Monocular deprivation (MD) during the critical period reduces the visual cortical response to the deprived eye and causes the geniculocortical axons serving the deprived eye to retract. When MD is combined with a pharmacological inhibition of the visual cortex, the cortical neurons weaken their response to an open eye and the input axons serving the open eye retract. To determine whether the 2 types of ocular dominance (OD) plasticity reflect an experience-driven modification of neural circuits sharing the same developmental time course, we analyzed the OD plasticity in an inhibited visual cortex using cats at different ages. MD did not affect the OD distribution in the inhibited cortex of adults, confirming that the OD plasticity in the inhibited cortex represents a developmental plasticity. In developing animals, the OD plasticity in the inhibited cortex was observed at the late phase of the critical period (P40–46) but not at the early phase (P22–26). We found a retraction of input axons serving an open eye at the late phase, whereas those at the early phase were comparable to the axons of normal animals. Therefore, the maturation of visual circuits might include an experience-driven rearrangement of thalamocortical projections during the late phase of development.
In the mammalian visual system, monocular deprivation (MD) during development causes the loss of cortical responses to the deprived eye (ocular dominance [OD] plasticity) (Wiesel and Hubel 1963). Anatomically, afferent axons from the lateral geniculate nucleus (LGN) serving the deprived eye retract (Antonini and Stryker 1993, 1996) and ultimately lose their cortical territory (Shatz and Stryker 1978; LeVay et al. 1980).
Previous works have demonstrated the role of the activity of cortical cells in OD plasticity. When MD is combined with a pharmacological inhibition of the visual cortical neurons by administering muscimol, a GABAA receptor agonist, leaving the activity of the geniculocortical afferents intact, the cortical neurons weaken their responses to the open eye and active afferents serving the open eye selectively retract (Reiter and Stryker 1988; Hata and Stryker 1994; Hata et al. 1999).
Although the 2 types of OD plasticity, one in the normal cortex and the other in the inhibited cortex, both reflect an experience-driven modification of neural circuits, previous studies demonstrated their distinct characteristics. An imbalance of inputs from the 2 eyes is necessary for OD plasticity in the normal cortex because the cortical responses and input axons remain mostly intact after binocular deprivation (Wiesel and Hubel 1965; Antonini and Stryker 1998). In contrast, in the inhibited cortex, the afferents serving an open eye significantly retract, regardless of whether the other eye is closed (Haruta and Hata 2007). In addition, OD plasticity in the normal cortex requires the activation of an intracellular signaling cascade involving the cAMP/PKA pathway (Beaver et al. 2001; Fischer et al. 2004), whereas OD plasticity in the inhibited cortex can be induced in the presence of a PKA inhibitor (Shimegi et al. 2003). These studies suggest that the 2 types of OD plasticity may represent distinct components of the experience-dependent development of the visual system. Alternatively, however, the reverse OD plasticity in the artificially inhibited cortex might be caused by an unknown mechanism that is not related to developmental plasticity. Therefore, it is important to determine whether the reverse OD plasticity in the inhibited cortex operates only in developing animals.
In the normal cortex, MD affects visual responsiveness in a restricted period of development called the critical period with its peak at the fourth week of age in cats (Olson and Freeman 1980). Therefore, we analyzed the OD of cortical neurons and morphology of geniculocortical axons in a pharmacologically inhibited visual cortex using cats at around the peak of the critical period (early phase, postnatal days [P] 22–26), at the late phase of this period (P40–46) and in adults to determine whether the OD plasticity in the normal and inhibited cortices shares the same developmental time course.
We demonstrate that the reverse OD plasticity is not observed in adult animals, confirming that the plasticity in the inhibited cortex reflects an aspect of developmental plasticity. Furthermore, we demonstrate that the reverse OD plasticity operates only in the late phase of the critical period but not at the peak of it. These observations suggest that the critical period may consist of multiple stages that have distinct time course.
Materials and Methods
All of the cats in this study were born in the breeding colony of Tottori University. The experimental procedures used met the guidelines of the animal care committee of Tottori University.
A stainless steel cannula connected to an osmotic minipump (Alzet 2001 or 2002, Durect, CA, United States of America) was implanted in the left hemisphere of the primary visual cortex (stereotaxic location: A −3.0 mm, L 2.0 mm, depth from cortical surface 2.0 mm) of animals of either sex at the early phase (P22–26) and the late phase (P40–46) of the critical period and adults (>P500), and a muscimol solution was infused continuously (30 mM in Ringer's solution, 1.0 or 0.5 µL/h, Sigma-Aldrich, MO, United States of America or Tocris Bioscience, MO, United States of America). During the same surgery, biotinylated dextran amine (BDA-10 000, Molecular Probes, OR, United States of America) was injected at lamina A of the LGN to label the geniculocortical axons. A glass pipette filled with BDA solution was inserted stereotaxically to locate a clear visual response from lamina A of the LGN, which is ipsilateral to the infusion cannula. BDA was iontophoretically injected (pipette positive current of 3–5 µA, 2 s on/off pulse, 50–70 times) at 3–5 sites in each LGN. After 1–2 days, either eye was deprived of vision by eyelid suture so that the labeled LGN axons represent inputs from either an open or closed eye. BDA was also injected in several normal animals at P22–23 and P41–43. All of the surgical procedures were performed using sterile conditions under anesthesia with 1.5–3.5% isoflurane in a 1:1 mixture of N2O and O2. All of the incisions were infiltrated with local anesthetics (Xylocaine pump spray, AstraZeneca, Japan). The animals were given an antimicrobial agent (enrofloxacin, 5 mg/kg, Baytril, Bayer, Germany) every day after the surgery.
Following 6 days of MD (7 or 8 days of muscimol infusion) or at the corresponding age in the case of the normal animals, the single-unit activity was recorded from the primary visual cortex (early phase, P28–34; late phase, P47–53). Anesthesia was induced with 4–5% isoflurane in N2O and O2 (1:1) and maintained with pentobarbital sodium (2–4 mg/kg/h i.v., Nembutal, Dainippon Sumitomo Pharma, Japan). The pupils were dilated with 0.5% tropicamide and 0.5% phenylephrine hydrochloride (Mydrin-P, Santen Pharma, Japan), and contact lenses were placed on the corneas. The animals were paralyzed with gallamine triethiodide (10 mg/kg/h, i.v., Sigma-Aldrich) or pancuronium bromide (0.2 mg/kg/h, i.v., Mioblock, Nihon Organon, Japan) and maintained under artificial respiration. The end-tidal CO2 concentration and body temperature were maintained at 4% and 38°C, respectively. All of the incisions were infiltrated with local anesthetics. The cortical area that was inhibited by muscimol was delineated by recording the cortical cell activity at various distances to the infusion cannula using a tungsten microelectrode, and then the pump was removed. After the recovery of cortical activity from the inhibitory effect of muscimol, the visual response of cortical neurons was recorded, and the OD of individual neurons was determined in both hemispheres on the basis of a conventional 7-point classification (Hubel and Wiesel 1962).
After the recording, a needle mark was made in the cortex at the border of the inhibited region. In the normal animals, 2 needle marks were made with 1 mm separation. The needle marks and cannula tracks were used to calculate the shrinkage correction in the analysis of the axon morphology. The animals were euthanized with an overdose of pentobarbital sodium (100 mg/kg, i.v.) and perfused transcardially with cold phosphate-buffered saline and 4% paraformaldehyde in 0.1 M phosphate buffer. Tissue blocks containing the LGN and entire caudal pole of the cortex were cut using a freezing microtome in the frontal plane (50 µm thickness). All of the sections were processed using the standard ABC kit (Vector Laboratories, CA, United States of America) to visualize the tracer. Selected sections containing the visual cortex or LGN were stained with cresyl violet for the localization of the layer boundaries and injection sites.
Axonal Arbor Analysis
All of the injection sites were well confined to lamina A of the LGN (Fig. 2A,B). When an injection site invades the lamina A1 representing the ipsilateral eye, we eliminated all of the samples from the same animal from the arbor reconstruction. The BDA-filled arbors were reconstructed from serial sections in 3 dimensions with the aid of computer software (Neurolucida, Microbrightfield, VT, United States of America) and then corrected for tissue shrinkage. We calculated 2 measures, the total length and branch point number, to quantify the size and complexity of the individual axonal arbors. The total length was obtained by adding the length of all of the axonal segments constituting the terminal field of an arbor. The branch point number is the number of axon bifurcations in the terminal arborization. Only the portion of the arbor located in layers II/III and IV was considered for these analyses. Very short endings <5 µm were omitted from the analyses.
Age-Dependence of the Physiological OD Plasticity in the Inhibited Visual Cortex
We infused one hemisphere of the visual cortex of young (early phase of the critical period, P22–26; late phase of the critical period, P40–46) and adult (>P500) cats with muscimol, and they were monocularly deprived for 6 days. MD caused a loss of visual response to the deprived eye in the control hemisphere, which is the untreated hemisphere contralateral to the muscimol-infusion site, of kittens at both the early and the late phase of the critical period but not in adults (Fig. 1A, “MD”), as demonstrated previously (Olson and Freeman 1980). We calculated the CBI (see the Materials and Methods section) for the individual hemispheres to evaluate the degree of OD plasticity. For the normal animals, the CBI values were 0.53 ± 0.05 (mean ± SD, n = 3 animals) in the early-phase group and 0.54 ± 0.04 (n = 4) in the late-phase group, reflecting a slight natural bias toward the contralateral eye (Fig. 1A, “normal”). The CBIs of the control hemispheres of young animals (early, 0.09 ± 0.06; late, 0.08 ± 0.04) were significantly deviated toward the ipsilateral open eye versus those of the normal animals in the 2 age groups (Fig. 1A, “MD”, and Fig. 1B).
In the inhibited cortex, the cortical neurons responded preferentially to the ipsilateral deprived eye (reverse OD plasticity) in the kittens at the late phase of the critical period (Fig. 1A, “Late”, “MD + muscimol”). Their CBIs (mean ± SD = 0.43 ± 0.10) were significantly different from those of the control hemisphere, showing that the OD shift toward an open eye had been blocked (Fig. 1B, “Late”). Furthermore, the CBIs in the inhibited cortex were significantly deviated from those of the normal animals toward the deprived eye (Fig. 1B, “Late”). In the kittens at the early phase of the critical period, most neurons in the inhibited cortex responded to both eyes equally (Fig. 1A, “Early”, “MD + muscimol”). Their CBIs (0.51 ± 0.03) were significantly higher than those of the control hemisphere, showing a blockage of the OD shift toward an open eye (Fig. 1B, “Early”). Unlike the late-phase group, however, the OD distribution in the inhibited cortex did not show a shift toward the deprived eye and was comparable to that of the normal animals (Fig. 1B, “Early”). The direct comparison of the OD distribution in the inhibited cortex showed a significant difference between the early- and late-phase groups (Fig. 1A, early vs. late in “MD + muscimol”, P < 0.05, χ2 test).
The adult animals preserved the binocular responses in both the control (Fig. 1, “MD”, CBI = 0.55 ± 0.04) and the inhibited cortices (“MD + muscimol”, CBI = 0.53 ± 0.04), confirming that the reverse OD plasticity can be expressed only in the developing brain, similar to the OD plasticity in the intact cortex. Therefore, the reverse OD plasticity in the inhibited cortex should reflect a type of developmental plasticity that operates only in the late phase of the critical period.
Experience-Driven Retraction of Geniculocortical Axons in the Late Phase of the Critical Period
Previous studies demonstrated that input axons from the LGN carrying information from an open eye significantly retract in the inhibited cortex (Hata et al. 1999; Haruta and Hata 2007). To determine the age dependence of the structural remodeling of input axons, we injected an anterograde tracer, BDA, into lamina A of the LGN and deprived vision in the ipsilateral eye, so as to label the axons serving the open eye (Fig. 2A,B). The cortical arbors of the geniculocortical axons in the inhibited cortex were labeled well with BDA, as shown in Figure 2C,D. We found no noticeable difference in the quality of labeling between the axons in the 2 age groups.
Axonal arbors serving the open eye in the inhibited cortical region were reconstructed from the animals at the early phase (n = 18 arbors from 8 animals) and the late phase (n = 11 arbors from 4 animals) of the critical period and compared with those of the age-matched normal animals (early, n = 9 arbors from 3 animals; late, n = 6 arbors from 2 animals). Examples of arbors from each group are shown in Figure 2E,F. We quantitatively evaluated the morphology of the individual axons by measuring 2 parameters: The total length of axonal segments and the number of branch points of the cortical arbors (Fig. 3). In the animals at the late phase of the critical period, the open-eye arbors in the inhibited cortex were significantly shorter and had fewer branch points than those in the age-matched normal animals (total length, P < 0.005; branch points, P < 0.01, Student's t-test) (Fig. 3, “Late”).However, the open-eye arbors in the inhibited cortex at the early phase of the critical period were preserved and similar in total length and branching number to those in the normal animals (not significant, Student's t-test) (Fig. 3, “Early”). We had previously reported that the arbors serving the deprived eye in the muscimol-treated cortex do not show a retraction and are comparable to normal axons in animals at the late phase of the critical period (Hata and Stryker 1994; Haruta and Hata 2007). Thus, we further determined whether visual inputs influence axonal morphology in the inhibited cortex of animals at the early phase of the critical period. By labeling the LGN neurons in lamina A and depriving the contralateral eye of vision, we reconstructed the deprived-eye arbors in the muscimol-treated cortex of early-age animals (10 arbors from 3 animals; Fig. 2E). We found no significant difference in the number of branching or total length between the deprived-eye arbors and open-eye arbors in the muscimol-treated cortex or between the deprived-eye arbors in the inhibited cortex and normal arbors (not significant, Student's t-test; Fig. 3, “Early”). Therefore, experience-dependent retraction of LGN axons in the inhibited cortex should occur at the late phase of the critical period but not around the peak of it.
In this study, the reverse OD plasticity in the inhibited cortex was observed only at the late phase of the critical period. Consistently, we found a retraction of the LGN axons serving an open eye in the inhibited cortex at the late critical period, whereas the axons at the early phase were comparable to those in the age-matched normal animals. Three previous studies have demonstrated reverse OD plasticity in the inhibited cortex (Reiter and Stryker 1988; Hata and Stryker 1994; Shimegi et al. 2003). The lack of reverse OD plasticity in the early-phase group of the present study does not contradict the previous results because those studies had used older animals (age at MD: P30–35 [Reiter and Stryker 1988], P30–37 [Hata and Stryker 1994], and P43–45 [Shimegi et al. 2003]). Among them, Reiter and Stryker (1988) had begun the MD at P30–35 and recorded at P35–43, whereas we recorded in the early-phase group of the present study at P30–34, which corresponds to the age of the beginning of MD in the previous study. Thus, the lack of reverse OD plasticity in the early-phase group in the present study suggests that the beginning of the period of reverse OD plasticity would be later than P30. Regarding the other 2 previous studies, Hata and Stryker (1994) had used a longer MD (4 weeks from P30 to 37) which completely covered the MD period of the late-phase group, and Shimegi et al. (2003) had used a similar protocol as the present late-phase group (MD from P43–45 to P48–50). Therefore, reverse OD plasticity can be observed until approximately P50, though we do not assert that the period terminates at approximately P50. Although the OD plasticity of cats declines gradually with age and the age of termination is not precisely determined, it is possible that we could observe reverse OD plasticity in animals older than those in the present study, considering that the OD plasticity in the normal cortex can be observed even at 1 year of age (Daw et al. 1992).
OD plasticity is observed in many species including rodents, carnivores, and primates. In the mouse, Gordon and Stryker (1996) defined the critical period of OD plasticity as P19–32 with its peak at around P28 using single-unit analysis, which is comparable to the critical period in rats (Fagiolini et al. 1994). However, subsequent studies using visually evoked potentials to evaluate OD demonstrated that some degree of OD plasticity can be observed in older animals up to P55 in rats and even in adults in mice (Guire et al. 1999; Sawtell et al. 2003). Also, geniculocortical axons extend and elaborate their cortical arbors in experience-dependent manner even after P40 in mice (Antonini et al. 1999). Therefore, some aspects of OD plasticity can proceed in the late- or postcritical period of rodents. On the other hand, the visual system of monkeys appears to develop earlier than that of cats and rodents. Anatomically, the OD columns are formed in utero and appear like adult at birth (Rakic 1976, 1977; Horton and Hocking 1996), suggesting an important role of spontaneous activity in the OD column formation. Postnatally, however, visually driven activity modifies the visual neural circuits. MD just after birth induces OD shift and a change in OD column width (LeVay et al. 1980; Horton and Hocking 1997). Therefore, the critical period appears to begin with no significant delay after birth in the monkey. Assessed by the effect of MD on the OD columns, the critical period of monkeys is considered to last until 10–12 weeks of age (LeVay et al. 1980; Horton and Hocking 1997).
The absence of reverse OD plasticity at the early phase of the critical period may simply reflect a less effective pharmacological inhibition in those animals than in the older ones because the degree of reverse OD plasticity is reported to be proportional to the size of the inhibited cortical area (Reiter and Stryker 1988). In the present study, we found no significant difference between the early- and late-age groups with regard to the size of the inhibited cortical area (early, 4.92 ± 3.19 mm; late, 5.66 ± 1.74 mm, Student's t-test) or in the distance from the cannula to the recording sites (mean ± SD: Early, 1.25 ± 0.36 mm; late, 1.14 ± 0.50 mm, Student's t-test). In addition, we found no significant difference among the arbor groups in the inhibited cortex with regard to the distance from the cannula to the reconstructed arbors (early open, 1.05 ± 0.88 mm; early closed, 1.59 ± 1.09 mm; late open, 1.49 ± 0.86 mm, Student's t-test).
Alternatively, the cortical neurons at the early phase of the critical period may be less sensitive to muscimol than those at the late phase due to an immaturity of the GABAergic inhibitory mechanisms. Actually, the expression levels of GABAA receptors and GABA-synthesizing enzymes increase during early postnatal development in the cat visual cortex (Shaw et al. 1986; Guo et al. 1997). Furthermore, the subunit composition of the GABAA receptor changes to the mature type, which contains the α1 subunit during the critical period of the cat visual cortex (Chen et al. 2001). Therefore, we examined the effect of muscimol on the activity of the visual cortical neurons using an animal at P19, which is younger than the age at the start of the muscimol infusion of the early-phase group. We observed no spontaneous or visually evoked activity in the region around the muscimol infusion site 2 h after starting the infusion. This result is consistent with a previous report showing that an iontophoretic application of GABA inhibited visual cortical activity even in kittens at 6–13 days old (Sato and Tsumoto 1984). Thus, the muscimol application in the present study should have inhibited the cortical activity effectively at both early and late phases of the critical period.
In this study, neither the normal nor reverse OD plasticity was observed in the adults. Thus, both types of OD plasticity should represent a developmental plasticity of the visual cortex. In the developing animals, the OD shift toward an open eye had been blocked in the inhibited cortex of the both early- and late-age animals, suggesting that cortical activity is necessary for the induction of normal OD plasticity in favor of the open eye. However, reverse OD plasticity was observed only at the late phase of the critical period. Considering that PKA activation is necessary for the OD plasticity in the normal cortex (Beaver et al. 2001; Fischer et al. 2004), but not for the reverse OD plasticity in the inhibited cortex (Shimegi et al. 2003), the mechanisms of reverse OD plasticity would be distinct from those of the normal OD plasticity and operate selectively at the late phase of the critical period. Interestingly, several genes encoding synaptic and signaling molecules are regulated by visual experience just after the end of the physiologically defined critical period in the mouse visual cortex (Majdan and Shatz 2006).
Regarding axonal rearrangement, the activity of the cortical neurons should be crucial for the retraction of the axons serving the deprived eye in the normal cortex because the deprived-eye axons retract when their target neurons are activated by visual inputs from the other eye but not when they are inhibited pharmacologically (Hata et al. 1999) or both eyes are deprived of vision (Antonini and Stryker 1998). Conversely, in the inhibited cortex, the afferent axons serving an open eye retract, regardless of whether the other eye is closed (Haruta and Hata 2007). Furthermore, presynaptic mechanisms should mainly underlie the retraction of open-eye axons in the inhibited cortex. Geniculocortical axons exhibit an experience-driven retraction even in the cortex treated with botulinum toxin which prevents synaptic transmission (Watanabe et al. 2009). Thus, the activity of afferent axons may directly regulate their own retraction. Although the mechanism of axon retraction in the inhibited cortex is yet unknown, the visually driven activity might recruit intracellular signals leading to morphological changes such as Ca2+ influx at the terminals, which can regulate axonal behavior (Henley and Poo 2004).
Taken together, the retraction of the open-eye axons in the inhibited cortex represents a developmental mechanism that prunes axons when there is no postsynaptic response. Such a mechanism is suitable for maintaining the OD columns by pruning the axon branches serving the wrong eye, as demonstrated in the eye-specific segregation of retinogeniculate axons (Chapman 2000). It is noteworthy that a large-scale reorganization of the orientation and OD columns occurs in the intact kitten visual cortex at the late phase of the critical period (after postnatal week 7) (Kaschube et al. 2009). Therefore, the structural reorganization of neural circuits, including thalamocortical projections, may occur during the late period of postnatal maturation by pruning the inappropriate connections.
This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas—Mesoscopic neurocircuitry—from the Ministry of Education, Culture, Sports, Science and Technology of Japan (22115010 to Y.H.).
Conflict of Interest: None declared.