9-Aminoacridine blocks NMDA and AMPA receptors by different mechanisms

Tricyclic mono- and dicationic compounds (derivatives of 9-aminoacridine) antagonize AMPA and NMDA glutamate receptors. The aim of the present study was to compare mechanisms of the 9-aminoacridine action on AMPA and NMDA receptors. Experiments were carried out by whole-cell patch-clamp technique on native receptors from rat brain neurons. An important peculiarity of the 9-aminoacridine action on NMDA receptors is the large slope of the concentration dependence, which suggests the binding of two molecules in the channel. AMPA receptors blockade also demonstrated interesting features. In contrast to the NMDA receptor channel block, inhibition of AMPA receptors is voltage-independent. 9-Aminoacridine and its dicationic analog demonstrated similar anti-AMPA activity. For classical AMPA-receptor channel blockers (derivatives of adamantane and phenylcyclohexyl) it was demonstrated that dicationic analogs are much more potent than monocationic analogs. We conclude that 9-aminoacridine binds to a specific site in AMPA receptors. This finding opens a possibility to develop a new family of non-competitive antagonists of AMPA receptors.     

Characterization of ionotropic glutamate receptors in insect neuro-muscular junction

Pharmacological properties of ionotropic glutamate receptors from Calliphora vicina larvae neuro-muscular junction (C. vicina iGlurs) were studied by two-electrode voltage-clamp technique. Characteristics of the ion channel pore were analyzed using a 26-member series of channel blockers, which includes mono- and dicationic derivatives of adamantane and phenylcyclohexyl. Structure-activity relationships were found to be markedly similar to the Ca2+-permeable AMPA receptors (AMPAR) but not NMDA receptors (NMDAR) channel subtype seen in vertebrates. Like AMPARs the channels of C. vicina iGlurs are sensitive mainly to dicationic compounds with 6-7 spacers between hydrophobic headgroup and terminal aminogroup. Study of the voltage dependence of block demonstrated that, like AMPARs, the C. vicina iGlur channels, are permeable to organic cations with dimensions exceeding 10 A. Concentration dependence of block suggests the presence of two distinct channel populations with approximately 20-fold different sensitivity to cationic blockers. The recognition domain properties are more complex. Besides glutamate, the channels can be activated by kainate, quisqualate and domoate. Competitive antagonists of AMPAR and NMDAR are virtually inactive against the C. vicina iGlurs as well as allosteric modulators GYKI 52466 and PEPA. Surprisingly, the responses were potentiated 3 times by 100 mkM of cyclothiazide. We conclude that the channel-forming domain of C. vicina iGlurs is AMPAR-like, whereas the recognition domain is specific.     

Voltage-dependent and -independent block of α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor channels

Polyamine-containing toxins and synthetic dicationic derivatives of adamantane and phenylcyclohexyl selectively antagonize Ca(2+)-permeable α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor channels. These compounds demonstrate voltage-dependent open-channel block and are trapped by closed channels. In this study, we describe an alternative mechanism of non-competitive AMPA receptor inhibition caused by 9-aminoacridine and some of its derivatives. These compounds exhibit similar potency against Ca(2+)-permeable and Ca(2+)-impermeable AMPA receptors. The inhibition is largely voltage-independent, binding and unbinding do not require presence of agonist. We conclude that 9-aminoacridine binds to a shallow site in the AMPA receptor, which is located above the activation gate. A comparison of three-dimensional structures of the antagonists suggests that the 'V-like' shape of the hydrophobic headgroup favors voltage-dependent binding to the deep site in the channel pore, whereas the compounds possessing flat aromatic headgroups preferably bind to the shallow site. The characterization of the novel mechanism of AMPA receptor channel antagonism opens a way to develop a new family of pharmacological agents, which can be of scientific and practical importance.     

Determinants of trapping block of N-methyl-d-aspartate receptor channels

Open channel blockers of NMDA receptors interact with the channel gate in different ways. Compounds like MK-801 and phencyclidine exhibit pronounced trapping block, whereas 9-aminoacridine and tetrapentylammonium cannot be trapped. Some blockers such as memantine and amantadine exhibit intermediate properties, so called 'partial trapping'. To analyze the determinants of trapping we have synthesized a series of mono- and dicationic derivatives of phenylcyclohexyl. The blocking action of these compounds as well as that of amantadine has been studied on native NMDA receptors of hippocampal pyramidal neurons. Use-dependence and kinetics of the blockade have been analyzed to estimate the degree of trapping. Dimensions of the blocking molecules apparently do not correlate with their trapping. However, the degree of trapping is voltage-dependent and correlates with the kinetics of unblock. For instance, amantadine behaved as non-trapping blocker at positive voltages, but demonstrated significant trapping at negative voltages. The data may be explained by the model in which the NMDA receptor channel has two binding sites: the shallow and deep ones. Binding to the deep but not to the shallow site allows trapping of the blockers.     

Structural characteristics of ionotropic glutamate receptors revealed by channel blockade

The topography of the channel binding site in glutamate receptors (AMPA and NMDA types of rat brain neurons, receptors of molluscan neurons and insect muscle), and in two subtypes of nicotinic cholinoreceptors (in frog muscle and cat sympathetic ganglion), has been investigated by comparison of the blocking effects of mono- and dicationic derivatives of adamantane and phenylcyclohexyl. The channels studied can be divided into two groups. The first one includes AMPA receptor and glutamate receptors of mollusc and insect, and is characterised by the absence of activity of monocationic drugs and the strong dependence of dicationic once on the internitrogen distance in the drug molecule. The second group includes NMDA receptor and both nicotinic cholinoreceptors. Contrary, here the blocking potency of monocations and dications are practically equal irrespective of molecule length. The data obtained suggest that hydrophobic and nucleophilic components of the binding site are located close to each other in the channels of the NMDA receptor type but are separated by approximately 10 A in the AMPA receptor channel.     

Structure of glutamate receptor ion channels and mechanisms of their blockade by organic cations

Structural determinants of blocking the glutamate receptors of AMPA and NMDA subtypes, were studied. Close location of hydrophobic and ammonium groups is necessary for affective blocking of the NMDA receptor channels, whereas blockers of the AMPA receptor channels have a distance of about 10 angstroms between these two groups. Models of the channels meeting these topographic data have been devised using a molecular mechanics approach. The accomplished studies revealed molecular basis of channel blockade of the NMDA and AMPA receptors. This may allow designing predictable new blocking compounds with a desired selectivity.     

Increase of desensitization as an additional mode of action for classical NMDA receptor channel blockers

It is well known that not all effects of such classical NMDA receptor channel blockers as spermine and phylantotoxins are madiated via channel blockade. To determine if these side effects have any functional role we studied inhibition of NMDA receptors by derivatives of phenylcyclohexyl and clinically approved memantine. We showed that these drugs not only blocked the channel pore but also potentiated desensitization of NMDA receptors. Apparently, these effects are not interrelated because there was no correlation between a degree of “trapping” and a level of channel desensitization induced by these blockers. Yet, in contrast to channel blockade, which was strongly voltage-dependent, these compounds affected NMDA receptor desensitization in a voltage-independent manner. Based on these facts, we suppose the existence of two different binding sites for memantine and functionally related compounds, one being responsible for channel blockade and another being coupled to channel desensitization. This possibility should be taken into account when interpreting experimental and clinical effects of these channel blockers.     

Effects of ionotropic glutamate receptor channel blockers on the development of audiogenic seizures in Krushinski-Molodkina rats

The action of noncompetitive blockers of glutamate receptors has been investigated on Krushinski-Molodkina rats genetically-prone to audiogenic seizures. The selective blockers of NMDA receptor channels, memantine and IEM-1921, and their dicationic homologues, IEM-1925 and IEM-1754, capable of blocking in varying degrees both NMDA and Ca-permeable AMPA receptor channels, were studied. The drugs were injected intramuscularly to rats with the different time intervals (30 min, 1, 2 or 3 hours) before sound signal. The effects of the drugs on latent period of initial locomotor activity provoked by audio stimulation (8 kHz sine-wave tone, 90 dB volume), the appearance of clonic convulsions of different intensities, and, finally, tonic convulsions with limb and tail extension were evaluated. Within 30 min after injection IEM-1921 at a dose of 5 mg/kg, 33% of rats manifested a complete absence of convulsive reactions to sound, and in 59% of rats audiogenic seizures occured only in the form of motor excitation without a generalized clonic-tonic convulsions. Memantine at a dose of 5 mg/kg did not cause a complete blockade of seizures, but after 1 h of injection in 50% of the rats and after 2 h in 70% of rats a weakening of the audiogenic seizures to the level of motor excitation only was observed. After 3 hrs after administration of blockers its anticonvulsive action weakened significantly (p < 0.01). Dicationic blockers that block both NMDA and AMPA/kainate receptors, IEM-1925 (in doses of 0.001-20.0 mg/kg) and IEM-1754 (0.025-50.0 mg/kg), did not affect audiogenic clonic-tonic convulsive reactions. The involvement of activation of NMDA and calcium permeable AMPA/kainate receptors in the pathogenesis of audiogenic seizures is discussed.     

Ion channels of glutamate receptors of nerve-muscle junction of larva of the fly <Emphasis Type="Italic">Calliphora vicina</Emphasis> demonstrate a high structural homology with vertebrate AMPA-channels

In experiments on the nerve-muscle junction of larvae of the fly Calliphora vicina, regularities of the blocking action of organic cations on ion channels of glutamate postsynaptic receptors have been studied. The measurements were performed by potential fixation on the muscle cell membrane. In total, effects of 26 compounds were studied. The following regularities of structural-functional relations have been revealed: (1) the channels are not blocked by monocation compounds; (2) bication derivatives block efficiently the channels with a certain distance between hydrophobic group and terminal amino group; (3) bication compounds with trimethylammonium terminal group are significantly more efficient than compounds with non-substituted amino group. All these regularities are characteristics of blockade of the AMPA channels, but not of the vertebrate-type NMDA channels. Earlier it was shown that differences in structural-functional relations during blockade of the AMPA and MNDA channels were determined by different location of the hydrophobic and hydrophilic components of the binding area as well as by different diameter of the channels. The fact that channels of the fly larva receptor demonstrate the same regularities of blockade as the vertebrate AMPA channels indicates their structural similarity that is a consequence of their high homology.     

Role of NMDA and AMPA glutamate receptors in the mechanism of korazol-induced convulsions in mice

The potency of mono- and dikationic derivatives of adamantane and phenylcyclohexyl to prevent seizures induced in mice by intraperitoneal administration of 80 mg/kg pentylenetetrazol (corazol), was studied. Monocationic derivatives of phenylcyclohexyl, being the selective channel blockers of NMDA glutamate receptors, as well memantine and MK-801 in micromolar concentrations, prevented both clonic and tonic components of corazol-induced convulsions. Their dicatonic derivatives which are channel blockers of NMDA and AMPA types of glutamate receptors, failed to prevent clonic seizures but at submicromolar concentrations prevented the tonic extensions provoked by corazol. Evidently, convulsive action of corazol originating from suppression of GABA-ergic inhibition is realized through activation of glutamergic synaptic transmission, and NMDA receptors are mainly involved in genesis of clonic seizures whereas activation of AMPA receptors is important for the tonic component of the corazol-induced syndrome.     

Study functional architecture of NMDA receptor channels with their blockers

Blockade of ionic currents through NMDA receptor channels in acutely isolated rat hippocampal neurons by tetraalkylammonium compounds, 9-aminoacridine and Mg2+ was studied using whole-cell patch-clamp technique. The currents through NMDA channels were elicited by 100 microM aspartate application in a Mg(2+)-free 3 microM glycine-containing solution. An analysis of the kinetics, charge transfer and dependencies of the stationary current inhibition on the membrane potential and the agonist and the blocker concentrations showed that the blockers affect NMDA channel closure, desensitization and the agonist dissociation in different ways. The size of the blocker proved to be the determinant of the blocker action on the NMDA channel gating machinery: large blockers prevented the channel closure and/or desensitization, smaller ones only partly affected these processes, while the smallest did not affect at all. It was shown that the apparent blocker affinity to the channel, 1/IC50, depended not only on the microscopic dissociation constant, Kd, but also on the number of the blocker binding sites, their mutual dependence, and, which is much more important, on the blocker interaction with the channel gating machinery. Based upon the data obtained, there was advanced hypotheses on the NMDA channel geometry and the structure of its gating machinery. The diameter of the channel at the level of the activation gate was estimated as 11 A.     

Intrinsic versus extrinsic voltage sensitivity of blocker interaction with an ion channel pore

Many physiological and synthetic agents act by occluding the ion conduction pore of ion channels. A hallmark of charged blockers is that their apparent affinity for the pore usually varies with membrane voltage. Two models have been proposed to explain this voltage sensitivity. One model assumes that the charged blocker itself directly senses the transmembrane electric field, i.e., that blocker binding is intrinsically voltage dependent. In the alternative model, the blocker does not directly interact with the electric field; instead, blocker binding acquires voltage dependence solely through the concurrent movement of permeant ions across the field. This latter model may better explain voltage dependence of channel block by large organic compounds that are too bulky to fit into the narrow (usually ion-selective) part of the pore where the electric field is steep. To date, no systematic investigation has been performed to distinguish between these voltage-dependent mechanisms of channel block. The most fundamental characteristic of the extrinsic mechanism, i.e., that block can be rendered voltage independent, remains to be established and formally analyzed for the case of organic blockers. Here, we observe that the voltage dependence of block of a cyclic nucleotide–gated channel by a series of intracellular quaternary ammonium blockers, which are too bulky to traverse the narrow ion selectivity filter, gradually vanishes with extreme depolarization, a predicted feature of the extrinsic voltage dependence model. In contrast, the voltage dependence of block by an amine blocker, which has a smaller “diameter” and can therefore penetrate into the selectivity filter, follows a Boltzmann function, a predicted feature of the intrinsic voltage dependence model. Additionally, a blocker generates (at least) two blocked states, which, if related serially, may preclude meaningful application of a commonly used approach for investigating channel gating, namely, inferring the properties of the activation gate from the kinetics of channel block.     

Comparison of the anticonvulsant activity of organic mono- and di-cations and their potential to inhibit NMDA and AMPA glutamate receptors

Effects of mono- and dicationic derivatives of adamantine and phenylcyclohexyl were studied on: (i) open channels of NMDA and AMPA glutamate receptors in the experiments on the isolated rat brain neurones, and (ii) convulsions induced by intraventricular injections of NMDA or kainate in mice. Monocations inhibited the NMDA receptors in vitro and prevented convulsions induced by NMDA in vivo, but failed to affect both the AMPA receptors and kainite-induced convulsions. Dications (IEM-1754 and IEM-1925) revealed both anti-NMDA and anti-AMPA potency in vitro, were highly effective against kainite-induced convulsions and excelled monocations in preventing the NMDA-induced ones. Evidently some steps connected with the AMPA receptor activity are involved in the genesis of the NMDA-induced convulsions. Anticonvulsant potency of IEM-1754 and IEM-1925 is comparable with those of known NMDA receptor inhibitors: memantine and MK-801. The IEM-1754 and IEM-1925 show no side effects. An incomplete correspondence between the activity in vitro and in vivo found studying some derivatives, may be due to peculiarities of their pharmacokinetics.     

Involvement of ionotropic glutamate receptors in the genesis of arecoline-induced tremor

The muscarinic agonist arecoline (6 mg/kg, subcutaneously in mice) induced a long-lasting tremor. The inhibitory potency of non-competitive antagonists of ionotropic glutamate receptors has been studied. These antagonists are the derivatives of adamantane and phenylcyclohexyl. A part of them: monocationic compounds, selectively block the NMDA-receptor channels, their dicationic analogues affecting both channels of the NMDA- and the AMPA-glutamate receptors. Monocationic blockers effectively reduced the arecoline-evoked tremor and their potency correlated with ability to block the NMDA-receptor channels. Dicationic blockers revealed protective effect only in low range doses (0.0001-0.01 microM/kg). Further increase of the dose reduced or completely abolished this effect. This suggests that the NMDA-receptors are involved in the genesis of arecoline-evoked tremor. The only moderate blockade of the AMPA-receptors potentiates the drug blocking action but the prevalent blockade of these receptors impedes the effect on arecoline-evoked tremor.     

Na channel activation gate modulates slow recovery from use-dependent block by local anesthetics in squid giant axons.

The time course of recovery from use-dependent block of sodium channels caused by local anesthetics was studied in squid axons. In the presence of lidocaine or its quaternary derivatives, QX-222 and QX-314, or 9-aminoacridine (9-AA), recovery from use-dependent block occurred in two phases: a fast phase and a slow phase. Only the fast phase was observed in the presence of benzocaine. The fast phase had a time constant of several milliseconds and resembled recovery from the fast Na inactivation in the absence of drug. Depending on the drug present, the magnitude of the time constant of the slow phase varied (for example at -80 mV): lidocaine, 270 ms; QX-222, 4.4 s; QX-314, 17 s; and 9-AA, 14 s. The two phases differed in the voltage dependence of recovery time constants. When the membrane was hyperpolarized, the recovery time constant for the fast phase was decreased, whereas that for the slow phase was increased for QX-compounds and 9-AA or unchanged for lidocaine. The fast phase is interpreted as representing the unblocked channels recovering from the fast Na inactivation, and the slow phase as representing the bound and blocked channels recovering from the use-dependent block accumulated by repetitive depolarizing pulse. The voltage dependence of time constants for the slow recovery is consistent with the m-gate trapping hypothesis. According to this hypothesis, the drug molecule is trapped by the activation gate (the m-gate) of the channel. The cationic form of drug molecule leaves the channel through the hydrophilic pathway, when the channel is open. However, lidocaine, after losing its proton, may leave the closed channel rapidly through the hydrophobic pathway.     

Effects of blockade of ionotropic glutamate receptors on the development of pentylenetetrazole kindling in mice

Effects of mono- and dicationic derivatives of adamantane and phenylcyclohexyl on the petyleneterazole-induced (35 mg/kg i. p.) kindling were studied in the experiments on mice. Monocationic derivative of phenylcyclohexyl IEM-1921, effectively retarded the development of kindling beginning the dose 0.0001 microM/kg. Memantine: derivative of adamantane (derivative of adamatane) produced the same effect with 100-fold increased dose. Dicationic derivative ofphenylcyclohexyl: IEM-1925, is able to block equally the open channels of both NMDA and subtype of Ca-permeable AMPA receptors. Its effect on kindling differed markedly from selective NMDA antagonists (IEM-1921 and memantine) in more complicated dose-dependence. The retardation of kindling IEM-1925 was induced at 0.001 microM/kg. On the contrary, a 10-time lower dose: 0.0001 microM/kg, facilitated the development of kindling. The observed difference in the activity of selective NMDA antagonists and the drugs combining anti-NMDA and anti-AMPA potency indicates that both types of ionotropic glutamate receptors are involved in the mechanism of petyleneterazole-induced kindling. The integral effect of channel blockade evoked by drugs seems to be dependent not only upon the ratio of the receptor types but on the kinetics of drug action, too.     

Kinetics of 9-aminoacridine block of single Na channels

The kinetics of 9-aminoacridine (9-AA) block of single Na channels in neuroblastoma N1E-115 cells were studied using the gigohm seal, patch clamp technique, under the condition in which the Na current inactivation had been eliminated by treatment with N-bromoacetamide (NBA). Following NBA treatment, the current flowing through individual Na channels was manifested by square-wave open events lasting from several to tens of milliseconds. When 9-AA was applied to the cytoplasmic face of Na channels at concentrations ranging from 30 to 100 microM, it caused repetitive rapid transitions (flickering) between open and blocked states within single openings of Na channels, without affecting the amplitude of the single channel current. The histograms for the duration of blocked states and the histograms for the duration of open states could be fitted with a single-exponential function. The mean open time (tau o) became shorter as the drug concentration was increased, while the mean blocked time (tau b) was concentration independent. The association (blocking) rate constant, kappa, calculated from the slope of the curve relating the reciprocal mean open time to 9-AA concentration, showed little voltage dependence, the rate constant being on the order of 1 X 10(7) M-1s-1. The dissociation (unblocking) rate constant, l, calculated from the mean blocked time, was strongly voltage dependent, the mean rate constant being 214 s-1 at 0 mV and becoming larger as the membrane being hyperpolarized. The voltage dependence suggests that a first-order blocking site is located at least 63% of the way through the membrane field from the cytoplasmic surface. The equilibrium dissociation constant for 9-AA to block the Na channel, defined by the relation of l/kappa, was calculated to be 21 microM at 0 mV. Both tau -1o and tau -1b had a Q10 of 1.3, which suggests that binding reaction was diffusion controlled. The burst time in the presence of 9-AA, which is the sum of open times and blocked times, was longer than the lifetime of open channels in the absence of drug. All of the features of 9-AA block of single Na channels are compatible with the sequential model in which 9-AA molecules block open Na channels, and the blocked channels could not close until 9-AA molecules had left the blocking site in the channels.     

Mechanism of the voltage sensitivity of IRK1 inward-rectifier K+ channel block by the polyamine spermine

IRK1 (Kir2.1) inward-rectifier K+ channels exhibit exceedingly steep rectification, which reflects strong voltage dependence of channel block by intracellular cations such as the polyamine spermine. On the basis of studies of IRK1 block by various amine blockers, it was proposed that the observed voltage dependence (valence approximately 5) of IRK1 block by spermine results primarily from K+ ions, not spermine itself, traversing the transmembrane electrical field that drops mostly across the narrow ion selectivity filter, as spermine and K+ ions displace one another during channel block and unblock. If indeed spermine itself only rarely penetrates deep into the ion selectivity filter, then a long blocker with head groups much wider than the selectivity filter should exhibit comparably strong voltage dependence. We confirm here that channel block by two molecules of comparable length, decane-bis-trimethylammonium (bis-QA(C10)) and spermine, exhibit practically identical overall voltage dependence even though the head groups of the former are much wider ( approximately 6 A) than the ion selectivity filter ( approximately 3 A). For both blockers, the overall equilibrium dissociation constant differs from the ratio of apparent rate constants of channel unblock and block. Also, although steady-state IRK1 block by both cations is strongly voltage dependent, their apparent channel-blocking rate constant exhibits minimal voltage dependence, which suggests that the pore becomes blocked as soon as the blocker encounters the innermost K+ ion. These findings strongly suggest the existence of at least two (potentially identifiable) sequentially related blocked states with increasing numbers of K+ ions displaced. Consequently, the steady-state voltage dependence of IRK1 block by spermine or bis-QA(C10) should increase with membrane depolarization, a prediction indeed observed. Further kinetic analysis identifies two blocked states, and shows that most of the observed steady-state voltage dependence is associated with the transition between blocked states, consistent with the view that the mutual displacement of blocker and K+ ions must occur mainly as the blocker travels along the long inner pore.     

Effects of NMDA receptor antagonists on nociceptive responsesin vivo: Comparison of antagonists acting at the glycine site with uncompetitive antagonists

Summary We have shown that members of a new series of tricyclic pyridophthalazine diones, defined as glycine_B site NMDA antagonistsin vitro, are selective and systemically active NMDA antagonistsin vivo. In electrophysiological tests in -chloralose anaesthetised rats, these compounds reduced nociceptive reflex responses. In conscious rats they displayed analgesic properties. These glycine_B antagonists were compared electrophysiologically with several uncompetitive NMDA channel blockers. The degree of voltage dependence previously reportedin vitro related to the effectiveness of the agents against different amplitude nociceptive responses of spinal cord neuronesin vivo.     

Early activation of Ca2+‐permeable AMPA receptors reduces neurite outgrowth in embryonic chick retinal neurons

Calcium entry through Ca²⁺‐permeable AMPA/kainate receptors may activate signaling cascades controlling neuronal development. Using the fluorescent Ca²⁺‐indicator Calcium Green 1‐AM we showed that the application of kainate or AMPA produced an increase of intracellular [Ca²⁺] in embryonic chick retina from day 6 (E6) onwards. This Ca²⁺ increase is due to entry through AMPA‐preferring receptors, because it was blocked by the AMPA receptor antagonist GYKI 52466 but not by the N‐methyl‐D ‐aspartic acid (NMDA) receptor antagonist AP5, the voltage‐gated Ca²⁺ channel blockers diltiazem or nifedipine, or by the substitution of Na⁺ for choline in the extracellular solution to prevent the depolarizing action of kainate and AMPA. In dissociated E8 retinal cultures, application of glutamate, kainate, or AMPA reduced the number of neurites arising from these cells. The effect of kainate was prevented by the AMPA/kainate receptor antagonist CNQX and by GYKI 52466 but not by AP5, indicating that the reduction in neurite outgrowth resulted from the activation of AMPA receptors. Blocking Ca²⁺ influx through L‐type voltage‐gated Ca²⁺ channels with diltiazem and nifedipine prevented the effect of 10–100 μM kainate but not that of 500 μM kainate. In addition, joro spider toxin‐3, a blocker of Ca²⁺‐conducting AMPA receptors, prevented the effect of all doses of kainate. Neither GABA, which is depolarizing at this age in the retina, nor the activation of metabotropic glutamate receptors with tACPD mimicked the effects of AMPA receptor activation. Calcium entry via AMPA receptor channels themselves may therefore be important in the regulation of neurite outgrowth in developing chick retinal cells. © 2001 John Wiley & Sons, Inc. J Neurobiol 49: 200–211, 2001