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Effects of blocking non-N-methyl-d -aspartate receptors on visual responses of neurons in the cat visual cortex
Neuroscience Vol. 94, No. 3, pp. 697–703, 1999 697 Copyright q 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/99 $20.00+0.00
Function of non-NMDA receptors in cat visual cortex
Pergamon PII: S0306-4522(99)00334-6
EFFECTS OF BLOCKING NON-N-METHYL-d-ASPARTATE RECEPTORS ON VISUAL RESPONSES OF NEURONS IN THE CAT VISUAL CORTEX H. SATO,*†‡ Y. HATA* and T. TSUMOTO* *Department of Neurophysiology, Osaka University Medical School, Yamadaoka, Suita, Osaka, Japan †School of Health and Sport Sciences, Osaka University, 1-17 Machikaneyama, Toyonaka, 560-0043 Osaka, Japan
Abstract—To elucidate the function of non-N-methyl-d-aspartate types of glutamate receptors in the primary visual cortex of the adult cat, we studied the effects of the iontophoretically applied glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3dione and d-amino-5-phosphonovalerate. Antagonists were applied with ejecting currents that selectively blocked non-N-methyl-daspartate receptors. Among 93 cells in which stable recordings were obtained, 6-cyano-7-nitroquinoxaline-2,3-dione reduced the visual response in all cells. The average response magnitude during 6-cyano-7-nitroquinoxaline-2,3-dione administration was reduced to 11.7% of the control (average ejecting current: 41.2 nA). The effect of 6-cyano-7-nitroquinoxaline-2,3-dione was obvious throughout all cortical layers. The effect of d-amino-5-phosphonovalerate on the visual response was tested in 14 cells and it was also effective in blocking the visual response: the average response magnitude during d-amino-5-phosphonovalerate administration was 45.0% of the control (average ejecting current: 41.4 nA). The effect of 6-cyano-7-nitroquinoxaline-2,3-dione on the response was compared in individual cells at both high and low firing rates in order to determine whether a differential effect exists on the level of firing activity of cells due to secondary inactivation of voltage-dependent N-methyl-d-aspartate receptors. However, no indication of response dependency on firing rate was seen with 6-cyano-7-nitroquinoxaline-2,3-dione. We suggest that excitatory transmission at the geniculocortical and corticocortical synapses seems to be strongly dependent on non-N-methyl-d-aspartate receptors throughout the primary visual cortex of the adult cat, and that both non-N-methyl-d-aspartate and N-methyl-d-aspartate type glutamate receptors function additively. q 1999 IBRO. Published by Elsevier Science Ltd. Key words: non-NMDA receptor, cat area 17, iontophoresis of CNQX, visual response.
Glutamate is known to be a major excitatory transmitter in the neocortex. Ionotropic receptors for glutamate can be divided into N-methyl-d-aspartate (NMDA) receptors and a-amino-3hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate (non-NMDA) receptors. Excitatory synaptic transmission in the central visual pathway seems to be largely dependent on glutamatergic synapses. 4,8,10,11,21,22,27–29 NMDA receptors are activated in a voltage-dependent manner, and are known to play a role in controlling plasticity in young animals 1,6,14,19 and in amplification of the visual response in the adult brain. 5 However, continuously applying an NMDA receptor antagonist as used in kittens, it is concluded that NMDA receptors make a major contribution to excitatory transmission in the visual cortex of the adult cat. 17 A similar view was also extended to the kitten visual cortex. 13 In the present study, we tested the effect of blocking nonNMDA receptors on visual responses with iontophoretically administered 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) in the primary visual cortex of the cat to elucidate the essential roles of non-NMDA receptors, and the possible functional interaction between non-NMDA and NMDA receptors. Preliminary results of this work have already appeared elsewhere. 20
EXPERIMENTAL PROCEDURES
Preparation Twelve adult cats, each weighing 2–5 kg, were used in this study. Dexamethasone acetate (Decadron-A, Banyu Pharm.) was injected (0.1 mg, i.m.) 24–36 h before the start of the experiment. Atropine (0.02 mg/kg, i.m.) and hydroxyzine hydrochloride (Atarax-P, Pfizer, 1 mg/kg, i.m.) were injected 20 min before surgery. All efforts were made to minimize animal suffering and to reduce the number of animals used. For that purpose, the depth of anesthesia was carefully checked throughout the experiments and every effort was made to collect as much data as possible from each animal after stable recording conditions were achieved. Owing to the nature of this study, the use of alternatives to in vivo techniques was not possible. Surgical procedures were all in accordance with NIH guidelines for the care of experimental animals (National Institute of Health, Committee on Care and Use of Laboratory Animals, 1985) and the regulations of the Animal Care Committee of Osaka University Medical School. Animals were anesthetized with ketamine (5 mg/kg, i.m.), followed by a mixture of halothane (2–3%) or isoflurane (2–3%) and N2O/O2 (2:1), and the trachea and femoral vein were cannulated. In the present study, ketamine, which is known to block NMDA receptors, 26 was used for induction of anesthesia. However, ketamine is known to be a very short-lasting anesthetic. For example, the effects of intravenously injected ketamine (4 mg/kg) on the electroencephalogram of the cat 2 or that of intramuscularly injected ketamine (10 mg/kg) on the consciousness of human subjects 3 disappear within 30 min and our recording experiment started more than 4 h after the injection of ketamine. Therefore, the residual effects of ketamine on neuronal activity during the recording experiment are assumed to be negligible. A local anesthetic, lidocaine, was given at pressure points and around surgical incisions. Monitoring of the electrocardiogram (ECG) and heart rate was started following induction of anesthesia. After the initial surgery, the animal was placed in a stereotaxic headholder, then paralysed with gallamine triethiodide (10 mg/kg/h, i.v.) or pancuronium bromide (0.08 mg/kg/h, i.v.), and a mixture of electrolytes, glucose (5%) and the antibiotic cefotiam dihydrochloride (Pansporin, Takeda, 80 mg/kg/ day), and maintained under artificial ventilation. The electrolyte solution used for the infusion was changed from Ringer’s solution (Na 1 147, K 1 4, Ca 21 4.5 and Cl 2 155.5 mEq/l) to a high K 1 solution (Na 1
‡To whom correspondence should be addressed at: the School of Health and Sport Sciences. Tel.: 181-6-6850-6021; fax: 181-6-6850-6030. E-mail address: [email protected] (H. Sato) Abbreviations: AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate; APV, d-amino-5-phosphonovalerate; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; ECG, electrocardiogram; GluR, glutamate receptor subunit; NMDA, N-methyl-d-aspartate; PBS, phosphate-buffered saline; PSTH, peristimulus time histogram; QA, quisqualate. 697
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Fig. 1. Example of spike histogram for selectivity test of CNQX. (A) The intensity of ejecting current for CNQX administration was determined to be that which selectively antagonized QA-induced firings. A non-NMDA receptor selective agonist, QA (Q), and NMDA-receptor selective agonist, NMDA (N), were administered alternately for 30 s, and 240 nA of CNQX and 240 to 250 nA of APV selectively blocked QA and NMDA, respectively. Recording for a layer III cell. Bin width: 2000 ms. (B, C) Examples of the increase in NMDA effect during repetitive administration which were successively recorded from two cells in the same penetration. NMDA was intermittently administered four times, indicated by thick bars (duration 30 s, interval 30 s and ejecting current 230 nA). Bin width: 1000 ms.
35, K 1 20, Cl 2 35 and l-lactate 20 mEq/l) every 3 h to adjust the electrolyte concentration in the blood. The infusion rate was 2 ml/ kg/h. The nictitating membrane was retracted and the pupil was dilated by topical application of a mixture of tropicamide (0.5%) and phenylephrine hydrochloride (0.5%) (Mydrin-P, Santen). The eyes were refracted using O2-permeable contact lenses so as to be focused on a tangent screen or CRT display at 57 cm in front of the animal. For the craniotomy, lidocaine was carefully injected subcutaneously over the skull before cutting the skin. A square-shaped hole was made by opening the skull, dura and arachnoid above the occipital cortex for insertion of the recording electrodes under anesthesia. The depth of anesthesia during this period was judged as adequate because a significant heart rate change (more than 10%) was not observed when the skin was cut. During recordings of neuronal activity, the concentration of halothane or isoflurane was reduced to 0.3–0.5% or 0.5–1.0%, respectively, in N2O/O2 (2:1). The halothane or isoflurane concentration was increased if the heart rate changed by more than 10% when the tail was pinched with the tip of a small, ribbed forceps. This test was repeated once every hour. Rectal temperature was maintained at 37–388C with a thermostatically controlled heating pad. End-tidal CO2 concentration was adjusted to 3.5–4.0%. ECG and heart rate were monitored continuously throughout the experiments. Throughout the course of experiments, which lasted 36–72 h, the monitored physiological parameters, ECG waveform, heart rate, end-tidal CO2 concentration and body temperature, were kept constant. Urination volume was monitored every 6 h and was found to be almost equal to the volume of the infused electrolyte solution. Visual stimulation The positions and sizes of the receptive fields were first plotted on a tangent screen. Next, receptive field properties, dominant eye, optimal orientation and direction of stimulus, response characteristics to a stationary flashed bar or spot, and tuning to stimulus length and
velocity were assessed with a hand-held Keeler Pantoscope. Then, visual stimuli generated by a computer (PC9801RA, NEC) were displayed on a CRT monitor (PC-TV455, NEC) using software originally written by Dr A. Mikami and modified by us for the precise and quantitative analysis of visual responses. Most of the experiments were conducted under moderately light-adapted conditions: luminances of visual stimuli and the background were 19–22 and 1.4–1.6 cd/m 2, respectively. For stimulation, a bright bar stimulus of preferred orientation and length was moved back and forth across the receptive field. The direction of motion was always orthogonal to the orientation of the bar. Stimulus velocity was 3.2–208/s. Recording of single-unit activity and iontophoresis Five-barrel glass micropipettes were used for extracellular recording of the action potentials of single neurons in area 17 and for iontophoretic administration of CNQX (Tocris, Buckhurst Hill, U.K.), quisqualate (QA; Research Biochemicals, Natick, MA, U.S.A.), d-amino5-phosphonovalerate (APV; Sigma, St Louis, MO, U.S.A.) and NMDA (Sigma) to the neurons under observation. The tip of the recording pipette protruded by 15–20 mm beyond the tip of the drug pipette. The tip diameter of each drug pipette was 2–4 mm. Drugs were dissolved in distilled water at the following concentrations (mM): CNQX, 20; QA, 20; APV, 50; NMDA, 20. The pH of all drug solutions was adjusted to 8.5 with HCl and NaOH. The recording pipette was filled with 0.5 M sodium acetate containing 4% Pontamine Sky Blue for histological identification of recording sites. Peristimulus time histograms (PSTHs) of unit responses to visual stimulation were constructed with a signal processor (7T17, NEC-San-ei) before, during and after the iontophoretic administration of drugs. Ejecting currents for CNQX, QA, APV and NMDA were between 25 and 280, 210 and 280, 220 and 280, and 210 and 280 nA, respectively. Retaining currents were between 15 and 120 nA. PSTHs during drug administration were constructed after the firing activity of the recorded cell reached a stable level, usually 2 or 3 min after the start of ejection. A control PSTH was constructed by
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between dye marks, based on the micrometer reading, by the actual distance measured between dye marks, and then multiplying by that ratio. RESULTS
Stable recordings were obtained from 93 cells in area 17, and these cells were tested with iontophoretically administered glutamatergic agonists and antagonists. Cells that did not show appropriate recovery (change in spike numbers of the response within ^25% of those of the control) from the drug administration were omitted from analysis. Selectivity test of 6-cyano-7-nitroquinoxaline-2,3-dione
Fig. 2. Effects of CNQX and APV on the visual response of a layer III complex cell. PSTHs of responses to the visual stimulus before, during and after iontophoretic administration of CNQX with a current of 215 nA or APV with 240 nA. A bright bar (orientation and direction of motion are shown at the top) was swept across the receptive field at a velocity of 4.08/s. Spikes were accumulated during 20 sweeps of the stimulus presentation. Both CNQX and APV reduced visual response and spontaneous firings. Bin width: 12 ms.
accumulating responses during 10–20 trials of visual stimulus presentation. Response magnitude was defined as the total number of spikes in the visual response evoked during 10–20 repetitions of the stimulus presentation. The possibility that CNQX might differentially exert an effect on the response at different firing frequencies was assessed in individual cells by comparing the number of response spikes elicited by movement in the preferred and non-preferred directions. Histology At the end of each penetration, dye marks were produced by applying tip-negative currents (intensity 7.5–10 mA, duration 1 s at 0.5 Hz, 100 pulses). Dye deposits were placed at the end point of each penetration and at a few points at an interval of 500–1000 mm along the course where the electrode was retracted. After the recording experiments, the animals were deeply anesthetized with sodium pentobarbital (40 mg/kg, i.v.) and perfused transcardially with buffered saline (pH 7.4) followed by 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS). Blocks of the occipital cortex were cut out and immersed in 30% sucrose in PBS for 48 h. Sixty-micrometer-thick frozen coronal sections were cut on a microtome and kept in PBS. Sections were stained with Cresyl Violet. The laminar location of recording sites was then identified under light microscopic observation. Shrinkage of cortical tissues was corrected by dividing the distance expected
At the beginning of each electrode penetration, we determined the appropriate dose of CNQX sufficient to selectively block non-NMDA receptors. At high concentrations, CNQX is known to affect not only non-NMDA receptors, but also NMDA receptors. 9,18 It is therefore necessary to administer CNQX in a dosage range that selectively and effectively blocks only non-NMDA receptors. Since the effective current for drug administration differs between electrodes depending on tip diameter, distance between the tips of the recording and drug pipettes, and other factors, we determined the effective drug currents for each electrode by testing the effects of iontophoresed antagonists on agonists with a time-course similar to that for the test of drug effects on the visual response (from approximately 2 to 6 min). An example of such a selectivity test for CNQX is shown in Fig. 1. The cell was located in layer III of the cortex. A non-NMDA receptor agonist, QA, and an NMDA receptor agonist, NMDA, were administered alternately for 30 s interspersed with a rest interval of 30 s. During CNQX administration with 240 nA, the QA-induced response was selectively antagonized while the NMDA response was spared. Conversely, during APV administration, the NMDA-induced response was selectively blocked while the QA response was spared. In Fig. 1, following CNQX administration, responses induced by NMDA increased gradually. It is unlikely that this is due to a non-specific augmenting effect of NMDA following the high CNQX dose, because the NMDA response continued to increase even after CNQX administration ceased and the QA response recovered, indicating a flushing out of CNQX. Rather, this can be explained by a gradual increase in agonist concentration at the tip of the drug pipette due to removal of the anion, which had soaked into the pipette when retaining current was passed. This explanation is supported by the fact that a gradual increase in the number of spike discharges was often observed during repetitive iontophoresis of NMDA (Fig. 1B, C). Therefore, for the penetration shown at Fig. 1A, we used CNQX and APV with an ejecting current of 240 and 250 nA, respectively. Ejecting current for CNQX was determined in a similar manner for all electrode tracks. Laminar difference in antagonists Iontophoretically applied CNQX reduced the visual response in all 93 cells recorded from all layers of the primary visual cortex. An example of the effect of the glutamatergic antagonists on the response of a layer III complex cell is shown in Fig. 2. Since this cell was tested with binocular stimulation, the
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Fig. 3. Laminar analysis of effect of CNQX on visual responses. Percentage of the number of spikes in the visual response during CNQX administration to that of the control was calculated for each cell. The spontaneous firing rate was subtracted. The layer in which the cell was located is shown on the left. The average percentage responses during CNQX administration and CNQX current (%; nA values in parentheses) for layers II/III, IV, V and VI were 4.5 ^ 9.8 (S.D.; 234.7), 19.7 ^ 22.5 (241.6), 7.0 ^ 7.0 (248.7) and 14.3 ^ 16.5 (244.3), respectively. Total number of cells was 58.
response to motion of the stimulus bar consisted of two response peaks, which correspond to ipsilateral eye response (the larger peak) and contralateral eye response (the smaller peak), respectively. The visual response and spontaneous firings were strongly reduced by 215 nA of CNQX, and 4 min after the start of ejection they were completely abolished (second PSTH from the top). Administration of the NMDA receptor antagonist APV by 240 nA also strongly reduced the visual response of this cell (fourth PSTH from top). These results suggest that the visual response of cells in the superficial layer depends on both non-NMDA and NMDA receptors, which is consistent with the findings of Fox et al. 5 The distribution of CNQX effects among layers is summarized in Fig. 3. Fifty-eight cells in which the laminar location could be clearly identified histologically were included in this analysis. In this figure, each dot corresponds to a percentage of the magnitude of response during CNQX administration to that of the control response for each cell. The effect of CNQX was most pronounced in layer II/III cells, where the average ratio of response magnitude in the presence of CNQX to that of the control was 4.5 ^ 9.8% (S.D.). CNQX was also effective to a certain extent in other layers, and the magnitude of the visual response in layers IV, V and VI was reduced to 19.7 ^ 22.5%, 7.0 ^ 7.0% and 14.3 ^ 16.5%, respectively. The laminar difference of the CNQX effects was statistically significant between layers II/III and IV (P , 0.02, t 2.56, d.f. 33), layers II/III and VI (P , 0.05, t 2.13, d.f. 30), and between layers II/III and other layers (P , 0.05, t 2.26, d.f. 56), but not significant between layers II/III and V (P . 0.4, t 0.64, d.f. 23). The average response magnitude of all the recorded cells (n 93) during CNQX administration was 11.7 ^ 17.6% (S.D.) of the control (average ejecting current: 241.2 ^ 22.0 nA). However, the suppressive effect of APV was substantially smaller than that of CNQX. Among 14 cells tested, the average response magnitude during APV administration was
Fig. 4. Effects of CNQX and APV on the visual response of a simple cell in layer IV PSTHs before, during and after administration of CNQX (210 nA) or APV (210 nA), or administration of both (210 nA of APV and 25 nA of CNQX). Both CNQX and APV reduced the visual response and spontaneous firings, and the effects of CNQX and APV were additive (fourth and fifth PSTHs from top). Stimulus velocity: 48/s. Other conventions are as in Fig. 2.
45.0 ^ 39.4% (S.D.) of the control (average ejecting current: 241.4 ^ 17.5 nA), and the difference in response magnitude between CNQX and APV was statistically significant (P , 0.001, t 5.39, d.f. 105, t-test). The average response magnitudes during APV application of cells recorded in layers II/III (n 4) and IV–VI (n 10) were 22.8 ^ 36.4% and 56.1 ^ 38.0%, respectively. The difference between these two values was not statistically significant (P . 0.1, t 1.50, d.f. 12, t-test), even though the sample size seemed to be too small for meaningful statistical analysis. An example of the fairly potent effect of APV in a simple layer IV cell is shown in Fig. 4. This cell was quite sensitive to CNQX and
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Fig. 5. Neuronal firing rate and effect of CNQX. The plot shows the firing frequency for the visual response of individual cells (n 48) in response to stimulus motion in the preferred and non-preferred directions. (A) Data for control trials. Regression line: f(x) 0.38x 1 3.32. Correlation coefficient: r 0.68. (B) Data for CNQX trials; f(x) 0.36x 1 0.76, r 0.58.
administration of CNQX with 210 nA almost completely abolished visual response (second PSTH from the top; response magnitude: 2.1% of the control). Administration of APV with 210 nA also reduced the visual response of this cell to 27.1% of the control. This result is inconsistent with the previous report by Fox et al., 4 in which APV was found to have no effect on the visual response of cells in layers IV–VI in the cat primary visual cortex. An additional administration of CNQX with 25 nA completely abolished both the visual response and spontaneous activity, confirming that the effects of APV and CNQX are due to antagonism of a different population of glutamate receptors and that the effects of the two drugs are additive. Firing frequency and effect of 6-cyano-7-nitroquinoxaline2,3-dione It is well known that activation of NMDA receptors exhibits a membrane voltage dependency, i.e. NMDA receptors are more active under depolarization. 16,24,25 There is a possibility that depolarization by non-NMDA receptor activation during visual inputs may enhance activation of the NMDA receptors in the observed visual responses. In this case, blockade of the non-NMDA receptors with CNQX should result in indirect suppression of the NMDA receptors. To clarify this point, we analysed the relationship between the firing frequency of the visual response and the effect of CNQX. To do this, we compared the firing frequency in response to stimulus motion in the preferred and non-preferred directions with or without the presence of CNQX for individual cells. Directional selectivity for each cell was defined based on the following relation: Direction Selectivity Index DSI 1 2 Rnp =Rp ; where Rp represents the number of spikes elicited in response to stimulus movement in the preferred direction at the optimal orientation and Rnp represents the number of spikes elicited to movement in the opposite direction. A cell was excluded from analysis if the cell was completely direction selective (i.e. DSI 1 under control conditions), or if the response to either the preferred or non-preferred stimulus direction was completely abolished by CNQX. The rationale for eliminating such
cells is that it is impossible to determine the extent to which the excitatory response was abolished by CNQX, since there were no spikes in the visual response. Forty-eight cells were included in this analysis. Figure 5 shows the firing frequencies of cells in response to stimulus motion in the preferred and non-preferred directions without (Fig. 5A) or with (Fig. 5B) CNQX. The average firing frequencies of the 48 cells to the preferred and non-preferred directions were 18.84 and 10.56 Hz in the control, and 6.46 and 3.10 Hz during CNQX administration, respectively. The average DSI values were 0.50 ^ 0.31 (S.D.) and 0.51 ^ 0.46 during the control phase and under CNQX administration, respectively, and these differences were not statistically significant. The slopes of the two regression lines for these two distributions (0.38 for the control and 0.36 for CNQX) were almost equal and there was no indication of a stronger suppression of response at higher firing frequency, i.e. in response to the motion stimulus in the preferred direction. Therefore, it seems unlikely that the blockade of non-NMDA receptors secondarily abolished NMDA receptor-mediated excitation of responses with higher firing frequencies. DISCUSSION
The blockade of non-NMDA receptors with iontophoretically administered CNQX eliminated nearly all neuronal activity in layers II–VI of the primary visual cortex of cats. This implies that activation of non-NMDA receptors is predominant for excitatory transmission at both geniculocortical and corticocortical synapses. However, the effect of CNQX seems to vary among cells, particularly in layers IV and VI (Fig. 3). This may be due partly to a difference in the subtypes of glutamate receptor expressed on the cells in each layer. There is evidence that glutamate receptor subtypes having different sensitivity to AMPA are differentially expressed among layers in the cat visual cortex; the GluR1 and GluR2/3 subunits are particularly abundant in non-pyramidal and pyramidal neurons, respectively. 7 This, in turn, suggests that the excitatory transmission of geniculocortical synapses in layer IV, which contains a larger population of stellate cells, is more dependent on GluR1 type receptors, while it is more dependent on GluR2 in other layers. However, we do
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not have a reasonable explanation for the variability of the effect of CNQX in layer VI. This point should be further assessed with a larger population of cortical cells. In the present study, an NMDA receptor antagonist, APV, also exerted a suppressive effect on the visual response of cells throughout all layers, and the average response magnitude during APV administration was 45.0% of the control. This result is partly consistent with the previous results of Miller et al. 17 that APV administered with an osmotic minipump profoundly suppressed visual responses of all cells within the APV-perfused area in the visual cortex of adult cats, even though the authors did not describe the laminar distribution of recorded cells. The substantial effects of APV found in the present study on visual responses in layers IV–VI is inconsistent with previous reports in which APV had no effect on visual responses in these layers. 4 In the light of the new findings, we suggest that non-NMDA and NMDA receptors function additively in the primary visual cortex. Consistent with this notion, NMDA receptors are hypothesized to be involved in the excitatory transmission not only in layers II/III, but also in granular and infragranular layers in in vivo 8 or in vitro experiments. 12,15,23
No evidence was seen that depolarization induced by activation of non-NMDA receptors secondarily evoked augmentation of visual responses through activation of voltagedependent NMDA receptors. However, this does not mean that NMDA receptors are not active within a range of membrane potential levels in vivo. Rather, as suggested previously, 5 the degree of activation of NMDA receptors is proportionally the same for small responses and large responses for an individual cell. Taken together, the available data are consistent with the idea that the effects of the activation of non-NMDA and NMDA receptors are additive in the generation of spike discharges.
Acknowledgements—We wish to thank Dr Nigel W. Daw for critical reading of an early version of the manuscript, Dr Hiroshi Tamura and Takafumi Akasaki for technical assistance, and William D. Stenson for editorial assistance. This work was supported by Grants-in-Aid for Scientific Research on Priority Areas 09268221 and 08458268 from the Japanese Ministry of Education, Science, Sports and Culture to H.S., “Research for the Future” Program JSPS-RFTF96L00201 from the Japan Society for the Promotion of Science to H.S. and also by a grant from the Kato Memorial Bioscience Foundation to H.S.
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25. Thomson A. M. (1986) Comparison of responses to transmitter candidates at an N-methyl-d-aspartate receptor mediated synapse, in slices of rat cerebral cortex. Neuroscience 17, 37–47. 26. Thomson A. M., West D. C. and Lodge D. (1985) An N-methyl-d-aspartate receptor-mediated synapse in rat cerebral cortex: a site of action of ketamine? Nature 313, 479–481. 27. Tsumoto T., Hagihara K., Sato H. and Hata Y. (1987) NMDA receptors in the visual cortex of young kittens are more effective than those of adult cats. Nature 327, 513–514. 28. Tsumoto T., Masui H. and Sato H. (1986) Excitatory amino acid transmitters in neuronal circuits of the cat visual cortex. J. Neurophysiol. 55, 469–483. 29. Tsumoto T. (1990) Excitatory amino transmitters and their receptors in neural circuits of the cerebral neocortex. Neurosci. Res. 9, 79–102. (Accepted 3 June 1999)
Function of non-NMDA receptors in cat visual cortex
Pergamon PII: S0306-4522(99)00334-6
EFFECTS OF BLOCKING NON-N-METHYL-d-ASPARTATE RECEPTORS ON VISUAL RESPONSES OF NEURONS IN THE CAT VISUAL CORTEX H. SATO,*†‡ Y. HATA* and T. TSUMOTO* *Department of Neurophysiology, Osaka University Medical School, Yamadaoka, Suita, Osaka, Japan †School of Health and Sport Sciences, Osaka University, 1-17 Machikaneyama, Toyonaka, 560-0043 Osaka, Japan
Abstract—To elucidate the function of non-N-methyl-d-aspartate types of glutamate receptors in the primary visual cortex of the adult cat, we studied the effects of the iontophoretically applied glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3dione and d-amino-5-phosphonovalerate. Antagonists were applied with ejecting currents that selectively blocked non-N-methyl-daspartate receptors. Among 93 cells in which stable recordings were obtained, 6-cyano-7-nitroquinoxaline-2,3-dione reduced the visual response in all cells. The average response magnitude during 6-cyano-7-nitroquinoxaline-2,3-dione administration was reduced to 11.7% of the control (average ejecting current: 41.2 nA). The effect of 6-cyano-7-nitroquinoxaline-2,3-dione was obvious throughout all cortical layers. The effect of d-amino-5-phosphonovalerate on the visual response was tested in 14 cells and it was also effective in blocking the visual response: the average response magnitude during d-amino-5-phosphonovalerate administration was 45.0% of the control (average ejecting current: 41.4 nA). The effect of 6-cyano-7-nitroquinoxaline-2,3-dione on the response was compared in individual cells at both high and low firing rates in order to determine whether a differential effect exists on the level of firing activity of cells due to secondary inactivation of voltage-dependent N-methyl-d-aspartate receptors. However, no indication of response dependency on firing rate was seen with 6-cyano-7-nitroquinoxaline-2,3-dione. We suggest that excitatory transmission at the geniculocortical and corticocortical synapses seems to be strongly dependent on non-N-methyl-d-aspartate receptors throughout the primary visual cortex of the adult cat, and that both non-N-methyl-d-aspartate and N-methyl-d-aspartate type glutamate receptors function additively. q 1999 IBRO. Published by Elsevier Science Ltd. Key words: non-NMDA receptor, cat area 17, iontophoresis of CNQX, visual response.
Glutamate is known to be a major excitatory transmitter in the neocortex. Ionotropic receptors for glutamate can be divided into N-methyl-d-aspartate (NMDA) receptors and a-amino-3hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate (non-NMDA) receptors. Excitatory synaptic transmission in the central visual pathway seems to be largely dependent on glutamatergic synapses. 4,8,10,11,21,22,27–29 NMDA receptors are activated in a voltage-dependent manner, and are known to play a role in controlling plasticity in young animals 1,6,14,19 and in amplification of the visual response in the adult brain. 5 However, continuously applying an NMDA receptor antagonist as used in kittens, it is concluded that NMDA receptors make a major contribution to excitatory transmission in the visual cortex of the adult cat. 17 A similar view was also extended to the kitten visual cortex. 13 In the present study, we tested the effect of blocking nonNMDA receptors on visual responses with iontophoretically administered 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) in the primary visual cortex of the cat to elucidate the essential roles of non-NMDA receptors, and the possible functional interaction between non-NMDA and NMDA receptors. Preliminary results of this work have already appeared elsewhere. 20
EXPERIMENTAL PROCEDURES
Preparation Twelve adult cats, each weighing 2–5 kg, were used in this study. Dexamethasone acetate (Decadron-A, Banyu Pharm.) was injected (0.1 mg, i.m.) 24–36 h before the start of the experiment. Atropine (0.02 mg/kg, i.m.) and hydroxyzine hydrochloride (Atarax-P, Pfizer, 1 mg/kg, i.m.) were injected 20 min before surgery. All efforts were made to minimize animal suffering and to reduce the number of animals used. For that purpose, the depth of anesthesia was carefully checked throughout the experiments and every effort was made to collect as much data as possible from each animal after stable recording conditions were achieved. Owing to the nature of this study, the use of alternatives to in vivo techniques was not possible. Surgical procedures were all in accordance with NIH guidelines for the care of experimental animals (National Institute of Health, Committee on Care and Use of Laboratory Animals, 1985) and the regulations of the Animal Care Committee of Osaka University Medical School. Animals were anesthetized with ketamine (5 mg/kg, i.m.), followed by a mixture of halothane (2–3%) or isoflurane (2–3%) and N2O/O2 (2:1), and the trachea and femoral vein were cannulated. In the present study, ketamine, which is known to block NMDA receptors, 26 was used for induction of anesthesia. However, ketamine is known to be a very short-lasting anesthetic. For example, the effects of intravenously injected ketamine (4 mg/kg) on the electroencephalogram of the cat 2 or that of intramuscularly injected ketamine (10 mg/kg) on the consciousness of human subjects 3 disappear within 30 min and our recording experiment started more than 4 h after the injection of ketamine. Therefore, the residual effects of ketamine on neuronal activity during the recording experiment are assumed to be negligible. A local anesthetic, lidocaine, was given at pressure points and around surgical incisions. Monitoring of the electrocardiogram (ECG) and heart rate was started following induction of anesthesia. After the initial surgery, the animal was placed in a stereotaxic headholder, then paralysed with gallamine triethiodide (10 mg/kg/h, i.v.) or pancuronium bromide (0.08 mg/kg/h, i.v.), and a mixture of electrolytes, glucose (5%) and the antibiotic cefotiam dihydrochloride (Pansporin, Takeda, 80 mg/kg/ day), and maintained under artificial ventilation. The electrolyte solution used for the infusion was changed from Ringer’s solution (Na 1 147, K 1 4, Ca 21 4.5 and Cl 2 155.5 mEq/l) to a high K 1 solution (Na 1
‡To whom correspondence should be addressed at: the School of Health and Sport Sciences. Tel.: 181-6-6850-6021; fax: 181-6-6850-6030. E-mail address: [email protected] (H. Sato) Abbreviations: AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate; APV, d-amino-5-phosphonovalerate; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; ECG, electrocardiogram; GluR, glutamate receptor subunit; NMDA, N-methyl-d-aspartate; PBS, phosphate-buffered saline; PSTH, peristimulus time histogram; QA, quisqualate. 697
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Fig. 1. Example of spike histogram for selectivity test of CNQX. (A) The intensity of ejecting current for CNQX administration was determined to be that which selectively antagonized QA-induced firings. A non-NMDA receptor selective agonist, QA (Q), and NMDA-receptor selective agonist, NMDA (N), were administered alternately for 30 s, and 240 nA of CNQX and 240 to 250 nA of APV selectively blocked QA and NMDA, respectively. Recording for a layer III cell. Bin width: 2000 ms. (B, C) Examples of the increase in NMDA effect during repetitive administration which were successively recorded from two cells in the same penetration. NMDA was intermittently administered four times, indicated by thick bars (duration 30 s, interval 30 s and ejecting current 230 nA). Bin width: 1000 ms.
35, K 1 20, Cl 2 35 and l-lactate 20 mEq/l) every 3 h to adjust the electrolyte concentration in the blood. The infusion rate was 2 ml/ kg/h. The nictitating membrane was retracted and the pupil was dilated by topical application of a mixture of tropicamide (0.5%) and phenylephrine hydrochloride (0.5%) (Mydrin-P, Santen). The eyes were refracted using O2-permeable contact lenses so as to be focused on a tangent screen or CRT display at 57 cm in front of the animal. For the craniotomy, lidocaine was carefully injected subcutaneously over the skull before cutting the skin. A square-shaped hole was made by opening the skull, dura and arachnoid above the occipital cortex for insertion of the recording electrodes under anesthesia. The depth of anesthesia during this period was judged as adequate because a significant heart rate change (more than 10%) was not observed when the skin was cut. During recordings of neuronal activity, the concentration of halothane or isoflurane was reduced to 0.3–0.5% or 0.5–1.0%, respectively, in N2O/O2 (2:1). The halothane or isoflurane concentration was increased if the heart rate changed by more than 10% when the tail was pinched with the tip of a small, ribbed forceps. This test was repeated once every hour. Rectal temperature was maintained at 37–388C with a thermostatically controlled heating pad. End-tidal CO2 concentration was adjusted to 3.5–4.0%. ECG and heart rate were monitored continuously throughout the experiments. Throughout the course of experiments, which lasted 36–72 h, the monitored physiological parameters, ECG waveform, heart rate, end-tidal CO2 concentration and body temperature, were kept constant. Urination volume was monitored every 6 h and was found to be almost equal to the volume of the infused electrolyte solution. Visual stimulation The positions and sizes of the receptive fields were first plotted on a tangent screen. Next, receptive field properties, dominant eye, optimal orientation and direction of stimulus, response characteristics to a stationary flashed bar or spot, and tuning to stimulus length and
velocity were assessed with a hand-held Keeler Pantoscope. Then, visual stimuli generated by a computer (PC9801RA, NEC) were displayed on a CRT monitor (PC-TV455, NEC) using software originally written by Dr A. Mikami and modified by us for the precise and quantitative analysis of visual responses. Most of the experiments were conducted under moderately light-adapted conditions: luminances of visual stimuli and the background were 19–22 and 1.4–1.6 cd/m 2, respectively. For stimulation, a bright bar stimulus of preferred orientation and length was moved back and forth across the receptive field. The direction of motion was always orthogonal to the orientation of the bar. Stimulus velocity was 3.2–208/s. Recording of single-unit activity and iontophoresis Five-barrel glass micropipettes were used for extracellular recording of the action potentials of single neurons in area 17 and for iontophoretic administration of CNQX (Tocris, Buckhurst Hill, U.K.), quisqualate (QA; Research Biochemicals, Natick, MA, U.S.A.), d-amino5-phosphonovalerate (APV; Sigma, St Louis, MO, U.S.A.) and NMDA (Sigma) to the neurons under observation. The tip of the recording pipette protruded by 15–20 mm beyond the tip of the drug pipette. The tip diameter of each drug pipette was 2–4 mm. Drugs were dissolved in distilled water at the following concentrations (mM): CNQX, 20; QA, 20; APV, 50; NMDA, 20. The pH of all drug solutions was adjusted to 8.5 with HCl and NaOH. The recording pipette was filled with 0.5 M sodium acetate containing 4% Pontamine Sky Blue for histological identification of recording sites. Peristimulus time histograms (PSTHs) of unit responses to visual stimulation were constructed with a signal processor (7T17, NEC-San-ei) before, during and after the iontophoretic administration of drugs. Ejecting currents for CNQX, QA, APV and NMDA were between 25 and 280, 210 and 280, 220 and 280, and 210 and 280 nA, respectively. Retaining currents were between 15 and 120 nA. PSTHs during drug administration were constructed after the firing activity of the recorded cell reached a stable level, usually 2 or 3 min after the start of ejection. A control PSTH was constructed by
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between dye marks, based on the micrometer reading, by the actual distance measured between dye marks, and then multiplying by that ratio. RESULTS
Stable recordings were obtained from 93 cells in area 17, and these cells were tested with iontophoretically administered glutamatergic agonists and antagonists. Cells that did not show appropriate recovery (change in spike numbers of the response within ^25% of those of the control) from the drug administration were omitted from analysis. Selectivity test of 6-cyano-7-nitroquinoxaline-2,3-dione
Fig. 2. Effects of CNQX and APV on the visual response of a layer III complex cell. PSTHs of responses to the visual stimulus before, during and after iontophoretic administration of CNQX with a current of 215 nA or APV with 240 nA. A bright bar (orientation and direction of motion are shown at the top) was swept across the receptive field at a velocity of 4.08/s. Spikes were accumulated during 20 sweeps of the stimulus presentation. Both CNQX and APV reduced visual response and spontaneous firings. Bin width: 12 ms.
accumulating responses during 10–20 trials of visual stimulus presentation. Response magnitude was defined as the total number of spikes in the visual response evoked during 10–20 repetitions of the stimulus presentation. The possibility that CNQX might differentially exert an effect on the response at different firing frequencies was assessed in individual cells by comparing the number of response spikes elicited by movement in the preferred and non-preferred directions. Histology At the end of each penetration, dye marks were produced by applying tip-negative currents (intensity 7.5–10 mA, duration 1 s at 0.5 Hz, 100 pulses). Dye deposits were placed at the end point of each penetration and at a few points at an interval of 500–1000 mm along the course where the electrode was retracted. After the recording experiments, the animals were deeply anesthetized with sodium pentobarbital (40 mg/kg, i.v.) and perfused transcardially with buffered saline (pH 7.4) followed by 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS). Blocks of the occipital cortex were cut out and immersed in 30% sucrose in PBS for 48 h. Sixty-micrometer-thick frozen coronal sections were cut on a microtome and kept in PBS. Sections were stained with Cresyl Violet. The laminar location of recording sites was then identified under light microscopic observation. Shrinkage of cortical tissues was corrected by dividing the distance expected
At the beginning of each electrode penetration, we determined the appropriate dose of CNQX sufficient to selectively block non-NMDA receptors. At high concentrations, CNQX is known to affect not only non-NMDA receptors, but also NMDA receptors. 9,18 It is therefore necessary to administer CNQX in a dosage range that selectively and effectively blocks only non-NMDA receptors. Since the effective current for drug administration differs between electrodes depending on tip diameter, distance between the tips of the recording and drug pipettes, and other factors, we determined the effective drug currents for each electrode by testing the effects of iontophoresed antagonists on agonists with a time-course similar to that for the test of drug effects on the visual response (from approximately 2 to 6 min). An example of such a selectivity test for CNQX is shown in Fig. 1. The cell was located in layer III of the cortex. A non-NMDA receptor agonist, QA, and an NMDA receptor agonist, NMDA, were administered alternately for 30 s interspersed with a rest interval of 30 s. During CNQX administration with 240 nA, the QA-induced response was selectively antagonized while the NMDA response was spared. Conversely, during APV administration, the NMDA-induced response was selectively blocked while the QA response was spared. In Fig. 1, following CNQX administration, responses induced by NMDA increased gradually. It is unlikely that this is due to a non-specific augmenting effect of NMDA following the high CNQX dose, because the NMDA response continued to increase even after CNQX administration ceased and the QA response recovered, indicating a flushing out of CNQX. Rather, this can be explained by a gradual increase in agonist concentration at the tip of the drug pipette due to removal of the anion, which had soaked into the pipette when retaining current was passed. This explanation is supported by the fact that a gradual increase in the number of spike discharges was often observed during repetitive iontophoresis of NMDA (Fig. 1B, C). Therefore, for the penetration shown at Fig. 1A, we used CNQX and APV with an ejecting current of 240 and 250 nA, respectively. Ejecting current for CNQX was determined in a similar manner for all electrode tracks. Laminar difference in antagonists Iontophoretically applied CNQX reduced the visual response in all 93 cells recorded from all layers of the primary visual cortex. An example of the effect of the glutamatergic antagonists on the response of a layer III complex cell is shown in Fig. 2. Since this cell was tested with binocular stimulation, the
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Fig. 3. Laminar analysis of effect of CNQX on visual responses. Percentage of the number of spikes in the visual response during CNQX administration to that of the control was calculated for each cell. The spontaneous firing rate was subtracted. The layer in which the cell was located is shown on the left. The average percentage responses during CNQX administration and CNQX current (%; nA values in parentheses) for layers II/III, IV, V and VI were 4.5 ^ 9.8 (S.D.; 234.7), 19.7 ^ 22.5 (241.6), 7.0 ^ 7.0 (248.7) and 14.3 ^ 16.5 (244.3), respectively. Total number of cells was 58.
response to motion of the stimulus bar consisted of two response peaks, which correspond to ipsilateral eye response (the larger peak) and contralateral eye response (the smaller peak), respectively. The visual response and spontaneous firings were strongly reduced by 215 nA of CNQX, and 4 min after the start of ejection they were completely abolished (second PSTH from the top). Administration of the NMDA receptor antagonist APV by 240 nA also strongly reduced the visual response of this cell (fourth PSTH from top). These results suggest that the visual response of cells in the superficial layer depends on both non-NMDA and NMDA receptors, which is consistent with the findings of Fox et al. 5 The distribution of CNQX effects among layers is summarized in Fig. 3. Fifty-eight cells in which the laminar location could be clearly identified histologically were included in this analysis. In this figure, each dot corresponds to a percentage of the magnitude of response during CNQX administration to that of the control response for each cell. The effect of CNQX was most pronounced in layer II/III cells, where the average ratio of response magnitude in the presence of CNQX to that of the control was 4.5 ^ 9.8% (S.D.). CNQX was also effective to a certain extent in other layers, and the magnitude of the visual response in layers IV, V and VI was reduced to 19.7 ^ 22.5%, 7.0 ^ 7.0% and 14.3 ^ 16.5%, respectively. The laminar difference of the CNQX effects was statistically significant between layers II/III and IV (P , 0.02, t 2.56, d.f. 33), layers II/III and VI (P , 0.05, t 2.13, d.f. 30), and between layers II/III and other layers (P , 0.05, t 2.26, d.f. 56), but not significant between layers II/III and V (P . 0.4, t 0.64, d.f. 23). The average response magnitude of all the recorded cells (n 93) during CNQX administration was 11.7 ^ 17.6% (S.D.) of the control (average ejecting current: 241.2 ^ 22.0 nA). However, the suppressive effect of APV was substantially smaller than that of CNQX. Among 14 cells tested, the average response magnitude during APV administration was
Fig. 4. Effects of CNQX and APV on the visual response of a simple cell in layer IV PSTHs before, during and after administration of CNQX (210 nA) or APV (210 nA), or administration of both (210 nA of APV and 25 nA of CNQX). Both CNQX and APV reduced the visual response and spontaneous firings, and the effects of CNQX and APV were additive (fourth and fifth PSTHs from top). Stimulus velocity: 48/s. Other conventions are as in Fig. 2.
45.0 ^ 39.4% (S.D.) of the control (average ejecting current: 241.4 ^ 17.5 nA), and the difference in response magnitude between CNQX and APV was statistically significant (P , 0.001, t 5.39, d.f. 105, t-test). The average response magnitudes during APV application of cells recorded in layers II/III (n 4) and IV–VI (n 10) were 22.8 ^ 36.4% and 56.1 ^ 38.0%, respectively. The difference between these two values was not statistically significant (P . 0.1, t 1.50, d.f. 12, t-test), even though the sample size seemed to be too small for meaningful statistical analysis. An example of the fairly potent effect of APV in a simple layer IV cell is shown in Fig. 4. This cell was quite sensitive to CNQX and
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Fig. 5. Neuronal firing rate and effect of CNQX. The plot shows the firing frequency for the visual response of individual cells (n 48) in response to stimulus motion in the preferred and non-preferred directions. (A) Data for control trials. Regression line: f(x) 0.38x 1 3.32. Correlation coefficient: r 0.68. (B) Data for CNQX trials; f(x) 0.36x 1 0.76, r 0.58.
administration of CNQX with 210 nA almost completely abolished visual response (second PSTH from the top; response magnitude: 2.1% of the control). Administration of APV with 210 nA also reduced the visual response of this cell to 27.1% of the control. This result is inconsistent with the previous report by Fox et al., 4 in which APV was found to have no effect on the visual response of cells in layers IV–VI in the cat primary visual cortex. An additional administration of CNQX with 25 nA completely abolished both the visual response and spontaneous activity, confirming that the effects of APV and CNQX are due to antagonism of a different population of glutamate receptors and that the effects of the two drugs are additive. Firing frequency and effect of 6-cyano-7-nitroquinoxaline2,3-dione It is well known that activation of NMDA receptors exhibits a membrane voltage dependency, i.e. NMDA receptors are more active under depolarization. 16,24,25 There is a possibility that depolarization by non-NMDA receptor activation during visual inputs may enhance activation of the NMDA receptors in the observed visual responses. In this case, blockade of the non-NMDA receptors with CNQX should result in indirect suppression of the NMDA receptors. To clarify this point, we analysed the relationship between the firing frequency of the visual response and the effect of CNQX. To do this, we compared the firing frequency in response to stimulus motion in the preferred and non-preferred directions with or without the presence of CNQX for individual cells. Directional selectivity for each cell was defined based on the following relation: Direction Selectivity Index DSI 1 2 Rnp =Rp ; where Rp represents the number of spikes elicited in response to stimulus movement in the preferred direction at the optimal orientation and Rnp represents the number of spikes elicited to movement in the opposite direction. A cell was excluded from analysis if the cell was completely direction selective (i.e. DSI 1 under control conditions), or if the response to either the preferred or non-preferred stimulus direction was completely abolished by CNQX. The rationale for eliminating such
cells is that it is impossible to determine the extent to which the excitatory response was abolished by CNQX, since there were no spikes in the visual response. Forty-eight cells were included in this analysis. Figure 5 shows the firing frequencies of cells in response to stimulus motion in the preferred and non-preferred directions without (Fig. 5A) or with (Fig. 5B) CNQX. The average firing frequencies of the 48 cells to the preferred and non-preferred directions were 18.84 and 10.56 Hz in the control, and 6.46 and 3.10 Hz during CNQX administration, respectively. The average DSI values were 0.50 ^ 0.31 (S.D.) and 0.51 ^ 0.46 during the control phase and under CNQX administration, respectively, and these differences were not statistically significant. The slopes of the two regression lines for these two distributions (0.38 for the control and 0.36 for CNQX) were almost equal and there was no indication of a stronger suppression of response at higher firing frequency, i.e. in response to the motion stimulus in the preferred direction. Therefore, it seems unlikely that the blockade of non-NMDA receptors secondarily abolished NMDA receptor-mediated excitation of responses with higher firing frequencies. DISCUSSION
The blockade of non-NMDA receptors with iontophoretically administered CNQX eliminated nearly all neuronal activity in layers II–VI of the primary visual cortex of cats. This implies that activation of non-NMDA receptors is predominant for excitatory transmission at both geniculocortical and corticocortical synapses. However, the effect of CNQX seems to vary among cells, particularly in layers IV and VI (Fig. 3). This may be due partly to a difference in the subtypes of glutamate receptor expressed on the cells in each layer. There is evidence that glutamate receptor subtypes having different sensitivity to AMPA are differentially expressed among layers in the cat visual cortex; the GluR1 and GluR2/3 subunits are particularly abundant in non-pyramidal and pyramidal neurons, respectively. 7 This, in turn, suggests that the excitatory transmission of geniculocortical synapses in layer IV, which contains a larger population of stellate cells, is more dependent on GluR1 type receptors, while it is more dependent on GluR2 in other layers. However, we do
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not have a reasonable explanation for the variability of the effect of CNQX in layer VI. This point should be further assessed with a larger population of cortical cells. In the present study, an NMDA receptor antagonist, APV, also exerted a suppressive effect on the visual response of cells throughout all layers, and the average response magnitude during APV administration was 45.0% of the control. This result is partly consistent with the previous results of Miller et al. 17 that APV administered with an osmotic minipump profoundly suppressed visual responses of all cells within the APV-perfused area in the visual cortex of adult cats, even though the authors did not describe the laminar distribution of recorded cells. The substantial effects of APV found in the present study on visual responses in layers IV–VI is inconsistent with previous reports in which APV had no effect on visual responses in these layers. 4 In the light of the new findings, we suggest that non-NMDA and NMDA receptors function additively in the primary visual cortex. Consistent with this notion, NMDA receptors are hypothesized to be involved in the excitatory transmission not only in layers II/III, but also in granular and infragranular layers in in vivo 8 or in vitro experiments. 12,15,23
No evidence was seen that depolarization induced by activation of non-NMDA receptors secondarily evoked augmentation of visual responses through activation of voltagedependent NMDA receptors. However, this does not mean that NMDA receptors are not active within a range of membrane potential levels in vivo. Rather, as suggested previously, 5 the degree of activation of NMDA receptors is proportionally the same for small responses and large responses for an individual cell. Taken together, the available data are consistent with the idea that the effects of the activation of non-NMDA and NMDA receptors are additive in the generation of spike discharges.
Acknowledgements—We wish to thank Dr Nigel W. Daw for critical reading of an early version of the manuscript, Dr Hiroshi Tamura and Takafumi Akasaki for technical assistance, and William D. Stenson for editorial assistance. This work was supported by Grants-in-Aid for Scientific Research on Priority Areas 09268221 and 08458268 from the Japanese Ministry of Education, Science, Sports and Culture to H.S., “Research for the Future” Program JSPS-RFTF96L00201 from the Japan Society for the Promotion of Science to H.S. and also by a grant from the Kato Memorial Bioscience Foundation to H.S.
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