmitter release sites (e.g., at the spine heads versus the cell body) may have a profound influence on properties of horizontal cell inhibition of cone response. Because of these important functional consequences, structurally realistic models of the horizontal cell network must incorporate spine properties.     

Monoclonal antibodies distinguish subtypes of retinal horizontal cells.

Sixteen hybridomas have been identified that secrete antibodies specific to horizontal cells in the carp retina. The hybridomas have been classified into three groups based on their antibody staining patterns: group I, staining associated with all horizontal cells; group II, staining associated with the most abundant subtype of horizontal cell (CH1); and group III, staining associated with other subtypes of horizontal cells. Most of the hybridomas fall in group II; some of these antibodies stain the entire horizontal cell, but others are specific only to the cell perikarya and do not stain axonal processes. Our results suggest that there are surface molecules specific (i) to all retinal horizontal cells, (ii) to individual subtypes of horizontal cells, and (iii) to portions of horizontal cells. Furthermore, a group II antibody, which recognizes a 48- to 50-kDa membrane protein, has been found to provide a substrate selective for horizontal cell growth. Horizontal cells plated on coverslips coated with this antibody remain healthy in culture and extend long and elaborate processes for at least 3 weeks.     

Dopamine decreases conductance of the electrical junctions between cultured retinal horizontal cells.

Horizontal cells from the white perch were isolated by enzymatic treatment and trituration of the retina and were maintained in culture for 1-5 days. Overlapping pairs of horizontal cells were identified, and the two cells were recorded from simultaneously, using whole-cell patch clamp techniques. Electrical coupling between cells was determined by passing current pulses into one cell, the driver cell, while (i) recording voltage changes in the other, follower cell, or (ii) measuring current flow into the follower cell. Most cell pairs of the same morphological type were coupled electrically, with coupling coefficients often greater than 0.9. Junctional resistance was typically found to be between 20 and 60 M omega and junctional conductance was between 150 and 500 nS. After application of 1-microliter pulses of dopamine (200 microM) to coupled pairs of cells, the coupling coefficient fell to approximately equal to 0.1, junctional resistance increased to 300-700 M omega, and junctional conductance decreased to 15-30 nS. Recovery of coupling took, for most cell pairs tested, 8-15 min after dopamine application. The exogenous application of 8-bromo-cyclic AMP (0.5-1 mM) also caused uncoupling of horizontal cell pairs; however, neither isoprenaline nor L-glutamate altered coupling significantly.     

Electrical synapses in retinal ON cone bipolar cells: subtype-specific expression of connexins

Retinal bipolar cells are known to form a complex, interconnecting network through electrical synapses that are either heterologous (with amacrine cells) or homologous (with other bipolar cells). These electrical synapses can be functionally as important as chemical synapses because their distinct properties provide a different character for the network. Much less is known, however, about electrical synapses in retinal bipolar cells than about chemical synapses. Here we report the molecular basis for electrical synapses in retinal bipolar cells, particularly ON cone bipolar cells. We have found variable connexin 36 (cx36) expression in different types of ON cone bipolar cells: cx36 message was found in some, but not all, ON cone bipolar cells (4 of 14 cells). In one specific type of ON cone bipolar cell (BPGus-GFP), however, cx36 was detected in 17 of 19 cells. Moreover, we have located cx36 puncta at the axonal terminals of BPGus-GFP cells, and we have found that these BPGus-GFP-associated cx36 puncta always colocalized with AII amacrine cell processes. Molecular and immunocytochemical evidence obtained in this study also shows that connexin 45 (cx45) is not present in BPGus-GFP cells. Taken together, our results suggest that connexins are expressed in bipolar cells in a neuronal subtype-specific manner and that cx36/cx36 gap junctions form the heterologous electrical synapses between AII amacrine cells and BPGus-GFP cells. Our findings imply that visual information can be differently processed by distinct subtypes of ON cone bipolar cells via electrical synapses.     

Ionotropic non-N-methyl-D-aspartate agonists induce retraction of dendritic spinules from retinal horizontal cells.

Horizontal cells invaginate the photoreceptors in the retina and form reciprocal synaptic connections in the cone pedicles. In fish retina the pattern of synaptic connections is plastic and modulated by the ambient light conditions. Numerous dendritic spinules protrude from the terminal horizontal-cell dendrites into the cone pedicle when the retina is light-adapted and are retracted during dark adaptation. The retraction of spinules can be induced during maintained illumination by an injection of the putative cone transmitter L-glutamate or its analogue kainic acid into the vitreous humor. The formation and the retraction of spinules have a time course of minutes. Activation of protein kinase C through phorbol esters initiates the formation of spinules, but the retraction has not yet been linked to a specific second messenger. Herein we report that physiological concentrations of the glutamate analogs quisqualic acid and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid induce retraction of spinules during maintained illumination. (+/-)-trans-1-Amino-1,3-cyclopentanedicarboxylic acid, an agonist for the metabotropic quisqualic acid receptor, was without effect on spinule retraction. N-Methyl-D-aspartate and L-2-amino-4-phosphonobutyric acid, agonists at other types of glutamate receptors, were also without any effect. The effects of the active agonists persisted when synaptic transmission was blocked. In the presence of the ionotropic quisqualate receptor antagonist 6-cyclo-7-nitro-quinoxaline-2,3-dione the effects of all active agonists were blocked. These results demonstrate that activation of ionotropic quisqualate receptors on the horizontal-cell membrane can induce dendritic spinule retraction, a process associated with dark adaptation.     

Dopamine modulates the kinetics of ion channels gated by excitatory amino acids in retinal horizontal cells.

Upon exposure to dopamine, cultured teleost retinal horizontal cells become more responsive to the putative photoreceptor neurotransmitter L-glutamate and to its analog kainate. We have recorded unitary and whole-cell currents to determine the mechanism by which dopamine enhances ion channels activated by these agents. In single-channel recordings from cell-attached patches with agonist in the patch pipette, the frequency of 5- to 10-pS unitary events, but not their amplitude, increased by as much as 150% after application of dopamine to the rest of the cell. The duration of channel openings also increased somewhat, by 20-30%. In whole-cell experiments, agonists with and without dopamine were applied to voltage-clamped horizontal cells by slow superfusion. Analysis of whole-cell current variance as a function of mean current indicated that dopamine increased the probability of channel opening for a give agonist concentration without changing the amount of current passed by an individual channel. For kainate, noise analysis additionally demonstrated that dopamine did not alter the number of functional channels. Dopamine also increased a slow spectral component of whole-cell currents elicited by kainate or glutamate, suggesting a change in the open-time kinetics of the channels. This effect was more pronounced for currents induced by glutamate than for those induced by kainate. We conclude that dopamine potentiates the activity of horizontal cell glutamate receptors by altering the kinetics of the ion channel to favor the open state.     

Effects of gamma-aminobutyric acid on skate retinal horizontal cells: evidence for an electrogenic uptake mechanism.

In the retinae of many vertebrates, there are classes of horizontal cell that probably utilize gamma-aminobutyric acid (GABA) as a neurotransmitter. As with other amino acid transmitter agents, the postsynaptic action of GABA is thought to be terminated by uptake into neurons and glia surrounding the release site. The present study examined whether an uptake system for GABA could be detected in isolated skate horizontal cells by means of electrophysiological methods. Pressure ejection of GABA onto voltage-clamped horizontal cells produced an inward current that showed no sign of desensitization regardless of the GABA concentration. The dose-response relationship followed simple Michaelis-Menten kinetics, with a half-maximal response elicited at approximately 110 microM. Nipecotic acid produced a similar current and reduced the responses to GABA when introduced in the bath solution prior to the GABA pulse. On the other hand, application of 500 microM muscimol or 1 mM baclofen, GABAA and GABAB receptor agonists, respectively, were completely without effect. The GABA-induced current was not blocked by superfusion with 500 microM bicuculline, 500 microM picrotoxin, or 500 microM phaclofen. However, the responses to GABA were abolished when the cells were superfused in Ringer's solution in which choline or lithium had been substituted for sodium, and were reduced when the extracellular chloride concentration was decreased from 266 mM to 16 mM. Current-voltage data showed a maximal response to GABA when the cells were held at or below their resting potential. At more depolarized levels, the inward current became progressively smaller until, near +50 mV, it could no longer be detected; over the range tested (-90 to +50 mV), the response never reversed into an outward current. These findings suggest that the GABA-induced currents in skate horizontal cells are mediated by an electrogenic uptake mechanism.     

Dopamine induces neurite retraction in retinal horizontal cells via diacylglycerol and protein kinase C.

Dopamine causes a significant retraction of neurites of bull-head catfish horizontal cells maintained in culture. The effects of dopamine are blocked by haloperidol and SCH 23390, a D1 antagonist, but not by sulpiride, a D2 antagonist. The dopamine-induced morphological changes were mimicked by SKF 38393, a D1 agonist, but not by quinpirole, a D2 agonist. Kainate also caused process retraction, but other neuroactive substances tested including glutamate, 5-hydroxytryptamine, N-methyl-D-aspartate, gamma-aminobutyric acid, and glycine caused only minor changes in neurite length. Cyclic AMP analogues do not induce neurite retraction in horizontal cells, indicating that this effect of dopamine is not mediated by cyclic AMP. However, a protein kinase C activator (phorbol 12-myristate 13-acetate) and synthetic diacylglycerol analogs (1-oleoyl-2-acetyl-sn-glycerol and dioctanoglycerol) caused marked neurite retraction. Their effects, as well as the dopamine-induced changes, were blocked by staurosporine, a potent protein kinase antagonist. The results suggest that dopamine causes neurite retraction by the activation of protein kinase C via diacylglycerol.     

Calcium influx selects the fast mode of endocytosis in the synaptic terminal of retinal bipolar cells

To investigate the regulation of endocytosis by Ca(2+), we have made capacitance measurements in the synaptic terminal of depolarizing bipolar cells from the retina of goldfish. After a brief depolarization, all of the excess membrane was retrieved rapidly (tau approximately 1 s). But when the rise in free [Ca(2+)] was reduced by the introduction of Ca(2+) buffers [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate (BAPTA) or EGTA], a large fraction of the membrane was retrieved by a second, slower mechanism (tau > or = 10 s). The block of fast endocytosis by EGTA could be overcome by increasing the amplitude of the Ca(2+) current, demonstrating that Ca(2+) influx was the trigger for fast endocytosis. These manipulations of the Ca(2+) signal altered the relative proportions of fast and slow endocytosis but did not modulate the rate constants of these processes. A brief stimulus that triggered fast endocytosis did not generate a significant rise in the spatially averaged [Ca(2+)], indicating that Ca(2+) regulated endocytosis through an action close to the active zone. The slow mode of retrieval occurred at the resting [Ca(2+)]. These results demonstrate that Ca(2+) influx couples fast endocytosis and exocytosis at this synapse.     

Glutamate and 2-amino-4-phosphonobutyrate evoke an increase in potassium conductance in retinal bipolar cells.

Although there is general agreement that L-glutamate can produce a depolarizing inward current to account for the hyperpolarizing (OFF) bipolar cell response, the conductance mechanism underlying the depolarizing (ON) response has been difficult to establish satisfactorily. To investigate the ionic bases of the center responses, we studied the whole-cell currents controlled by L-glutamate and its analogues in solitary bipolar cells from salamander retina. We report here two groups of isolated bipolar cells: one group responded to L-glutamate with the previously described inward current [Attwell, D., Mobbs, P., Tessier-Lavigne, M. & Wilson, M. (1987) J. Physiol. (London) 387, 125-161] and a second group showed an outward current that reversed at about -70 mV. Both were associated with an increase in membrane conductance. In addition, DL-2-amino-4-phosphonobutyrate, a compound diagnostic for ON-bipolar cell activity [Slaughter, M. M. & Miller, R. F. (1981) Science 211, 182-185], elicited outward currents that closely resembled those seen in response to L-glutamate and, furthermore, that were shown to arise from an increase in conductance to potassium ions. Thus the presence of two distinct conductances controlled by L-glutamate in solitary cells would provide one mechanism for generating the ON and OFF light responses at the bipolar cell level in the intact retina.     

A circadian clock regulates rod and cone input to fish retinal cone horizontal cells.

In the vertebrate retina, the light responses of post-receptor neurons depend on the ambient or background illumination. Using intracellular recording, we have found that a circadian clock regulates the light responses of dark-adapted fish cone horizontal cells. Goldfish were maintained on a 12-hr light/12-hr dark cycle. At different times of the day or night, retinas were superfused in darkness for 90 min ("prolonged darkness"), following which horizontal cells were impaled without the aid of any light flashes. In some of the experiments, fish were kept in constant darkness for 3-48 hr prior to surgery. After prolonged darkness during the night, but not during the day, the light responses of L-type cone horizontal cells resembled those of rod horizontal cells with respect to threshold, waveform, intensity-response functions, and spectral sensitivity. Following light sensitization during the night and day, the light responses of rod and cone horizontal cells were clearly different with respect to threshold, waveform, intensity-response functions, and spectral sensitivity. Under conditions of constant darkness for two full light/dark cycles, average responses of cone horizontal cells to a bright light stimulus during the subjective day were greater than during the subjective night. Prior reversal of the light/dark cycle reversed the 24-hr rhythm of cone horizontal cell responses to bright lights. In addition, following one full cycle of constant darkness, average cone horizontal cell spectral sensitivity during the subjective night closely matched that of rod horizontal cells, whereas average cone horizontal cell spectral sensitivity during the subjective day was similar to that of red (625 nm) cones. These results indicate that the effects of dark adaptation depend on the time of day and are regulated by a circadian clock so that cone input to cone horizontal cells predominates in the day and rod input predominates in the night.     

Feedback from luminosity horizontal cells mediates depolarizing responses of chromaticity horizontal cells in the Xenopus retina.

It has been proposed that the depolarizing responses of chromaticity horizontal cells (C-HCs) to red light depend on a feedback signal from luminosity horizontal cells (L-HCs) to short-wavelength-sensitive cones in the retinas of lower vertebrates. In this regard we studied the C-HCs of the Xenopus retina. C-HCs and L-HCs were identified by physiological criteria and then injected with neurobiotin. The retina then was incubated with peanut agglutinin, which stains red-but not blue-sensitive cones. Electron microscopic examination revealed that L-HCs contact all cone classes, whereas C-HCs contact only blue-sensitive cones. Simultaneous recordings from C-HC/L-HC pairs established that when the L-HC was saturated by a steady bright red light, C-HCs alone responded to a superimposed blue stimulus. In response to red test flashes, the C-HC response was delayed by approximately 30 msec with respect to the L-HC response. Isolated HCs of both subtypes were examined by whole-cell patch clamp. Both responded to kainate with sustained inward currents and to quisqualate or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) with desensitizing currents from a negative holding potential; i.e., both have AMPA-type glutamate receptors. gamma-Aminobutyric acid or glycine opened a chloride channel in the L-HC, whereas the C-HC was unresponsive to either inhibitory amino acid. Since glycine has been shown to abolish selectively the depolarizing response of the C-HC, this finding and other pharmacological data strongly implicate the L-HC in the underlying circuit. Moreover, because the C-HC does not respond to gamma-aminobutyric acid, the neurotransmitter of the L-HC, by elimination, a feedback synapse from L-HC to blue cone is the most plausible mechanism for the creation of depolarizing responses in C-HCs.     

Dopamine alters glutamate receptor desensitization in retinal horizontal cells of the perch (Perca fluviatilis).

The patch-clamp technique in combination with a fast liquid filament application system was used to study the effect of dopamine on the glutamate receptor desensitization in horizontal cells of the perch (Perca fluviatilis). Kinetics of ligand-gated ion channels in fish horizontal cells are modulated by dopamine. This modulation is presumably mediated by a cAMP-dependent protein phosphorylation. Before incubation with dopamine, the glutamate receptors of horizontal cells activate and desensitize with fast time constants. In the whole-cell recording mode, fast application of the agonists L-glutamate, quisqualate, or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid prior to the dopamine incubation gives rise to fast transient currents with peak values of about 200 pA that desensitize within 100 ms. Kainate as agonist produced higher steady-state currents but no transient currents. After incubation of the cells with dopamine for 3 min, the desensitization was significantly reduced and the agonists L-glutamate, quisqualate, or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid induced steady-state currents with amplitudes that were similar to the previously observed transient currents. Kainate-induced currents were only slightly affected. Fast desensitizing currents upon fast application of L-glutamate were also recorded from outside-out patches that were excised from horizontal cells before incubation with dopamine. The currents from excised patches desensitized to a steady-state level of about 0.2 of the peak amplitude with time constants of less than 2 ms. When the outside-out patches were excised from cells after dopamine incubation, steady-state currents were enhanced and no transient currents were observed. The results may indicate that the dopamine-dependent modulation of glutamate-induced currents, which is presumably mediated by a protein phosphorylation, is due to an alteration of the desensitization of the glutamate receptors.     

TRPM1 is a component of the retinal ON bipolar cell transduction channel in the mGluR6 cascade

An essential step in intricate visual processing is the segregation of visual signals into ON and OFF pathways by retinal bipolar cells (BCs). Glutamate released from photoreceptors modulates the photoresponse of ON BCs via metabotropic glutamate receptor 6 (mGluR6) and G protein (Go) that regulates a cation channel. However, the cation channel has not yet been unequivocally identified. Here, we report a mouse TRPM1 long form (TRPM1-L) as the cation channel. We found that TRPM1-L localization is developmentally restricted to the dendritic tips of ON BCs in colocalization with mGluR6. TRPM1 null mutant mice completely lose the photoresponse of ON BCs but not that of OFF BCs. In the TRPM1-L-expressing cells, TRPM1-L functions as a constitutively active nonselective cation channel and its activity is negatively regulated by Go in the mGluR6 cascade. These results demonstrate that TRPM1-L is a component of the ON BC transduction channel downstream of mGluR6 in ON BCs.     

Neurotransmission plays contrasting roles in the maturation of inhibitory synapses on axons and dendrites of retinal bipolar cells

Neuronal output is modulated by inhibition onto both dendrites and axons. It is unknown whether inhibitory synapses at these two cellular compartments of an individual neuron are regulated coordinately or separately during in vivo development. Because neurotransmission influences synapse maturation and circuit development, we determined how loss of inhibition affects the expression of diverse types of inhibitory receptors on the axon and dendrites of mouse retinal bipolar cells. We found that axonal GABA but not glycine receptor expression depends on neurotransmission. Importantly, axonal and dendritic GABA_A receptors comprise distinct subunit compositions that are regulated differentially by GABA release: Axonal GABA_A receptors are down-regulated but dendritic receptors are up-regulated in the absence of inhibition. The homeostatic increase in GABA_A receptors on bipolar cell dendrites is pathway-specific: Cone but not rod bipolar cell dendrites maintain an up-regulation of receptors in the transmission deficient mutants. Furthermore, the bipolar cell GABA_A receptor alterations are a consequence of impaired vesicular GABA release from amacrine but not horizontal interneurons. Thus, inhibitory neurotransmission regulates in vivo postsynaptic maturation of inhibitory synapses with contrasting modes of action specific to synapse type and location.Interneurons of the CNS control neuronal excitability through release of γ-aminobutyric acid (GABA) and glycine. How inhibition modifies neuronal output depends largely on the types of presynaptic interneurons making synapses onto a postsynaptic cell, and the location and densities of these synapses (1–3). Moreover, inhibitory receptor types with distinct transmitter affinities and kinetics present on the axon or dendrites of an individual neuron can critically shape its output (3–6). Although much is known about how different inhibitory synapses shape the spatiotemporal activity patterns of mature neurons, it is less clear what factors regulate the expression of inhibitory receptors at these synapses during development in vivo. Is the expression of distinct inhibitory receptor types within a cellular compartment (axon or dendrite) regulated coordinately or independently? Conversely, is the expression of the same receptor type at different cellular compartments of an individual neuron regulated by common or separate factors?To answer these questions, we assessed expression of inhibitory receptors on the axon and dendrites of individual glutamatergic retinal neurons in mice with genetically suppressed inhibition. We generated retina-specific knockouts of the vesicular inhibitory amino acid transporter (VIAAT), which mediates uptake of GABA or glycine into synaptic vesicles (7, 8). We perturbed inhibition because it has been found previously to influence pre- and postsynaptic maturation of GABAergic synapses (9–12). However, whether inhibitory receptor expression at the “input” and “output” compartments of an individual neuron is coordinately regulated by activity remains unknown. We focused on retinal bipolar cells (BCs) because of the rich variety of inhibitory synapses found on these neurons. Moreover, the many types of BCs enabled us to determine whether inhibitory transmission plays a uniform or diverse role in regulating inhibitory synapses across cell types that signal in parallel. We compared the postsynaptic maturation of GABA- and glycinergic synapses on cone BCs (CBCs) versus rod BCs (RBCs) that operate at different light levels. Among CBCs, we analyzed both ON and OFF BC types, which depolarize or hyperpolarize to light increments, respectively (13).     

Vesicle association and exocytosis at ribbon and extraribbon sites in retinal bipolar cell presynaptic terminals

Synaptic vesicles release neurotransmitter by following a process of vesicle docking and exocytosis. Although these steps are well established, it has been difficult to observe and measure these rates directly in living synapses. Here, by combining the direct imaging of single synaptic vesicles and synaptic ribbons, I measure the properties of vesicle docking and evoked and spontaneous release from ribbon and extraribbon locations in a ribbon-type synaptic terminal, the goldfish retinal bipolar cell. In the absence of a stimulus, captured vesicles near ribbons associate tightly and only rarely undock or undergo spontaneous exocytosis. By contrast, vesicle capture at outlier sites is less stable and spontaneous exocytosis occurs at a higher rate. In response to a stimulus, exocytic events cluster near ribbons, but show no evidence of clustering away from ribbon sites. Together, the results here indicate that, although vesicles can associate and fuse both near and away from synaptic sites, vesicles at synaptic ribbons associate more stably and fusion is more tightly linked to stimuli.     

Immunological synapse arrays: patterned protein surfaces that modulate immunological synapse structure formation in T cells

T cells are activated by recognition of foreign peptides displayed on the surface of antigen presenting cells (APCs), an event that triggers assembly of a complex microscale structure at the T cell-APC interface known as the immunological synapse (IS). It remains unresolved whether the unique physical structure of the synapse itself impacts the functional response of T cells, independent of the quantity and quality of ligands encountered by the T cell. As a first step toward addressing this question, we created multicomponent protein surfaces presenting lithographically defined patterns of tethered T cell receptor (TCR) ligands (anti-CD3 "activation sites") surrounded by a field of tethered intercellular adhesion molecule-1 (ICAM-1), as a model substrate on which T cells could be seeded to mimic T cell-APC interactions. CD4(+) T cells seeded on these surfaces polarized and migrated; on contact with activation sites, T cells assembled an IS with a structure modulated by the physical pattern of ligand encountered. On surfaces patterned with focal spots of TCR ligand, T cells stably interacted with activation sites, proliferated, and secreted cytokines. In contrast, T cells interacting with activation sites patterned to preclude centralized clustering of TCR ligand failed to form stable contacts with activation sites, exhibited aberrant PKC- clustering in a fraction of cells, and had significantly reduced production of IFN-gamma. These results suggest that focal clustering of TCR ligand characteristic of the "mature" IS may be required under some conditions for full T cell activation.