Effect of the Group II Metabotropic Glutamate Agonist, 2R,4R-APDC, Varies With Age, Layer, and Visual Experience in the Visual Cortex

C. J. Beaver, Q.-H. Ji, and N. W. Daw

Department of Ophthalmology and Visual Science, Yale University School of Medicine, New Haven, Connecticut 06520-8061


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Beaver, C. J., Q.-H. Ji, and N. W. Daw. Effect of the Group II Metabotropic Glutamate Agonist, 2R,4R-APDC, Varies With Age, Layer, and Visual Experience in the Visual Cortex. J. Neurophysiol. 82: 86-93, 1999. Group II metabotropic glutamate receptors (mGluR 2/3) are distributed differentially across the layers of cat visual cortex, and this distribution varies with age. At 3-4 wk, mGluR 2/3 receptor immunoreactivity is present in all layers. By 6-8 wk of age, it is still present in extragranular layers (2, 3, 5, and 6) but has disappeared from layer 4, and dark-rearing postpones the disappearance of Group II receptors from layer 4. We examined the physiological effects of Group II activation, to see if these effects varied similarly. The responses of single neurons in cat primary visual cortex were recorded to visual stimulation, then the effect of iontophoresis of 2R,4R-4 aminopyrrolidine-2,4-decarboxylate (2R,4R-APDC), a Group II specific agonist, was observed in animals between 3 wk and adulthood. The effect of 2R,4R-APDC was generally suppressive, reducing both the visual response and spontaneous activity of single neurons. The developmental changes were in agreement with the immunohistochemical results: 2R,4R-APDC had effects on cells in all layers in animals of 3-4 wk but not in layer 4 of animals >6 wk old. Moreover, the effect of 2R,4R-APDC was reduced in the cortex of older animals (>22 wk). Dark-rearing animals to 47-54 days maintained the effects of 2R,4R-APDC in layer 4. The disappearance of Group II mGluRs from layer 4 between 3 and 6 wk of age is correlated with the segregation of ocular dominance columns in that layer, raising the possibility that mGluRs 2/3 are involved in this process.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

During postnatal development of the visual cortex, the expression of many factors is regulated by age and/or by visual experience and may be distributed differentially across the cortical layers (for a review, see Daw 1995). The pattern of expression of some of these factors is correlated with certain aspects of visual cortical development and the critical period for plasticity in this system while others are not. For example, the basal level of cAMP in the visual cortex peaks at the same time as the critical period (Reid et al. 1996). The level of GAP-43, an extracellular matrix protein, in the visual cortex also changes during development. In contrast to cAMP levels, however, its levels slowly decrease after birth to reach adult levels and do not match the profile of the critical period (McIntosh et al. 1990; Mower and Rosen 1993).

Dark-rearing, the procedure whereby one raises an animal in complete darkness from shortly after birth, is known to interfere with the development of various visual properties such as the segregation of ocular dominance columns in layer 4 (Mower et al. 1985; Stryker and Harris 1986; Swindale 1981, 1988) and the development of normal orientation selectivity (Blakemore and van Sluyters 1975; Buisseret and Imbert 1976; Imbert and Buisseret 1975), as well as postponing the beginning and end of the critical period for the effects of monocular deprivation (Cynader and Mitchell 1980; Mower 1991). One expects that the anatomic distribution and physiological function of a molecule involved in some aspect of visual cortical development should be affected by dark-rearing similarly to that aspect of development, and this is used as one test for the involvement of a molecule in development.

The role of the glutamatergic system in the visual cortex has received particular attention. Glutamate activates two categories of receptors; ionotropic and metabotropic. The ionotropic receptors [N-methyl-D-aspartate (NMDA), AMPA, and kainate] are ligand-operated channels the roles of which in the visual cortex have been studied extensively (for a review, see Daw 1994). The eight members of the G-protein-coupled metabotropic glutamate receptors (mGluRs) can be divided into three groups on the basis of their activation of different intracellular second-messenger systems and their affinity for different agonists (Conn and Pin 1997; Pin and Duvoisin 1995). Group I (mGluRs 1 and 5) and Group II receptors (mGluRs 2 and 3) are activated preferentially by the agonist 1S,3R-1-amino-1,3-cyclopentane-dicarboxylic acid (ACPD). Group III receptors (mGluRs 4, 6, 7, and 8) do not respond to 1S,3R ACPD but are activated preferentially by L(+)-amino-4-phosphonobutyric acid (L-AP4). In general, Group I mGluRs are linked to increased phosphoinositide (PI) turnover, whereas Group II and Group III mGluRs are coupled negatively to adenylate cyclase (Conn and Pin 1997; Pin and Duvoisin 1995). mGluRs have been implicated as playing a role in long-term potentiation and depression (LTP and LTD) in the hippocampus (Bashir and Collingridge 1994; Bashir et al. 1993; Bortolotto et al. 1994; Bortolotto and Collingridge 1995; Holsher et al. 1997; Huang et al. 1997a,b; Manahan-Vaughan 1997; Oliet et al. 1997). With respect to the visual system, there is disagreement as to their role in certain aspects of plasticity (Haruta et al. 1994; Hensch and Stryker 1996; Huber et al. 1998; Kato 1993).

The level of expression of mGluRs 1, 5, and 2/3 receptor types in the visual cortex as determined by Western blots is downregulated during development (Reid et al. 1995b, 1997). Each of the receptors also is distributed differentially across the layers through development (Reid et al. 1995a,b, 1997). mGluR 1 receptors are found predominantly in all layers except layer 4, and this pattern does not change during development nor is it affected by dark-rearing. Shortly after birth, mGluR 5 receptor immunoreactivity is found in all layers of the cortex. At around 5 wk of age, however, the receptors become concentrated around the layer 4/5 border, and this distribution is maintained into adulthood. Dark-rearing postpones the redistribution of mGluR 5 receptors from 5 wk until around 3-4 mo of age (Reid et al. 1997). The distribution of mGluR 2/3 receptors also changes during development and is postponed by dark-rearing (Reid et al. 1995b). In animals of 3-4 wk of age, immunoreactivity is found in all layers of the cortex and is strongest in layer 4. Between the ages of 4 and 6 wk immunoreactivity in layer 4 disappears but remains in the extragranular layers. Dark-rearing postpones the disappearance of mGluR 2/3 receptors from layer 4 until between 6 and 13 wk of age.

The role of mGluRs in the cortex in vivo has not been examined extensively. Iontophoresis of ACPD onto cells in the somatosensory cortex has both excitatory and depressive effects that vary with layer: excitatory effects are found primarily in infragranular layers, whereas depressive effects are found in all layers (Cahusac 1994; Taylor and Cahusac 1994). In the visual cortex of animals between the ages of 6 and 13.5 wk, the effect of ACPD also varies by layer (Reid and Daw 1997). In superficial layers (2 and 3), iontophoresis of ACPD suppresses both the spontaneous activity and the visual response of neurons. In deeper layers, ACPD also suppresses the visual response, but the spontaneous activity of some neurons in deeper layers is increased significantly. These results have been interpreted as indicating a mixture of effects at both Group I and II mGluRs. The ability to ascribe a particular effect to the physiological function of a specific receptor, however, has been difficult due to the lack of specific agonists and antagonists. Recently advances have been made in the field of new agonists and antagonists to mGluRs (Conn and Pin 1997). 2R,4R-aminopyrrolidine-2,4-decarboxylate (2R,4R-APDC) is a novel agonist at Group II mGluRs with no effect at Group I receptors (Monn et al. 1996; Schoepp et al. 1995, 1997). The aims of this study were as follows: to use 2R,4R-APDC to examine the specific effects of Group II activation on the visual response and spontaneous activity of area 17 neurons, to determine whether the effect of 2R,4R-APDC changes with age in different layers in a manner that matches the observed changes in immunoreactivity, and, if there are laminar differences, to observe whether they are absent from the visual cortex of dark-reared kittens >6 wk.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Rearing

Light reared animals (n = 18) were kept from birth in colony rooms with 12:12 light:dark cycles. Kittens were housed with their mother until 6-8 wk of age. Dark-reared animals (n = 3) were placed with their mothers in a dark room within 48-72 h of their birth. Animals were placed in a stainless steel cage (105 × 60 × 60 cm) that could be accessed through two doors in the front of the cage. The cages were made light-tight by sealing all seams and doors with black electrical tape. Animals were checked daily and cages changed once a week by using a Hi-Tek G-201 infrared viewer (Hi-Tek Intl., Redwood City, CA).

Surgery

Animals were sedated with acepromazine (0.1 mg/kg) and given a preanesthetic dose of atropine (0.04 mg/kg). Anesthesia was induced with halothane (4%) in a mixture of 67% nitrous oxide-33% oxygen and maintained with 0.4-1.0% halothane afterward. Dark-reared animals were injected in the dark with a mixture of ketamine (25 mg/kg) and xylazine hydrochloride (Butler, Columbus, OH; 2.2 mg/kg). Once the animal was anesthetized, it was brought to the laboratory for the remainder of the procedure. After a tracheotomy and insertion of a cannula into the femoral vein, the skull was opened up over the lateral gyrus, and a small hole made in the dura for insertion of the electrode. All wound margins were treated with lidocaine. The eyes were covered with contact lenses of zero power, and curvature appropriate to focus the retina on a tangent screen at 57 in. After surgery, the animal was paralyzed by intravenous infusion of pancuronium bromide at 0.6-1.5 mg/h (Elkins-Sinn, Cherry Hill, NJ). Heart rate and end tidal CO2 were monitored continuously, and CO2 was maintained at 3.8-4.3% by adjusting the respirator.

Electrodes and recording

Single-unit recordings were made with a carbon-fiber microelectrode with six side barrels for iontophoresis. During the course of an experiment, at least four of the iontophoretic barrels of the microelectrode were filled with drugs. Electrodes were advanced through the hole in the dura and traversed all layers of the cortex. No layer was targeted during a penetration. After isolation of the unit, the receptive field was mapped on a tangent screen with a hand-held projector to determine the preferred orientation, optimal size, and velocity of the stimulus. Subsequently, computer-generated stimuli were used to stimulate the cell through the dominant eye with the preferred parameters. The stimulus rested at first for 1 s outside of the receptive field, then swept across the receptive field, resting for 1 s on the far side, and swept back, resting 1 s more. This procedure was repeated three to five times, depending on the velocity of the stimulus, for one group of records every minute. Amplified spikes were discriminated with a voltage window and monitored for amplitude and waveform on a storage oscilloscope. The computer also was used to store spike discharge times and to construct a peristimulus-time histogram on-line as spikes came in. These data were stored on a hard disk for subsequent off-line analysis using custom-written ASYST programs (Asyst Software, Rochester, NY).

Control groups of records were taken until six consecutive groups showed a set of stable responses. Then 2R,4R-APDC (Eli Lilly, Indianapolis, IN), at a concentration of 10 mM and a pH 7.8-8.0, was iontophoresed for 3 min at -20 nA. To prevent the 2R,4R-APDC from leaking out of the barrels between drug applications, a retaining current of +5 to +10 nA was applied to all barrels. As the cell recovered, records were taken every minute until six consecutive groups showed a consistent set of responses. Data were used for analysis only if the response after recovery was >= 70% of the response before drug application.

A concentration of 10 mM for the drug in the pipette, roughly 3 log units higher than the EC50 for 2R,4R-APDC in vitro (Schoepp et al. 1997), was chosen to attempt to ensure a concentration at the level of the EC50 of the drug at receptors in vivo. The exact concentration of the 2R,4R-APDC at the tip of the electrode in our experiments is unknown, but experiments involving iontophoresis of 50 mM catecholamines have found that a 10-nA ejecting current results in a tip concentration of ~5 × 10-5 M (Armstrong-James et al. 1981). Moreover, the concentration of a drug falls a further 2 log units over a distance of 230 µm in vitro (Fox et al. 1989). However, because of restricted diffusion in vivo, the concentration over the same area would be expected to be higher. Fox et al. (1989) estimated that iontophoresis of a 100 mM solution of (D)-2-amino-5-phosphonovaleric acid (APV) in the visual cortex in vivo reached an effective concentration of 25 µM between 100 and 200 µm from the electrode tip. Thus a pipette concentration of 10 mM APDC is likely to maintain an effective concentration of 10 µM only over a local area around the cell that is being recorded. A -20-nA ejection current was selected for comparing between animals after initially testing a range of ejection currents (-10 to -70 nA) as it produced a reliable effect in a majority of cells without causing a near complete reduction from which the cell's response didn't recover. The efficacy of the drug was judged by comparing responses within a penetration. That is, if a cell within a particular layer did not show an effect, cells in other layers within the same penetration typically did.

Data analysis

Firing rates were averaged over the peak of the visual response. Visual responses were expressed as average firing rate during the response minus the spontaneous activity. The firing rate for the preferred direction was taken in the case of cells with a preferred direction, and the firing rates in both directions were analyzed for cells responding to both forward and reverse movement. The effect of the 2R,4R-APDC on the visual response was estimated by taking the firing rate during the second and third minute of drug application as a percentage of control. Control firing rates were calculated by averaging the firing rates during the control and recovery periods.

Lesions and histology

At least two lesions were made in each penetration through the recording electrode, using 3.5-4.5 µA DC current for 10 s. On completion of the experiment, the animal was anesthetized deeply with 4% halothane and perfused through the heart with 0.066 M phosphate-buffered saline (PBS) followed by 4% paraformaldehyde. The lateral gyrus was removed and allowed to sink in a 30% sucrose/4% paraformaldehyde solution. Frozen sections (60 µm) were cut and stained with methylene blue-azure. The electrode tracks were reconstructed and cells were assigned to layers according to layering criteria described by Kelly and Van Essen (1974). Cells were assigned to the borders of layers when the histology indicated that their inclusion in layers 2 and 3, 4, 5, or 6 was ambiguous.

The procedures used in this study were approved by the Animal Care and Use Committee at Yale University and conform to National Institutes of Health guidelines.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of 2R,4R-APDC in normal animals

The effect of 2R,4R-APDC was examined on 242 cells in the visual cortex of light-reared animals. Iontophoresis of 2R,4R-APDC onto cells in area 17 typically caused a reduction in their overall firing rate, reducing both their visual response and spontaneous activity. An example of APDC's effect is shown for two cells from 3- to 4-wk-old normal animals (Fig. 1). Peristimulus-time histograms are shown for the control condition (top), the last 2 min of a 3-min application of 10 mM 2R,4R-APDC at an ejection current of -20 nA (middle), and the recovery period (bottom). The left side of the figure shows the effect of 2R,4R-APDC iontophoresis on a layer 5 cell. This cell had a spontaneous activity of 0.5 spikes/s and responded almost equally well to a bar moving in both directions at a velocity of 2°/s. During 2R,4R-APDC application, both the visual response and the spontaneous activity of this cell were reduced by ~90 and 75%, respectively. For comparison, the typical effect of 2R,4R-APDC on a layer 4 cell of an animal of the same age is shown in the graphs at the right side of the figure. This cell gave a more vigorous response to a bar moving in the forward direction at 5°/s than to movement in the reverse direction and had a spontaneous activity of 2.3 spikes/s. 2R,4R-APDC reduced the visual response by 70% and the spontaneous activity by 90%. The suppressive effect of 2R,4R-APDC was observed also in layers 2 and 3. 



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 1. Peristimulus-time histograms of a layer 5 cell (A-C) and a layer 4 cell (D-F) from 2 3- to 4-wk-old light-reared animals. Layer 5 cell responded best to a bar of light that was moving along the 0°/180° axis (0° taken as vertical) at a velocity of 2°/s. Layer 4 cell responded best to a bar of light moving along the 135°/315° axis at a velocity of 5°/sec. A and D: control response. B and E: response during application of 2R,4R-4 aminopyrrolidine-2,4-dicarboxylate (2R,4R-APDC) at -20 nA. C and F: response after recovery from APDC. Ramps under the figures indicate the movement of the stimulus.

In older animals (6-11 wk) the effect of 2R,4R-APDC was also suppressive but this effect was not consistent in all layers of the cortex. Figure 2, A-C, shows an example of the effect of 2R,4R-APDC on a layer 5 cell of an animal of 54 days old that responded best to movement of a bar at 2°/s. In this case, iontophoresis of 2R,4R-APDC caused the visual response to be reduced by 72% and the spontaneous activity by 99%. This type of effect also was observed in layers 2 and 3 of animals of this age. In contrast to the younger animals, however, 2R,4R-APDC typically had a negligible effect on both the spontaneous activity and the visual response in layer 4. An example is shown in the histograms in Fig. 2, D-F. In this case, the visual response and the spontaneous activity were reduced by 3 and 0%, respectively. This trend also was observed in recordings made from animals older than 22 wk. The lack of effect in layer 4 was not due to a biased sampling as no layer was targeted during a penetration. Furthermore when a cell or cells in layer 4 showed no effect, cells in other layers within the same penetration typically did.



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 2. Typical examples of peristimulus-time histograms of a layer 5 cell (A-C) and a layer 4 cell (D-F) from 6- to 11-wk-old light-reared animals. Layer 5 cell responded best to a bar of light that moved along the 90°/270° axis at a velocity of 2°/s. Layer 4 cell responded best to a bar of light that was moving along the 0°/180° axis at a velocity of 5°/s. A and D: control response. B and E: response during application of 2R,4R-APDC at -20 nA. C and F: response after recovery from APDC.

To summarize the development of laminar differences in the effect of APDC, Fig. 3 plots the mean effect (±SE) of 2R,4R-APDC on the spontaneous activity (A-C) and the visual response (D-F) as a function of layer in animals of 3-4 wk (left), 6-11 wk (middle), and 22 wk to adulthood (right). In the younger animals (Fig. 3, A and D), the effect of 2R,4R-APDC was similar across all layers of the cortex: the average reduction of the spontaneous activity and visual response of neurons was 73.9 ± 7.8 and 45.7 ± 13.3%, respectively. In contrast, for the 6-11 wk old animals (Fig. 3, B and E), the effect of 2R,4R-APDC was not consistent in all layers. As in the younger animals, cells in layers 2, 3, 5, and 6 showed a decrease of both the visual response and the spontaneous activity. The reductions in spontaneous activity in extragranular layers in the older animals were similar to those observed in the younger animals. Spontaneous activity was reduced by 56.5 ± 5.8% in layers 2/3, 60.8 ± 6.4% in layer 5, and 71.8 ± 4.5% in layer 6. The visual response was suppressed by 37.3 ± 4.3% in layers 2/3, 39.8 ± 5.8% in layer 5, and 39.1 ± 3.6% in layer 6. In layer 4, however, the effect of 2R,4R-APDC was reduced in comparison with the effects observed in layer 4 of the younger animals. The reductions of the visual response and spontaneous activity were only 2.6 ± 5.6% and 10.2 ± 29.0%, respectively, as compared with 66.3 ± 9.3% and 39.9 ± 7.6% for the younger animals. No significant difference in the reduction of spontaneous activity (P = 0.064) or visual response (P = 0.086) was found in comparisons of layer 4 with extragranular layers in the younger animals. In contrast, significant differences were observed between layer 4 and extragranular layers in the 6- to 11-wk-old animals for both the spontaneous activity (P < 0.001) and visual response (P < 0.001).



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 3. Summary graphs showing the mean effect (±SE) of 2R,4R-APDC on the spontaneous activity (A-C) and the visual response (D-F) as a function of layer in animals of 3-4 wk (A and D), 6-11 wk (B and E), and >22 wk (C and F). Numbers in parentheses above each point indicates the number of cells in each layer grouping.

In terms of the effect of 2R,4R-APDC on spontaneous activity (Fig. 3C), the animals in the oldest group (22 wk to adulthood) also showed a similar laminar trend to that observed for the 6- to 11-wk-old animals, however there was no effect of layer. Unlike the 6- to 11-wk-old animals, 2R,4R-APDC had little effect on the visual response of neurons in any layer (Fig. 3F).

Effect of 2R,4R-APDC in dark-reared animals

The developmental change in the efficacy of 2R,4R-APDC to produce suppression of the visual response and spontaneous activity of neurons in layer 4 correlates well with the disappearance of mGluR 2/3 immunoreactivity in that layer: the effect of 2R,4R-APDC is reduced in layer 4 at the same age when the mGlu 2/3 receptors are no longer present. As the disappearance of mGluR 2/3 from layer 4 is postponed in the visual cortex of dark-reared animals, we sought to examine whether the physiological effect of 2R,4R-APDC matched the anatomic changes. To this end, we recorded from area 17 of three dark-reared animals between the ages of 47 and 54 days, at which age mGluR 2/3 receptor immunoreactivity still is present in layer 4. Typically, cells recorded from the visual cortex of the three dark-reared animals were not orientation or direction selective, which is in accord with previous reports of the stimulus selectivity of cells in dark-reared kittens (Imbert and Buisseret 1975). However, in all three animals that we recorded, the cells gave vigorous responses to moving bars that did not habituate to multiple presentations of the stimulus.

Figure 4 presents peristimulus histograms from a layer 6 and a layer 4 cell of a dark-reared animal showing the response before, during, and after an application of 10 mM 2R,4R-APDC at -20 nA for 3 min. Both cells responded to movement in all directions. The layer 6 cell responded best to movement at 4°/s, whereas the layer 4 cell responded best to movement at 5°/s. Iontophoresis of 2R,4R-APDC onto both cells caused a reduction of both spontaneous activity and the visual response. The spontaneous activity and visual response were reduced to 38.9 and 35.3% of control, respectively, for the layer 6 cell, whereas they were reduced to 29.1 and 48.0% of control, respectively, for the layer 4 cell.



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 4. Examples of peristimulus-time histograms of a layer 6 cell (A-C) and a layer 4 cell (D-F) from 6- to 8-wk-old dark-reared animals. Layer 6 cell was stimulated with a bar of light that was moving along the 45°/225° axis at a velocity of 4°/s. Layer 4 cell was stimulated with a bar of light that was moving along the 90°/270° axis at a velocity of 5°/s. A and D: control response. B and E: response during application of 2R,4R-APDC at -20 nA. C and F: response after recovery from APDC.

The two graphs of Fig. 5 show the mean effect (±SE) of 2R,4R-APDC on the spontaneous activity (top) and visual response (bottom) as a function of layer for the three animals that were dark-reared to 47-54 days and should be compared with the graphs in Fig. 3, B and E. It is evident that 2R,4R-APDC had similar effects on cells in all layers of the cortex of dark-reared animals of this age. The average reduction of the spontaneous activity was 53.2 ± 5.2% in layers 2/3, 70.1 ± 3.9% in layer 5, and 51.5 ± 7.4% in layer 6. The mean suppression of the visual response was 39.4 ± 4.8% in layers 2/3, 49.2 ± 7.9% in layer 5, and 58.6 ± 4.2% in layer 6. This is similar to the effects observed for light-reared animals in extragranular layers. However, in contrast to the results from cells from layer 4 of light-reared animals of the same age (Fig. 3, B and E), 2R,4R-APDC had a greater effect in layer 4 of dark-reared kittens, reducing the spontaneous activity and the visual response on average by 54.4 ± 2.8% and 30.9 ± 2.2%.



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 5. Summary graphs showing the mean effect (±SE) of 2R,4R-APDC on the spontaneous activity and the visual response as a function of layer in dark-reared animals of 6-8 wk of age. Numbers in parentheses above each point indicates the number of cells in each layer grouping.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The results of this study can be summarized as follows: 1) the general effect of Group II mGluR activation by 2R,4R-APDC is to suppress both the visual response and spontaneous activity of neurons in area 17 of kittens and adult cats; 2) the effect of 2R,4R-APDC is similar in all layers of young animals (3-4 wk old); 3) APDC's effect is reduced in the granular layer of animals that are >6 wk of age, and this reduction in APDC's efficacy matches the decrease in the level of mGluR 2/3 receptors in layer 4 as shown by immunohistochemical methods (Reid et al. 1995b); and 4) dark-rearing until 47-54 days, which postpones the disappearance of Group II receptor immunoreactivity from layer 4, maintains the suppressive effect of Group II activation in layer 4.

The suppressive effect of 2R,4R-APDC on both the visual response and the spontaneous activity is consistent with previous reports of APDC's effect (Dubé and Marshall 1997; Lovinger and McCool 1995). Group II receptor activation has been shown to reduce glutamate release at cortico-striatal synapses (Lovinger and McCool 1995) and in the locus coeruleus (Dubé and Marshall 1997). Furthermore 2R,4R-APDC has been shown to reduce veratridine-induced release of excitatory amino acids in the striatum (Battaglia et al. 1997). At the present time, the precise mechanism underlying the suppression of responses in the visual cortex remains unknown. However, it has been shown that Group II receptors are able to modulate Ca2+ channel activity in other systems to reduce neurotransmitter release (Conn and Pin 1997; Pin and Duvoisin 1995). mGluR activation by ACPD has been shown to reduce excitatory transmission in neurons from rat visual cortical slices (Sladeczek et al. 1993). The effects of ACPD were blocked by the potassium channel blocker 4-aminopyridine, which suggests that mGluRs also could act to open presynaptic potassium channels and thereby decrease transmitter release (Sladeczek et al. 1993).

The physiological results presented here are also in agreement with the immunohistochemical results (Reid et al. 1995b) and with other studies indicating a primarily presynaptic localization of Group II receptors (Conn and Pin 1997; Pin and Duvoisin 1995; Shigemoto et al. 1997). Neki et al. (1996), however, have reported previously that, although it was difficult to distinguish axons from cell bodies and dendrites in most brain regions, Group II receptors were found postsynaptically on Golgi cells in the cerebellum. It cannot be ruled out, therefore, that APDC acts to reduce excitation via a postsynaptic mechanism, possibly the modulation of calcium channels. However, results from an in vitro slice preparation of rat visual cortex indicate that the effect of 2R,4R-APDC remains during recordings in which GDP-beta -S is included in the patch pipette (Flavin and Daw 1998). Thus we believe that 2R,4R-APDC is primarily acting presynaptically to reduce glutamate release.

The effects of 2R,4R-APDC contrast with those of the specific Group I and Group III agonists, (S)-3,5-dihydroxyphenylglycine (DHPG) and L-AP4 (Beaver et al. 1997, 1998). Group I mGluRs have been shown to potentiate NMDA responses at certain synapses (Aniksztejn et al. 1991; Fitzjohn et al. 1996) and to reduce transmission presynaptically at others (Gereau and Conn 1995; Manzoni and Bockaert 1995). In area 17, iontophoresis of DHPG in vivo has mixed facilitory and suppressive effects on individual neurons in all layers of area 17 (Beaver et al. 1997). Likewise, Group III mGluRs are known to inhibit glutamatergic transmission presynaptically in the spinal cord (Ishida et al. 1993) and hippocampus (Bushell et al. 1995; Macek et al. 1996; Vignes et al. 1995), In slices of rat visual cortex L-AP4 reduces the size of excitatory postsynaptic potentials elicited by stimulating the predominant afferent input (Jin and Daw 1998). In contrast to these results, L-AP4 activation of Group III receptors in vivo has facilitatory effects on the spontaneous activity and visual response of neurons in the cat visual cortex (Beaver et al. 1998). Similar disinhibitory effects have been observed in the somatosensory cortex of rats in vivo (Wan and Cahusac 1995). As the effects of 2R,4R-APDC differ from those of the other specific agonists, they appear to be specific for Group II mGluR activation and not due to a general depressive effect resulting from mGluR activation.

The suppressive effect of 2R,4R-APDC shown in this study extends the results previously obtained using iontophoresis of the general group I and II mGluR agonist ACPD in the visual cortex (Reid and Daw 1997). Both the visual response and spontaneous activity of neurons in the superficial layers (2/3) of 6- to 13.5-wk-old animals are suppressed by ACPD. It is in these layers that Group II mGluRs levels are high, whereas those of mGluR 1 and mGluR 5 are low. Iontophoresis of ACPD in the deeper layers of the cortex reduces the visual response but increases spontaneous activity. It is likely that activation of Group II receptors is responsible for the suppressive effects and possible that Group I receptors are responsible for the facilitatory effects on spontaneous activity observed in deeper layers. However, because the Group I receptor agonist, DHPG, can have the mixed effects mentioned earlier, the situation may be more complicated than this. A better understanding of the precise pre- and postsynaptic location of different mGluRs in the visual cortex and of the nature of interactions between the different mGluRs will be necessary to put these results together.

Possible relation of Group II mGluRs to geniculocortical afferent segregation

Geniculocortical afferents from the two eyes are intermingled in layer 4 at 3 wk of age and segregate into ocular dominance columns between 3 and 6 wk of age (LeVay et al. 1978). This period of development (3-6 wk of age) is precisely the period during which the effect of Group II mGluR activation is reduced. Thus there is a strong temporal correlation between these two processes, implying that Group II receptors may play a role in ocular dominance segregation. Dark-rearing is known to affect ocular dominance segregation, although there is still some debate as to whether dark-rearing abolishes the segregation (Mower et al. 1985; Swindale 1981, 1988) or simply slows it down (Stryker and Harris 1986). In any case, the effect of dark-rearing on ocular dominance segregation and on Group II mGluR inhibition is similar. Furthermore segregation of geniculocortical afferents fails to occur in layer 4 of dark reared kittens removed from the dark at 4 mo of age and given binocular or monocular visual experience (Mower et al. 1985). Although dark-rearing postpones the decrease in Group II receptor immunoreactivity in layer 4, this redistribution of mGluRs 2/3 has occurred by 4 mo of age, at which time segregation of geniculocortical afferents no longer occurs.

To date, no one has examined directly the specific role of Group II receptors in ocular dominance formation. Studies that have examined the role of mGluRs in visual system plasticity have found that, although synapse depotentiation (Hensch and Stryker 1996) and LTD in vitro (Haruta et al. 1994) are blocked by the general mGluR antagonist alpha -methyl-4-carboxyphenylglycine (MCPG), ocular dominance plasticity in vivo is not (Hensch and Stryker 1996). Studies that utilize MCPG in vivo are difficult to interpret, however, as MCPG is neither a selective nor a very potent antagonist (Pin and Conn 1997). Moreover, MCPG has been shown to block 1S,3R ACPD-induced increases in phosphoinositides but not glutamate-induced phosphoinositide turnover in the visual cortex (Huber et al. 1998). In the hippocampus, Group I and II mGluRs have been shown to play different roles in the induction of LTP and LTD in vitro (Huang et al. 1997a,b) and in vivo (Holsher et al. 1997; Manahan-Vaughan 1997). The effect of MCPG on Group I and II receptors therefore may have opposing effects that cancel one another.

Group II mGluRs could influence segregation through their action in controlling glutamate release and consequently the degree of glutamate receptor activation. An alternate possibility is that Group II mGluRs may act on second messengers to influence plasticity. Previous work from our laboratory has shown a strong correlation between the critical period for monocular deprivation and increased levels of both basal and mGluR-induced increases in cAMP (Reid et al. 1996). Consequently, Group II receptors instead may act to influence plasticity through their modulatory effect on cAMP levels as has been implicated in hippocampus (Bailey et al. 1996).

In conclusion, we have shown that the effect of the specific Group II agonist, 2R,4R-APDC, causes suppression of the visual response and spontaneous activity of neurons in the primary visual cortex of the cat. The effect of 2R,4R-APDC varies with layer and with age in a manner that is correlated strongly with changes in the distribution of Group II receptor immunoreactivity with layer, age, and visual experience. The disappearance of Group II receptors in layer 4 between the ages of 3 and 6 wk is correlated with the time of geniculocortical afferent segregation (LeVay et al. 1978). With the advent of more selective and potent antagonists (Ornstein et al. 1998), we should now be able to address the question of the relationship between Group II mGluRs and geniculocortical afferent segregation in the visual cortex.


    ACKNOWLEDGMENTS

The authors thank Dr. D Schoepp and the Lilly Co. for the generous gift of the 2R,4R-APDC and Drs. X.-T. Jin, Helen Flavin, Donald Mitchell, and Peter Kind for comments on the manuscript.

This work was supported by National Eye Institute Grant EY-00053 to N. W. Daw. C. J. Beaver was supported by the Ziegler Foundation.


    FOOTNOTES

Address for reprint requests: C. Beaver, Dept. of Ophthalmology and Visual Science, Yale University School of Medicine, 330 Cedar St., New Haven, CT 06520-8061.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 27 May 1998; accepted in final form 4 March 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society