Ординатура / Офтальмология / Английские материалы / Eye, Retina, and Visual System of the Mouse_Chalupa, Williams_2008

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The development of neural circuits and of behavior is regulated by the interaction between genetically coded innate programs and experience. The prevailing consensus that genes and the environment work in concert in shaping brain development is the result of the nature versus nurture debate (for a review, see Krubitzer and Kahn, 2003), in which genetic and environmental influences have long been considered mutually exclusive in regulating mammalian brain development. This position has mostly resulted from the relatively recent (1960s) ability to quantify and measure environment-induced changes in the brain. Beginning in the early 1960s, brain development ceased to be considered an experience-independent process, and its mature structure has proved not to be immutable.

The mouse is used as a privileged model for studies aimed at investigating the influence of the environment on brain and behavior, thanks to the detailed knowledge available on its central nervous system (CNS) functions and the remarkable possibility afforded by the application of genetic engineering techniques in this species, allowing targeting of specific molecules in various transgenic lines. Considerable advances in this field have been obtained with the environmental enrichment (EE) paradigm.

Environmental enrichment was first defined by Rosenzweig et al. (1978) as “a combination of complex inanimate and social stimulation.” It is a relative concept: an environmental setting is enriched with respect to other environmental settings. In studies on rodents under laboratory conditions, animals receiving EE are reared in large groups (8–12 individuals per cage) and in cages of large dimensions, with a variety of toys, tunnels, nesting material, and staircases available and changed frequently. In addition to the exploratory activity promoted by the presence of new objects, an essential component of EE is the opportunity for animals to attain high levels of voluntary physical activity on running wheels. For comparison, in the standard laboratory condition, animals are reared in small groups of 3–5 individuals in small cages where no particular items other than nesting material and food and water are present. At the opposite end of an ideal continuum of enrichment is the impoverished condition, in which even normal social interactions are prevented because animals are reared alone in separate cages.

It has often been suggested that EE in laboratory conditions is simply a way of rearing the animals in a setting more similar to the conditions in the wild, where animals explore the environment to find food, engage in physical activity both while foraging and while escaping predators, and cope with several challenges. However, observation of mice and rats that play in the EE and choose when and how much to run on the wheel, explore, and interact with the new objects suggests a different idea, namely, that EE is not just a way to reproduce more natural life conditions. Rather, EE animals are voluntarily exploring a rich environment without having to face the challenges present in the wild. One could speculate that the activity of mice and rats in the wild is mostly driven by necessity, whereas in an EE it is driven by curiosity (and perhaps pleasure). It is as if the ingenuity stimulated by challenge were exchanged for ingenuity stimulated by play.

Rosenzweig and colleagues originally showed that the morphology, chemistry, and physiology of the brain can be altered by exposure to EE (Rosenzweig, 1966; Rosenzweig and Bennett, 1969). Since then, a number of studies have been performed showing that EE can elicit various plastic responses in the brain at molecular, anatomical, and functional levels (for reviews, see Rosenzweig and Bennett, 1996; van Praag et al., 2000; Diamond, 2001).

The use of mice in these studies has made it possible to advance our knowledge of the processes underlying brain responses to the external world, and of the molecules involved. One field that has particularly benefited from studying mice in EE paradigms is visual system plasticity. The visual system is classically considered to be a paradigmatic model for the experience-dependent development and plasticity of the brain, and for the analysis of factors restricting environmental influence to specific developmental time windows, known as critical periods (Wiesel, 1982; Berardi et al., 2003; Hensch, 2005). The fields of visual cortical plasticity and EE, until recently separated, have been combined in the novel approach of investigating the effects of enhanced sensorimotor stimulation on the processes governing ex- perience-dependent development in the visual system. In this analysis, visual system development serves as a model to study the effects of environment on brain development

and plasticity, allowing researchers to uncover previously unknown effects of environmental experience on neural circuit development. At the same time, EE turned out to be a useful tool for probing visual circuit plasticity and for discovering previously unknown aspects of developmental visual plasticity. Use of the mouse in such experiments has often led to a characterization of the underlying molecular factors.

This chapter first surveys the vast literature on the effects of EE on the brain at anatomical, molecular, and behavioral levels. We have highlighted those studies in which the analysis particularly benefited from use of the mouse model. We then discuss the influence of early polysensorial stimulation on neural and behavioral development by reviewing data on the effects of maternal care and precocious novelty exposure in rodents. Finally, we present studies ongoing in our laboratory on the influence of EE on the development and plasticity of the visual system.

Influence of environment on brain and behavior: Neural consequences of environmental enrichment

Environmental enrichment has a variety of effects on the brain, and these effects have been found in several species of mammals, from mice and rats to gerbils, ground squirrels, cats, and monkeys (Rosenzweig and Bennett, 1969). At the anatomical level, which was the first to be investigated, exposure to an enriched living condition leads to a robust increase in cortical thickness and weight compared with those same parameters in animals reared under standard laboratory conditions (Rosenzweig et al., 1964). These changes occur in the entire dorsal cortex, including frontal, parietal, and occipital cortex. Since this initial finding was published, many studies have reported various anatomical changes associated with enriched living conditions, including an increase in soma size and in the size of nerve cell nucleus (Diamond, 1988), increased dendritic arborization (Holloway, 1966; Globus et al., 1973; Greenough et al., 1973), increased length of dendritic spines and increased synaptic size and number (Mollgaard et al., 1971; Turner and Greenough, 1985; Black et al., 1990), increased postsynaptic thickening (Diamond et al., 1964), and gliogenesis (Diamond et al., 1966). Recent studies have shown that exposure to an EE increases hippocampal neurogenesis (Kempermann et al., 1997) and reduces apoptotic cell death (Young et al., 1999).

One of the most striking properties of EE is the capacity to modify behavior, with the general rule that the best characterized effects are seen on tasks involving superior cognitive functions, mostly learning and memory (for a review, see Renner and Rosenzweig, 1987). EE enhances spatial learning and memory on the Morris water maze (Pacteau et al., 1989; Tees et al., 1990; Falkenberg et al., 1992; Paylor

et al., 1992; Moser et al., 1997; Van Praag et al., 1999; Tees, 1999; Williams et al., 2001), reducing the cognitive decline in spatial memory typically associated with aging (for a review, see Winocur, 1998). The effects of EE on learning and memory are not limited to spatial abilities but extend to visual recognition memory and to classic conditioning, as shown by Rampon et al. (2000b).

Efforts aimed at understanding possible molecular mechanisms underlying the reported changes operated by EE on brain and behavior started very precociously, prompted by an interest in finding molecules that could be manipulated to reproduce the beneficial effects of the enriched experience. Early studies by Rosenzweig et al. (1962, 1967) found an increase in acetylcholinesterase activity, suggesting an effect on the cholinergic system, with subsequent studies confirming and extending this initial observation to the other neurotransmitter systems that have diffuse projections to the entire brain, such as the serotoninergic system (Rasmuson et al., 1998) and the noradrenergic system (Escorihuela et al., 1995; Naka et al., 2002). A large number of genes were found to change their expression levels in response to EE, most of them in functional classes linked to neuronal structure, synaptic transmission and plasticity, neuronal excitability, and neuroprotection (Rampon et al., 2000a; Keyvani et al., 2004). One group of molecules particularly sensitive to environmental stimuli and exerting potent effects on the nervous system are the neurotrophins, a class of neurotrophic factors promoting neuronal development and survival, comprising nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neuro- trophin-4 (NT-4). Neurotrophins are strongly implicated in regulating structural and functional plasticity both during development and in the adult (reviewed in Bonhoeffer, 1996; Cellerino and Maffei, 1996; Berardi and Maffei, 1999; Thoenen, 2000; Berardi et al., 2003). EE increases neurotrophic factor expression (reviewed in Pham et al., 2002) in the visual cortex and hippocampus (Torasdotter et al., 1996, 1998). In addition, EE can promote brain uptake of physiologically relevant trophic factors, such as insulinlike growth factor I (IGF-I), levels of which are sensitive to the amount of voluntary physical exercise (Carro et al., 2000, 2001; Koopmans et al., 2006).

Effects of early-life stimulation on neuronal and behavioral development

Experiences acquired between birth and weaning age are essential in promoting and regulating neural development and behavioral traits in the newborn of most mammalian species (Fleming et al., 1999). During this critical period of high developmental plasticity, maternal influence is one of the most important sources of sensory experience for the developing subject (Hofer, 1984; Ronca et al., 1993;

Liu et al., 2000), directly regulating physical growth and promoting neural maturation of brain structures involved in cognitive functions (Fleming et al., 1999). This issue has been intensively studied in laboratory rodents, in which the maternal behavior consists of stereotyped modules that can be easily investigated and manipulated in controlled experimental conditions.

One of the best characterized effects of precocious maternal influence is that on the stress responses exhibited by the offspring when they become adult (for a review, see Francis and Meaney, 1999). It has been repeatedly demonstrated that the offspring of mothers exhibiting high levels of maternal care show reduced behavioral fearfulness and stress levels as adults, compared with the offspring of less active caregivers. These differences extend beyond the system underlying the stress response to involve systems known to mediate cognitive processing. In particular, the offspring of mothers that show high levels of maternal care have enhanced spatial learning and memory when tested as adults in the Morris water maze (Liu et al., 2000). Furthermore, the same animals show increased expression of NMDA receptor subunits and BDNF mRNA in the hippocampus (Liu et al., 2000). Recently it has been shown that mice that received enhanced levels of maternal care owing to the presence of more than one dam in the cage displayed (once adult) a higher propensity to interact socially, achieved more promptly the behavioral profile of either dominant or subordinate male, and had higher NGF and BDNF levels (Branchi and Alleva, 2006).

The reported differences in stress response between the offspring of mothers exhibiting high and low levels of maternal care were found to depend on differences in DNA methylation of the glucocorticoid receptor (GR) gene promoter at the hippocampal level (Weaver et al., 2004). DNA methylation is a stable epigenetic mark of gene regulatory sequences, strongly affecting levels of gene transcription, with hypomethylation being associated with active chromatin structure and transcriptional activity (Keshet et al., 1985; Razin, 1998). In offspring of mothers providing high levels of care, the promoter region of the GR gene in hippocampal neurons undergoes a selective demethylation (Weaver et al., 2004). This difference in DNA methylation is long-lasting, remaining consistent through adulthood. The reduced methylation levels of the GR gene promoter render this sequence more accessible to transcription factor binding, thus resulting in increased GR gene expression. The ensuing higher efficiency of the feedback for circulating stress hormones is enhanced in rats that have experienced intensive maternal care levels during infancy, which explains the typical phenotype of reduced stress response exhibited by these animals (Weaver et al., 2004).

These new results strongly demonstrate that early environmental experiences can profoundly affect the adult phe-

notype through epigenetic processes affecting structure and function of the chromatin.

Despite the large amount of data obtained from enriched adult animals, the possibility that an enhanced sensorimotor stimulation provided by EE early in life could induce neural and behavioral changes has been little investigated. The scant interest in early-life EE studies can be partially attributed to the fact that preweaning enrichment is characterized by very little voluntary physical exercise, as the pups are simply too small and inert to engage in sustained activities. Since early enrichment provides increased levels of polysensorial stimulation during a period of high anatomical and functional rearrangement of the cerebral cortex, however, it might be expected to elicit brain changes through experience-dependent plasticity processes. This supposition has been confirmed by a handful of reports in the literature. Indeed, more complex dendritic branching has been found in cortical pyramidal cells following EE occurring either in the postnatal day 10 (P10)–P24 period (Venable et al., 1989) or starting post-weaning (Kolb, 1995), and EE attenuates the effects of an early lesion of the motor cortex (Kolb and Gibb, 2001).

A significant increase in neuronal cytodifferentiation has also been found in the motor cortex of rats reared under EE conditions during the early period of P5–P21, with EE animals consistently performing better on many measures of behavioral adaptive responses, as measured in open field, narrow path crossing, hind limb support, and rope climbing (Pascual and Figueroa, 1996).

Environmental enrichment and visual system development and plasticity

Despite the vast literature on the effects of EE on the brain, until recently little was known about the influence exerted by the early environment on the development, physiology, and plasticity of sensory systems. Indeed, in statements such as “the development of visual functions is experience dependent,” experience generally refs to visual experience. Only recently has evidence been forthcoming that enhanced levels of sensorimotor stimulation in very young animals, such as that provided by an EE, affect the developmental plasticity of the visual system.

The most striking effect elicited by an EE paradigm starting at birth on visual system development is that it causes a marked acceleration in the maturation of visual acuity, a very sensitive and predictive index of the entire visual system development. Visual acuity development has been assessed in rats reared under EE conditions or in a standard laboratory cage (EE and non-EE rats) using electrophysiological recordings of visual evoked potentials (VEPs) from the binocular portion of the primary visual cortex (Landi et al., 2007b). A pronounced acceleration of visual acuity matura-

tion due to exposure to EE has been replicated in the mouse at the behavioral level, using the visual water box task (Cancedda et al., 2004). The effect of EE on visual acuity maturation is illustrated in figure 37.1A (see also color plate 28). The acceleration in visual acuity development elicited by EE is a robust and surprising effect. When the time course for visual acuity development in the rat is rescaled to human development (see Berardi et al., 2000), it is as a child reached his or her final visual acuity at around 3 years of age, about 2 years before the age at which children’s acuity development normally ends.

The ability of EE to profoundly affect the time course of visual function development underscores the importance of windows of high experience-dependent plasticity in the maturation of the cerebral cortex (Berardi et al., 2000).

These early sensitive phases, known as critical periods, are normally well fitted with the maturational necessities of the developing organism, allowing, for instance, auditory neural circuitries to compensate for progressive enlargement of distances between sensory detectors at the periphery. The very high plasticity of developing neural circuits serves the purpose of allowing experience to guide the maturation of neural connections. As experience promotes the maturation of a set of neural circuits and the corresponding neural function, circuits and function become progressively less modifiable by experience, and plasticity declines.

Not surprisingly, the effects of early EE on visual acuity development are paralleled by a profound influence on the time course of developmental visual cortical plasticity. First,

A

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Figure 37.1 Acceleration of visual system development by environmental enrichment. A, EE accelerates the maturation of visual acuity. Visual acuity of non-EE (white) and EE (black) rats has been assessed by means of visual evoked potentials (VEPs) recorded from the binocular portion of the visual cortex at different ages during postnatal development. VEP acuity is normalized to the acuity value at P44–P45 and is plotted as a function of age for each experimental group to show the leftward shift of the curve for EE animals. B, Higher levels of BDNF and GAD65/67 expression in EE pups. ELISA and Western blot analysis have been used to measure, respectively, BDNF and GAD65/67 protein levels in the visual cortex of EE and non-EE animals at different postnatal ages. Data are plotted as percentage of variation between the two groups,

with positive values indicating higher levels in EE mice. C, Accelerated CRE-mediated gene expression development in the visual cortex of EE mice. C1, Examples of brains at different ages (P10– P30) from CRE-lacZ transgenic mice reared in non-EE or EE conditions. X-gal histochemistry has been used to reveal the occurrence of CRE-mediated gene expression (black staining). C2, Quantification of the density of X-gal-positive cells for non-EE (white) and EE (black) mice at the indicated ages. Fields were chosen to sample layers II–VI of the binocular visual cortex. CRE-mediated gene expression is developmentally regulated in both groups, but its peak is accelerated in EE mice. See color plate 28. (A, Modified from Landi et al., 2007b. B and C, Modified from Cancedda et al., 2004.)

EE mice have an accelerated developmental decline of the long-term potentiation (LTP) of layer II/III field potentials induced by theta burst stimulation from the white matter (WM-layer II/III LTP) in their visual cortex. This kind of LTP is a well-established in vitro model of developmental plasticity (Kirkwood et al., 1995; Huang et al., 1999), since the susceptibility to potentiation of layer II/III synaptic responses after stimulation of the white matter is present only during a critical period in the early life of rodents, being absent in adult animals. Although at a very early age, substantial WM-layer II/III LTP is found in both EE and nonEE mice, the developmental decline in LTP magnitude is much faster under EE conditions (Cancedda et al., 2004). Second, EE rats have a precocious closure of the critical period for ocular dominance (OD) plasticity in response to monocular deprivation (MD) (Medini et al., 2008), a classic measure of developmental experience-dependent plasticity in the visual cortex. Therefore, it seems that the quality and intensity of environmental-dependent experience powerfully sculpt the development of the visual system, regulating the interplay between its critical period time course and the maturation of visual functional abilities.

Which molecular factors underly the effects of EE on visual plasticity and development? One important observation is that the acceleration of visual system development in EE mice closely resembles that previously reported in mice engineered to overexpress in their forebrain the neurotrophin BDNF. These mice also exhibit a pronounced acceleration in both the maturation of their visual acuity and in the time course of visual cortical synaptic plasticity (Huang et al., 1999). One possible model put forward to explain the accelerated visual acuity development and visual cortical plasticity decline in BDNF-overexpressing mice (Huang et al., 1999) is that higher BDNF levels would accelerate the development of the inhibitory GABAergic system, which, by affecting receptive field (RF) development and synaptic plasticity, could explain both the faster maturation of visual acuity and the precocious decline of cortical plasticity. Interestingly, the same model turned out to be valid in the case of EE mice. Mice reared from birth in an EE have higher levels of the BDNF protein in their visual cortex at P7 (figure 37.1B and color plate 28), an effect accompanied by increased expression of the GABA biosynthetic enzymes, GAD65/67, at both P7 and P15 (See figure 37.1B and color plate 28). Therefore, an important mediator of environmentally dependent BDNF action could be intracortical inhibition (Cancedda et al., 2004; Sale et al., 2004). Downstream in the BDNF signaling pathway, an effective role in the acceleration of visual cortical development has been reported for the cAMP/CREB system, which is known to be an important hub in the development and plasticity of sensory systems (Impey et al., 1996; Pham et al., 1999; Barth et al., 2000; Mower et al., 2002; Cancedda et al., 2003). Transgenic mice

carrying the lacZ reporter gene under the control of the CRE promoter (CRE-LacZ mice; Impey et al., 1996) offer the possibility to visualize the neural cells in which CREmediated gene expression occurs. In these animals the lacZ gene product, β-galactosidase, can be visualized as a blue precipitate using X-gal histochemistry. CRE-mediated gene expression is developmentally regulated in the mouse visual cortex, peaking at around P25 under normal rearing conditions (Cancedda et al., 2004). Notably, this peak of gene expression is markedly accelerated in EE subjects (Cancedda et al., 2004) (figure 37.1C1 and C2 and color plate 28). That this change in the time course of CRE-mediated gene expression is involved in the acceleration of visual system maturation found at the physiological level is demonstrated by an experiment in which this molecular pathway was pharmacologically enhanced through injections of rolipram (Cancedda et al., 2004), a specific inhibitor of the high-affinity phosphodiesterase type IV that activates the cAMP system via inhibition of cAMP breakdown, resulting in an increased phosphorylation of the transcription factor CREB (Tohda et al., 1996; Kato et al., 1998; Nakagawa et al., 2002). Mice injected with rolipram from P7 until P21 have accelerated development of visual acuity that mimics, at least in part, that found in EE mice (Cancedda et al., 2004).

These studies show that the environment sculpts the development of the visual system not only through changes in the levels of visual stimulation but also through factors activated even in the absence of vision. This is suggested by the observation that the increased cortical BDNF expression and the accelerated maturation of intracortical inhibition in EE animals are observed at very early ages, before eye opening, and by experiments that have used a temporally restricted EE protocol in which pups are maintained under enriched conditions only until P12, when they are transferred to a normal standard cage. In these animals, exposed to EE up to eye opening, there is a “priming” of visual acuity development: when tested at P25, they show a higher visual acuity than non-EE animals, although the effect is smaller that that found in animals left in EE up to P25 (Baldini et al., 2007). Thus, exposure to EE before eye opening can affect the much later occurring visual acuity development.

This raises the possibility that visual cortical development can be influenced by EE even in the complete absence of vision. This important issue has been addressed in the investigation of EE effects in dark-reared (DR) animals performed by Bartoletti et al. (2004).

Lack of visual experience from birth prevents maturation of the visual cortical circuits. In particular, visual connections do not consolidate, remaining plastic well after the end of the critical period, and visual acuity does not develop (Cynader and Mitchell, 1980; Mower, 1991; Fagiolini et al., 1994; Berardi et al., 2000). All these effects can be

completely counteracted by providing DR animals with the opportunity to experience high sensorimotor stimulation in an EE while in the dark (Bartoletti et al., 2004). DR rats exposed to EE show normal closure of the critical period for OD plasticity and normal visual acuity development. In particular, by assessing the effectiveness of MD in shifting the OD of visual cortical neurons after P50, it has been found that MD (1 week) was still effective in shifting the OD of visual cortical neurons in favor of the ipsilateral nondeprived eye in DR-non-EE rats, as expected (figure 37.2B and C, and color plate 29). However, MD from P50 was ineffective in DR-EE littermates of DR-non-EE rats placed in EE at P18, as is the case for rats with normal visual experience (see figure 37.2B and C and color plate 29).

The EE effect in this case is also very similar to that found in BDNF-overexpressing mice, where a rescue of DR effects

on visual acuity development and the critical period for OD plasticity has been reported (Gianfranceschi et al., 2003). The similarity between the effects obtained using either genetic engineering techniques to increase neurotrophin expression or conditions of increased environmental complexity offers a clear view of the interaction between nature and nurture during the development of sensory systems.

It is known that DR prevents the developmental organization into perineuronal nets of chondroitin sulfate proteoglycans (CSPGs), components of the extracellular matrix that have recently been shown to be important nonpermissive factors for visual cortical plasticity (Lander et al., 1997; Pizzorusso et al., 2002); indeed, removal of CSPGs restores OD plasticity to the adult visual cortex (Pizzorusso et al., 2002, 2006). Interestingly, EE greatly reduces the effects of

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Figure 37.2 Environmental enrichment promotes consolidation of visual cortical connections in dark-reared rats. A, Schematic diagram of the dark-rearing (DR) protocol combined with EE. Newborn rats were reared in complete darkness from P0 until P50. Together with DR, they were maintained, starting from P18, in either non-EE or EE conditions. Rats were then subjected to 1 week of MD, in normal light conditions. B and C, Normal closure of the critical period for MD in EE rats. B, Summary of MD effects in all DR animals. The OD distribution of each animal has been summarized with the contralateral bias index (CBI = {[N(1) − N(7)] + 1/2[N(2/3) − N(5/6)] + N(Tot)}/2N(Tot), where N(tot) is the total number of recorded cells and N(i) is the number of cells in class (i) (open diamonds denote individual data; circles denote mean of the group ± SE). CBI scores in DR-non-EE + MD rats differ from

those in DR-EE + MD rats, which do not differ from those in normal adults (shaded rectangle). C, Cumulative fractions for OD scores. For each cell, an OD score was computed as {[Peak(ipsi) − baseline(ipsi)] − [Peak(contra) − baseline(contra)]}/{[Peak(ipsi) − baseline(ipsi)] + [Peak(contra) − baseline(contra)]} (Rittenhouse et al., 1999). This score is −1 for class 1 cells, +1 for class 7 cells, and around 0 for class 4 cells. Only the curve for DR + non-EE MD animals significantly differs from that in normal rats. D, Examples of staining for Wisteria floribunda agglutinin, which labels perineural nets, in Oc1B of a normal rat, a DR-non-EE rat, and a DR-EE rat at P50. The decrease caused by DR in the number of perineural net–surrounded neurons is reduced in EE-DR animals. Calibration bar = 100 μm. See color plate 29. (Modified from Bartoletti et al., 2004.)

DR on the development of perineuronal nets (figure 37.2D and color plate 29). Bartoletti et al. (2004) also examined the effects of EE on the status of cortical inhibition, as there is growing evidence that the maturation of inhibition in the visual cortex is an important determinant of the critical period (Hensch, 2005). The expression of GAD65 in the presynaptic boutons of GABAergic interneurons around the soma of the target neurons is decreased by DR, as is already known for other markers of GABAergic function (Benevento et al., 1995). In DR-EE animals, however, GAD65 expression was normal.

The observation that EE promotes the development of visual acuity, closure of the critical period for OD plasticity, and the development of crucial determinants of developmental visual cortical plasticity (as perineuronal nets and intracortical inhibition in DR rats) shows that it is possible to modulate the outcome of visual deprivation by varying the environmental conditions in a mammalian species. This confirms the hypothesis, prompted by the precociousness of the molecular effects elicited by EE in the visual cortex, that factors not under the exclusive control of visual experience may contribute to visual cortical development. The possibility of affecting visual cortical development in the complete absence of vision underscores the importance of the interactions between distinct sensory experiences for the maturation of neural circuits.

Which kind of nonvisual experience, then, acts on pups reared in EE? The first 2 weeks of life in rodents are char-

acterized by a general absence of interaction between the newborn and the environment; pups remain in the nest, totally dependent on the mother, which can be considered the most important source of sensory experience for the developing pup (Hofer, 1984; Liu et al., 2000). It has therefore been suggested (Cancedda et al., 2004) that during the first 2 weeks of life, enriched stimuli present in the environment affect pup visual system development through maternal behavior: different levels of maternal care received by pups in different environmental conditions could act as an indirect mediator of the earliest effects of EE on visual system development. Indeed, a detailed quantitative analysis of maternal care behavior in either EE or standard condition showed that enriched pups receive higher levels of maternal care than those reared in the standard condition (Sale et al., 2004). In particular, EE pups experience continuous physical contact owing to the presence of adult females in the nest (figure 37.3A) and also receive higher levels of licking (figure 37.3B), two of the most critical maternal behaviors for the development of the newborn, which have been associated with hormonal regulation of growth and with the expression of neuroendocrine and behavioral responses to stress (Liu et al., 1997). It is likely that this sustained tactile stimulation can affect pup neural development, providing a source for the earliest changes observed in EE mice. This conclusion is supported by data showing that variations in maternal care can affect BDNF levels and the neural development of offspring (Liu et al., 2000).

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Figure 37.3 Maternal care in EE mice. Maternal care behavior was scored during six daily observation sessions of 75 minutes each for the first 10 days postpartum. The observation sessions occurred at 6:45 a.m., 9:00 a.m., 12:00 a.m., 3:00 p.m., 6:00 p.m., and 9:00 p.m.; the first and last sessions were during dark phase of the daily cycle and were performed under dim red light illumination. During each session the behavior of each adult female was scored every 3 minutes, recording whether or not a target behavior was present. A, Frequency of “pups alone” recordings during the first 10 days postpartum in non-EE (white) and EE (black) mice. The percentage of time spent by pups alone in the nest is dramatically lower in EE than in non-EE mice. Indeed, EE pups are virtually never alone in the nest, since when the mother is absent, she is always replaced

by another adult female. B, Frequency of “licking” recordings during the first 10 days postpartum in non-EE (white), EE (black), and EE in a simplified condition where no adult females other than the mother are present (EE without helpers, gray). In the EE condition, maternal and nonmaternal licking have been summed. EE pups receive the highest levels of licking, followed by pups in the EE without helper condition and then by non-EE pups. Interestingly, an analysis of licking behavior exhibited only by dams (excluding the contribution of helper females) shows that mothers in the EE without helper condition exhibit significantly more licking than both non-EE and EE mothers. (Modified from Sale et al., 2004.)

One protocol particularly informative of the effects of maternal care on pup growth entails maternal separation: pups are removed from the dam some times a day for a total of 3–6 hours during the first 2 weeks of postnatal age. This treatment has many detrimental consequences for newborn development. For instance, just 1 hour of maternal separation in the rat results in a decrease in the activity of ornithine decarboxylase (Wang et al., 1996) and growth hormone, both substances essential for normal somatic and neural development. This effect can be completely prevented through artificial tactile stimulation applied to separated pups with a brush at a frequency resembling that of maternal licking (Pauk et al., 1986). Artificial tactile stimulation during the first 10 days of life also attenuates the effects of a perinatal lesion of the motor cortex (Kolb and Gibb, 2001), reproducing the effects of EE.

If the enhanced tactile stimulation provided to the pups in EE is a crucial part of the “nonvisual experience” that has been shown to act on visual development, it might be possible to induce an acceleration in visual acuity development by providing artificial tactile stimulation to pups reared in a nonenriched condition. This hypothesis has been tested by applying to developing rats a stimulation that combines gentle stroking with a wet brush and a manual massage applied on the pup’s trunk and limbs from P1 to P12. These two treatments mimic maternal licking and grooming. The results show that this artificial tactile stimulation is quite effective in “priming” visual acuity development: when tested at P25 and P28 (both electrophysiologically and behaviorally), treated pups showed a higher visual acuity than control rats (Baldini et al., 2007). The acceleration produced by this treatment is less than that shown in agematched subjects receiving continuous EE from birth to the day of testing but is equal to that obtained using the temporally restricted EE protocol in which pups are maintained under enriched conditions only until P12. These results point toward maternal stimulation as the fundamental factor mediating the effects of EE on visual system development at very early ages, when pups do not engage in direct exploration of the surrounding environment (Sale et al., 2004). It would be important to know whether a similar cross-modal effect of tactile stimulation on visual development could be documented in human newborns. Results obtained in premature infants, in collaboration with the Department of Developmental Neuroscience, Stella Maris, Pisa, and the Neonatology Unit, Dipartimento di Medicina della Procreazione e dell’Età Evolutiva, Pisa University, go in this direction.

In sum, the available evidence has dissipated the mystery that once surrounded the remarkable ability of EE to strongly affect the brain—a phenomenon that in turn has caused EE to be viewed with mistrust as a scientific protocol for studying brain-environment interactions. EE acts during visual

system development by modulating well-known factors involved in controlling developmental cortical plasticity. Some of them, such as BDNF and the maturation of intracortical inhibitory circuitry, are precociously affected by EE through the different levels of maternal care experienced by EE pups; others, such as the CRE-CREB system and the perineural nets, are affected later in development and can therefore be modulated both by the direct interactions of pups with the EE and by events triggered by the early factors.

Besides confirming the role of already well known players in the development and plasticity of the rodent visual system, EE has allowed the identification of a new player. There is indeed one molecule whose critical role in mediating the influence of the environment on the development of the visual system has emerged through studies using the EE paradigm, insuline-like growth factor-I (IGF-I). IGF-I crosses the blood-brain barrier and, acting on neurons bearing its receptors, increases their electrical activity, inducing the production of other factors important for visual cortical plasticity, such as BDNF (Thoenen and Sendtner, 2002). IGF-I receptors are expressed in the occipital cortex (Frolich et al., 1998). Recent data suggest that IGF-I might be an important mediator of EE action on the development of the visual cortex. IGF-I is increased postnatally in the visual cortex of EE rats, and after weaning, administration of IGF-I in this structure through osmotic minipumps mimics EE effects on visual acuity maturation, leading to a marked acceleration that is particularly evident at P25 (Ciucci et al., 2007). Furthermore, blocking IGF-I action in the visual cortex of developing EE subjects through infusion of the IGF-I antagonist JB1 completely prevents EE effects on visual acuity development. Interestingly, a role for IGF-I in visual cortical plasticity was recently suggested following a complex genetic screening for factors controlled by visual experience during development: Tropea et al. (2006) demonstrated that MD increases the expression of IGF-I-binding protein and affects several genes in the IGF-I pathway, and that exogenous application of IGF-I prevents the physiological effect of MD on OD plasticity in vivo.

One important issue that has recently begun to be addressed is whether the influence of the environment during development is restricted to the visual cortex or whether structures considered less plastic than the visual cortex, such as the retina, can be affected by EE. It is commonly assumed that retinal development is independent of sensory inputs, leading to the notion that the retina is less plastic than the cortex or hippocampus, the very site of experiencedependent plasticity. For instance, visual deprivation such as MD or DR that are known to dramatically affect visual cortical acuity are virtually ineffective on retinal acuity in cat, rat, and humans (Baro et al., 1990; Fagiolini et al., 1994; Fine et al., 2003). However, it has recently been shown that

DR alters inner retinal development in mice (Tian and Copenhagen, 2001, 2003), preventing the segregation of ON and OFF pathways, at both electrophysiological and anatomical level. EE, which so powerfully affects visual cortical development, seemed a paradigm suitable for probing the actual sensitivity of retinal development to experience, and to gain insight into the factors involved.

To understand whether retinal functional development is a target of EE and to compare EE effects on cortical and retinal development, the development of retinal responses was assessed using pattern electroretinogram (P-ERG) (Landi et al., 2007a). P-ERG is a sensitive measure of the function of retinal ganglion cells (RGCs), the very output of

retinal circuitry (Maffei and Fiorentini, 1981). The results show that retinal acuity development is sensitive to EE on the same time scale as cortical acuity. In particular, retinal acuity development is strongly accelerated in EE rats compared with non-EE rats starting from P25, and this accelerated retinal development in EE animals is also found in rats exposed to EE for only the first 10 days of life, that is, before eye opening (figure 37.4A and B; Landi et al., 2007b). These experiments suggest that factors influenced by EE and sufficient to trigger the much later occurring retinal acuity development are affected during the first 10 days of life. One crucial factor in this process is BDNF: EE causes a precocious increase in BDNF expression in the RGC layer, and

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Figure 37.4 Retinal functional development is sensitive to environmental enrichment. A, Examples of steady-state pattern electroretinogram (P-ERG) signals recorded at P25 in response to visual stimulation with gratings of three different spatial frequencies in one non-EE (gray traces) and one EE (black traces) rat. The gratings were sinusoidally modulated at a temporal frequency of 4 Hz (period of 250 ms), and the principal component of the P-ERG response is on a temporal frequency twice the temporal frequency of the stimulus. A P-ERG recorded in response to a blank field is reported to show the noise level. A response to a pattern of 0.5 c/deg is still present in the EE rat but not in the non-EE rat. B, Acceleration of retinal acuity maturation in EE rats. Retinal acuity values were assessed at P25–P26 (small symbols denote individual data; larger symbols denote mean of the group) in non-EE rats (white), in rats enriched until P25 (EE, black), and in rats enriched until P10 (striped). Retinal acuity is higher in EE than in non-EE animals, and, interestingly, retinal acuity of EE-until-P10 rats does not differ from that in EE rats. This suggests that 10 days’

enrichment is sufficient to trigger EE effects on retinal functional development. C, BDNF is necessary for EE effects on retinal functional development. C1, Injections of BDNF antisense oligonucleotides block the accelerated maturation of retinal acuity observed in EE animals. Retinal acuity has been determined at P25 for both eyes in EE animals that are treated with BDNF antisense oligos in one eye and are left untreated in the other eye; for each animal, the retinal acuity of the treated and of the untreated eye are reported, joined by a dotted line. The acuity of the BDNF antisensetreated eye is significantly lower than that of the fellow eye in all animals. C2, Mean retinal acuity in EE, EE treated intraocularly with BDNF sense oligos (EE-S, control treatment), EE treated with antisense oligos (EE-AS), and non-EE rats. The EE and non-EE groups are as described in B. The retinal acuity of EE-AS rats differs from that of EE animals but not from that of non-EE animals, while the retinal acuity in EE-S rats differs from that of non-EE rats but not from that of EE rats. (Modified from Landi et al., 2007b.)