NT-4/5 prevents the loss of responses to a deprived eye
To determine whether exogenous NT-4/5 would prevent ocular dominance plasticity caused by monocular visual deprivation, two identical cannulas were implanted into the visual cortex, one in each hemisphere, in five cats at approximately postnatal day 28 (P28), near the peak of the critical period for ocular dominance plasticity. NT-4/5 was infused at 100 ng/hr from the experimental cannula into primary visual cortex, and vehicle solution was infused at the same rate into the control hemisphere. To allow the region affected by neurotrophin infusion to reach its steady-state size, the infusions proceeded for 2 d before we initiated a 2 d period of MD by unilateral eyelid suture. The protocol for this experiment is illustrated in . Although a blind procedure was used for pump implantation, in practice, the effect of NT-4/5 infusion was so striking that the identity of the neurotrophin-treated hemisphere always became evident during optical imaging or single-unit recording, whichever was done first. It is known from previous microelectrode recording and optical imaging experiments that 2 d of MD are sufficient to cause the deprived eye to lose the ability to drive nearly all cells in primary visual cortex, whereas the open eye continues to drive nearly all cells strongly (Olson and Freeman, 1975; Crair et al., 1997
). The effects of NT-4/5 infusion were then assessed by making microelectrode penetrations to record from isolated cortical cells within 1.5 mm from the experimental and control infusion sites. The ocular dominance histograms of , display the relative efficacy of the two eyes in driving cortical cells in control and experimental hemispheres. Responses of cells near the vehicle infusion site in control hemispheres () were strongly shifted to favor the open eye in all animals, as expected for monocularly deprived animals of this age, whereas cells near the NT-4/5 infusion site () were nearly all driven well by both eyes, with no tendency for the deprived eye to be less effective than the open eye. Thus, the loss of response to the deprived eye did not occur in areas in which NT-4/5 levels were high. For comparison, an ocular dominance histogram from animals of comparable age with normal visual experience and untreated cortices is shown in (data from Stryker and Harris, 1986
). Compared with either normal or deprived animals, very few cells were monocularly driven by either eye after NT-4/5 treatment.
Figure 1 NT-4/5 prevents ocular dominance plasticity. Ocular dominance histograms compiled from cells recorded in primary visual cortex of five animals that received the treatment protocol shown in a, in which drug infusion lasted for 4 d, the last 2 d concomitant (more ...)
A more complete picture of the pattern of cortical response may be revealed by recording intrinsic signal optical images of cortical reflectance changes under red light in response to visual stimulation (Bonhoeffer and Grinvald, 1991
). Images from such optical recordings were produced in control and experimental hemispheres. Four different stimulus orientations were presented to each eye, along with a series of blank-screen stimuli. The blank-normalized images shown in , represent the fractional change in reflectance produced by each stimulus in each eye compared with the blank no-stimulus condition; dark areas
indicate strong responses. The position of the cannula is indicated by the filled arrowhead
in the leftmost
images. Artifacts attributable to shadows of blood vessels like the one indicated by the curved arrow
also appear as dark
or light lines
in many of the images. The images from control hemisphere driven by the open (ND
) eye in show dark areas of strong response interdigitated with very light areas. The different stimulus orientations when seen through the open eye activate different patterns of response in this hemisphere, indicating that the cortex is selective for stimulus orientation and revealing the positions of the orientation columns. In the maps of deprived (D
) eye responses from this same hemisphere, the only areas activated strongly by visual stimulation are patches that occupy the same positions for all four stimuli and are therefore not selective for stimulus orientation. This pattern of response through the deprived and nondeprived eyes is typical of cortical maps in short-term deprived animals (Crair et al., 1997
Figure 2 Optical imaging and dose dependence of the NT-4/5 effect. a, b, Typical grayscale optical images for vehicle and NT-4/5 infusion, showing that NT-4/5 infusion causes response to the deprived (D) eye to more nearly equal response to the nondeprived (ND (more ...)
The images from NT-4/5-treated cortex () are quite different from the control images. Two qualitatively different regions, whose boundaries are demarcated by open arrowheads, can be seen in the images. Far from the cannula site, to the right of the line indicated by the open arrowheads, the response patterns are similar to those described in the paragraph above for control cortex, with clear areas of strong, orientation-selective response from the nondeprived eye, and with strong responses from the deprived eye only within patches that are not selective for stimulus orientation. Near the cannula, to the left of the open arrowheads, response patterns from the two eyes are similar to each other. In this area, there is much less modulation of response, and the patterns for the different stimulus orientations are also very similar to each other. The similarity between the response for the two eyes (primarily unpatterned in both cases) is consistent with the single-unit recordings made in this area () and suggests that the effects of MD were blocked by the neurotrophin infusion.
This apparent blockade of the effects of MD in the optical images might artifactually result if the neurotrophin had merely suppressed the responses of cortical neurons to stimuli through both eyes. This is clearly not the case because, although the majority of cells in untreated cortex typically do not respond at all to monocular stimulation of the deprived eye (and receive ocular dominance scores of 1), in NT-4/5-treated cortex, most cells did respond to deprived-eye stimulation; thus, the response to deprived-eye stimulation was greater than in normal cortices. We further examined the response to the two eyes in the hemisphere illustrated in by making electrode penetrations at successively greater distances from the infusion site. These experiments also revealed the dose dependence of the neurotrophin effect. Electrode penetrations were made at the positions indicated on the picture of the cortical surface (). The unit recordings were grouped based on whether they lay within the region near the cannula that appeared to be affected in the optical maps shown in , or outside of this region, where responses appeared to be normal. For this hemisphere, an intermediate group includes the cells encountered along an electrode penetration close to the apparent border of the effect. shows that, near the cannula, where NT-4/5 concentration is presumably highest, cells were driven well by both eyes. Cells farthest from the cannula, within the area that shows normal patterning in the optical maps, are almost completely dominated by the open eye and give rise to a histogram similar to that seen for control hemispheres, as shown in . The ocular dominance histogram for cells in an intermediate region is intermediate, with a bias toward the open eye but with substantial deprived-eye responses as well (). All four hemispheres tested this way showed the same effect, nearly equal responses to the two eyes near the cannula and a strong dominance of the nondeprived eye farther from the cannula, indicating a dose-dependent effect of NT-4/5.
Measurements of the relative efficacy of the two eyes from optical imaging experiments led to similar conclusions. shows optical responses from the two eyes in another case in which the cannula position is indicated by the filled arrowhead to the left. The ocular dominance ratio map shows an area of faint pattern near the cannula and a more strongly modulated pattern farther from the infusion site. The quantitative measure computed for the regions indicated, one near to and the other far from the cannula, were 0.49 and 0.77, respectively (“optical BIs,” in which 0.5 indicates equal responses to the two eyes and 1.0 indicates complete dominance by the open eye). Although absolute responsiveness in spikes per second cannot be measured in these optical maps, the optical response to visual stimulation by a set of gratings compared with interleaved stimulation by a blank screen of mean luminance gives an overall measurement of visual response and was calculated for the affected and the control regions. For the case in , the two regions showed similar reflectance changes in response to visual stimulation (8.3 · 10-4 in the affected portion of the map near to the cannula and 8.0 · 10-4 far from the cannula). Across the five hemispheres whose data appear in , the ratio of average visual response near to and far from the cannula was 0.99, indicating that overall optical response in areas affected by NT-4/5 was very nearly the same as that in areas that showed no effect of NT-4/5 infusion. This response is not visually apparent in the illustrations of the maps for two reasons: (1) the affected area is activated nearly uniformly rather than in a modulated pattern, like that of the orientation columns, and (2) the illustrations are high-pass filtered over a uniform 2.35 mm square kernel to render the columnar patterns on the limited contrast range available on paper.
Figure 3 Ocular dominance computed from the optical maps in NT-4/5-treated cortex show results similar to those obtained with single-unit recording. A, Ocular dominance ratio map showing an area of faint ocular dominance pattern near the cannula and more strongly (more ...)
NT-4/5 causes cortical cells to lose orientation selectivity
One of the striking features of the optically imaged response patterns near the infusion sites in the neurotrophin-treated hemispheres is the relatively weak modulation of response to stimulus orientation. uses the conventional pseudocolor images computed from the grayscale response patterns to show features of the orientation response. Two kinds of maps are presented. Both maps show the preferred orientation as the hue of each pixel. The polar maps show a second dimension, using lightness to code orientation selectivity; dark areas are broadly tuned, showing similar responses (which may be either strong or weak) to different stimulus orientations. The HLS maps show three dimensions: hue to encode preferred orientation, color saturation to encode the degree of orientation selectivity, and lightness to encode the magnitude of visual responses. A site that responds well to all orientations is nearly white in the HLS map, and areas that do not respond are dark; bright, saturated areas have strong and selective responses. , shows both polar and HLS maps for the two hemispheres illustrated in . In control hemisphere () and in the experimental hemisphere far anterior to the infusion site (), responses through the nondeprived eye gives rise to well tuned (bright) polar maps and to well tuned (saturated) and strongly responsive (bright) HLS maps. Response to the deprived eye in control areas is not seen in the polar maps, but the patches of strong and poorly orientation-selective deprived-eye response show up as white areas (one of which is indicated by the white arrow) on the HLS maps. This may be compared with the ocular dominance ratio maps shown in . Within the area of NT-4/5 infusion (to the left of the open arrowhead in ), the polar map for the open eye is dark and the HLS map is unsaturated, indicating that neither eye is capable of producing a selective orientation map. Similar effects of NT-4/5 infusion are also evident in . The optical maps indicate that NT-4/5 treatment causes a loss of orientation selectivity when the cortex is driven through either the nondeprived or the deprived eye.
Figure 4 Polar, HLS, and ocular dominance ratio maps for control (a-c) and experimental (d-f) hemispheres after 4 d NT-4/5 infusion, with 2 d MD (same hemispheres shown in ). In the color polar maps, hue encodes the stimulus orientation that best drives (more ...)
Poor selectivity in cortical maps could be attributable to either reduced selectivity in individual cells or a reorganization in which cells selective for the same orientation were no longer clustered. Single-unit recordings show that the disappearance of the orientation map in regions affected by the NT-4/5 infusion is attributable to a reduction of selectivity in individual cortical cells. Cells within the infusion area were generally not selective or at best poorly selective for stimulus orientation, whether tested with hand-plotted or with automated stimuli. compares the orientation tuning of cells within the infusion zone with that of cells in control area. , shows polar plots of orientation tuning from cells recorded in penetrations 1 and 2 at the positions illustrated in . As was common in this area, both eyes drove cells effectively, but the response was not tuned for orientation through either eye. The cells shown in , from an electrode penetration in a control area, were well tuned for stimulus orientation but responded only to stimulation through the nondeprived eye. These four cells were recorded in a single electrode penetration and therefore had similar preferred orientations, as expected for cells within a single orientation column. The visual cortices of all young animals tested experienced a loss of orientation tuning after 4 d of NT-4/5 infusion. shows similar experimental data from another case, in which the polar plots show clear visual responses above the spontaneous firing rates but with little or no selectivity for orientation. Although spontaneous firing rates in , are elevated over normal, the cells shown in exhibit background firing rates equal to those in control areas. No consistent effects of NT-4/5 infusion on spontaneous firing rates were seen in this study. Single-unit recording after NT-4/5 treatment was consistent with the optical maps in revealing a loss of orientation selectivity when cells were driven through either eye.
Figure 5 Orientation selectivity of individual cortical neurons is affected by NT-4/5 infusion. a, Cortical surface of imaged area, showing position of cannula and sites of penetrations 1 and 2, overlaid by vector polar map showing extent of effect (open arrows (more ...)
Although not all cells were studied quantitatively, a crude assessment of orientation selectivity on a three-point scale was made from hand plots for all visually responsive cells. In normal cat visual cortex, nearly all cells are well tuned for stimulus orientation and would receive a score of 2. For most of the cells found in experimental areas affected by NT-4/5, a preferred orientation could not be determined; these cells received an orientation selectivity score of 0. Cells that responded somewhat more strongly to some orientations than others were scored as 1. Within each electrode penetration, orientation selectivity scores were averaged to give an OSI for the penetration. Likewise, an index of the bias toward the open eye was calculated for the collection of cells recorded in each individual electrode penetration; a value >0.5 indicates a bias in favor of the open eye. shows summary data for the dependence of the NT-4/5 effect on distance from the infusion cannulas. Orientation selectivity was compromised near the experimental cannula in all cases and reached control values at a distance of 1.5-2 mm (). Ocular dominance was not biased toward the open eye near the experimental cannulas but was progressively more shifted with increasing distance from the infusion site (). At distances farther than 2 mm, neuronal populations were as in control hemispheres. A lack of bias toward the open eye in the population of cells near the cannulas could result from either individual cells in the population that were driven well by both eyes or similar numbers of cells that were monocularly driven by the deprived and nondeprived eyes. The monocularity index () answers this question by showing that the individual cells near the cannulas were driven binocularly (MI near 0). Although biological activity of the NT-4/5 could not be measured directly in cortical tissue, the correlations of orientation selectivity and open-eye bias with distance from the infusion site point to a concentration effect of the neurotrophin. shows the rather variable but significant tendency (p < 0.01; Mann-Whitney U) for responsiveness to the optimal stimulus to be reduced within the NT-4/5-treated area. This finding from single-unit recording is not in conflict with the demonstration by optical imaging that, in the same animals, the overall level of visual responsiveness was not affected by NT-4/5 because the imaging measured the average response to the entire set of visual stimuli at all orientations, whereas the single-unit measure considers only the response to the single optimal stimulus.
Figure 6 Summary figure of dose dependence of NT-4/5 effect on ocular dominance shift and on orientation selectivity in four animals, showing that the effect of NT-4/5 on ocular dominance shift, monocularity, and orientation selectivity decreases with distance (more ...)
Ligands for trkA and trkC do not mimic the effects of NT-4/5
Neurotrophin-4/5 belongs to the family of neurotrophins that also includes NGF and NT-3. NGF and NT-3 exert their effects principally through activation of the trkA and trkC receptors, respectively. NGF in particular has powerful effects on visual cortical plasticity in rodents (Maffei et al., 1992
), and much weaker effects of NGF infusion into the lateral ventricle in cats have been reported (Carmignoto et al., 1993
). We sought to determine the specificity of the NT-4/5 effects noted above by comparing them with the effects of similar infusions of NGF or NT-3. shows results from two animals treated with NGF, following the protocol of that described in (one animal received 0.2 mg/ml for 4 d with 2 d MD, and the other animal received 0.4 mg/ml for 7 d with 2 d MD). Single-unit recordings made within 1.5 mm of the experimental cannula () revealed no mitigation of the ocular dominance shift in the area immediately surrounding the infusion site compared with the area around the control cannula (). Lacking a reliable measure for absolute biological activity of the neurotrophin within the infused area, we perfused the animals and immunostained for NGF in the tissue in which recordings had been made. shows that NGF was present at levels far exceeding endogenous concentrations within a small zone near the infusion site that was approximately equivalent to the zone of effect we had seen previously with NT-4/5 infusion. Experiments to be reported elsewhere established that the NGF infused into the cortex in these experiments was biologically active (M. Silver, M. Fagiolini, D. Gillespie, and M. Stryker, unpublished observations).
Figure 7 Intracortical infusion of NGF did not prevent the ocular dominance shift in two animals, one of which received NGF infusion for 4 d and one for 7 d, both with 2 d MD over the final 2 d of infusion. a, b, Ocular dominance histograms constructed from cells (more ...)
The trkC ligand NT-3 also failed to mimic the effects of NT-4/5 infusion in preventing the ocular dominance shift or in causing a loss of orientation selectivity. shows optical imaging results, as well as ocular dominance histograms for two animals in which NT-3 was infused for 4 d with MD during the final 2 d of neurotrophin infusion. A result like that in control cortex was observed with both single-unit recording and optical imaging (). Although neither biological activity nor absolute levels of NT-3 could be measured, immunostaining of the tissue from which recordings were made indicates that NT-3 was present at increased levels near the infusion site.
Figure 8 Intracortical infusion of NT-3 did not prevent the ocular dominance shift in two animals with 4 d NT-3 infusion and 2 d MD. a, Polar maps of cortex in which NT-3 was infused, showing well organized signal up to the cannula when stimulated through the (more ...)
NT-4/5 restores deprived-eye responses after a previous ocular dominance shift
Because NT-4/5 infusion prevents the loss of response to inputs from the deprived eye, it was interesting to examine whether it might restore the function of deprived-eye inputs that had already lost their efficacy. Two additional animals were monocularly deprived by unilateral eyelid suture at P28 and P31 during the critical period. After 3 d of MD, a period sufficient to induce profound ocular dominance plasticity (Crair et al., 1997
), a pump and cannula infusing NT-4/5 were implanted. The MD continued for 4 more days concurrent with the NT-4/5 infusion, at the end of which optical imaging and microelectrode recording were performed. This protocol is shown schematically in . The polar maps of , illustrate these cases and show that the ocular dominance shift and strong orientation-selective responses are present only in the region most distant from the infusion cannula. Nearer to the cannula, the images show the loss of orientation selectivity expected from the effects of NT-4/5 noted above. In addition, they show similar responses to the two eyes.
Figure 9 NT-4/5 nullifies a previous ocular dominance shift. a, Schematic of protocol for the two hemispheres shown in this figure. b-d, NT-4/5-treated hemisphere of animal whose MD began at P31. b, Polar map showing extent of infusion effect. Locations of cannula (more ...)
Consistent with the imaging results, response properties of single units were poorly selective in the area affected by NT-4/5, with OSIs equal to 0.62 and 0.03 for the penetrations near the cannula compared with 1.5 and 1.1 for the penetrations in more distant regions. Most strikingly, neurons in the affected area responded nearly as well to the deprived eye as they did to the open eye, despite the prolonged period of MD. , shows the relatively balanced ocular dominance distributions constructed from neurons in penetrations near the cannula, and d
show the profound shift in ocular dominance in unaffected regions. Because previous work had established that deprived-eye responses were primarily lost after 2 d of MD (Crair et al., 1997
), these results indicate that NT-4/5 infusion actually restored deprived-eye responses even during a period of continuing deprivation, nullifying the effects of the previous MD.
Effects of NT-4/5 on ocular dominance and orientation selectivity cannot be accounted for by acute actions
Acute effects of neurotrophins at central synapses have been reported by several groups (Kang and Schuman, 1995; Figurov et al., 1996; Akaneya et al., 1997; Scharfman, 1997
). These acute effects in the literature raised the possibility that the effects described here might result from direct actions on synaptic transmission or excitability rather than from effects on the signaling systems that regulate growth and development. In four cases, we prepared the animal for optical imaging and single-unit recording and then immediately implanted a cannula for neurotrophin delivery (using the same concentration of neurotrophin as in the chronic experiments, delivered from osmotic minipumps in three cases and from a microliter syringe pump at a higher rate of infusion, 12 μ
l/hr, in one case). Single-unit responses made as close as possible to the cannula and intrinsic-signal optical responses were monitored at successive times after the onset of the infusion to allow us to detect possible acute effects of the neurotrophin as a recovery of response to the deprived eye or as a loss of orientation selectivity. The dura was left intact to protect the cortex until recordings were begun at different times after onset of NT-4/5 infusion in the different animals (0-2, 24, 31-36, and 48 - 60 hr). The results for a representative animal that was monocularly deprived 2 d before the induction of anesthesia and implantation of minipump are shown in . At the end of this imaging session, NT-4/5 had been continuously infused for 30 hr, and by the end of single-unit recording, this hemisphere had experienced NT-4/5 infusion for 36 hr. Both optical recording and extracellular unit recordings reveal a cortical response strongly shifted toward the open eye, as shown in . Optical imaging showed clear orientation columns (), and electrophysiology showed that the individual neurons were well tuned for stimulus orientation (OSI of 2.0 for the two penetrations shown). Staining for antibodies to NT-4/5 after perfusion demonstrated that a high level of NT-4/5 was present in the cortical area from which optical imaging and extracellular recordings were made, despite the lack of an effect on responses. Immunostaining for NT-4/5 nearly always revealed very sharp borders for the neurotrophin diffusion, suggesting that NT-4/5 had completely saturated the area in which recordings were made. Because the neurotrophin solution was pumped at a rate of 1 mm3
/hr and recordings were made within 0.5 mm of the infusion cannula, we are confident that synapses at the recording sites were exposed to the neurotrophin within the first hour of the infusion. For all periods of NT-4/5 infusion <60 hr in anesthetized animals, no effects of NT-4/5 infusion on cortical maps or on the receptive field properties of individual cells were apparent. In addition, the acute NT-4/5 infusions produced no detectable changes from control areas in neuronal responsiveness.
Figure 10 Acute administration of NT-4/5 does not cause noticeable effects in <2 d. The animal shown in a experienced MD (2 d), but no neurotrophin infusion, before physiological recording. Onset of neurotrophin infusion coincided with the beginning of (more ...)
Because ocular dominance plasticity does not occur in the anesthetized animal, the experiments above do not exclude the possibility that NT-4/5 infusion into visual cortex might have prompt effects on responses and the induction of plasticity in awake animals, even when it did not do so in anesthetized animals. In one case, a 2 d NT-4/5 infusion was begun in an alert animal simultaneously with the onset of a 2 d period of MD. Optical images shown in revealed that the cortex remained selective and was dominated by the open eye up to the infusion site at the left edge of the images. Thus, no prompt effect of this neurotrophin on the visual cortex was found. The earliest effects we found were not present within the first 2 d, although all of the effects noted above appeared within 4 d of the onset of infusion. We conclude that the effects of NT-4/5 are not acute effects on synaptic function. The latency of NT-4/5 effects is so long that they were not detected before 60 hr of treatment, for either the induction of plasticity in alert animals or the recovery from plasticity in anesthetized animals.
The effectiveness of NT-4/5 in altering cortical cell response properties is confined to a period early in life
Monocular deprivation causes plasticity of visual cortical responses only during a critical period in early life. If the NT-4/5 acted as a retrograde messenger to regulate the mechanisms responsible for this plasticity, it might be expected to be effective only during the critical period. In three adult animals (ages 6 months to 5 years), we tested the efficacy of NT-4/5 in altering response properties in visual cortex well past the critical period for plasticity. One animal was monocularly deprived at P28. At 6 months of age, a minipump and cannula were implanted to deliver twice the normal concentration of NT-4/5, and 4 d later, optical imaging and extracellular recording were performed. summarizes the results from this animal. The optical maps indicate that the cortex was completely shifted to the nondeprived eye and that orientation selectivity was strong, even very close to the cannula (). The ocular dominance histogram for the electrode penetration closest to the cannula likewise shows that these cells remained selective for the open eye (), despite the presence of NT-4/5 immunohistochemically demonstrated in . The single units in this penetration, all driven exclusively by the open eye, remained normally selective (OSI of 1.9). We also found no loss of selectivity for stimulus orientation or changes in ocular dominance in two additional adult animals (~2 and 5 years old, both without deprivation during the critical period), including one in which NT-4/5 was infused for 2 full weeks. These experiments indicate that NT-4/5 inf usion into adult cortex, even for three times the duration or twice the concentration that is effective during the critical period, appears to be without effect on any aspect of cortical responses that we measured. This finding is consistent with a temporally specific role for this neurotrophin in development.
Figure 11 NT-4/5 infusion has no effect on cortex of an animal 6 months of age. a, Polar maps from the hemisphere of a 6-month-old animal, monocularly deprived at P28, that received 4 d NT-4/5 infusion immediately before recording. Black dot indicates position (more ...)