Disrupted cortical function underlies behavior dysfunction due to social isolation

Stressful events during early childhood can have a profound lifelong influence on emotional and cognitive behaviors. However, the mechanisms by which stress affects neonatal brain circuit formation are poorly understood. Here, we show that neonatal social isolation disrupts molecular, cellular, and circuit developmental processes, leading to behavioral dysfunction. Neonatal isolation prevented long-term potentiation and experience-dependent synaptic trafficking of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors normally occurring during circuit formation in the rodent barrel cortex. This inhibition of AMPA receptor trafficking was mediated by an increase of the stress glucocorticoid hormone and was associated with reduced calcium/calmodulin-dependent protein kinase type II (CaMKII) signaling, resulting in attenuated whisker sensitivity at the cortex. These effects led to defects in whisker-dependent behavior in juvenile animals. These results indicate that neonatal social isolation alters neuronal plasticity mechanisms and perturbs the initial establishment of a normal cortical circuit, which potentially explains the long-lasting behavioral effects of neonatal stress.

Mental stress can have different effects on the nervous system. While brief mild stress can evoke emotional arousal and enhance learning (1), chronic elevated stress can induce deficits in neuronal function (2, 3). In particular, social isolation early in life caused by neglect (a form of child abuse) can induce mental stress and lead to various mental illnesses such as depression, drug addiction, and anxiety disorders (4, 5). Because of the complex and poorly understood regulation of neuronal functions by stress (6, 7), elucidation of how stress affects the nervous system at the molecular, cellular, and circuit levels is an important step in controlling stress disorders.

Experience early in postnatal life is important to the establishment of normal functioning cortical circuits (8, 9). During this period, sensory experience through whiskers, an important form of social communication in rodents (10, 11), leads to a several-fold increase in excitatory synaptic transmission in the barrel cortex (12).

Excitatory synaptic transmission in the central nervous system is mainly mediated by α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid–type (AMPA-type) glutamate receptors (13, 14). The number of AMPA receptors at synaptic sites determines the synaptic responses of postsynaptic neurons and controls neuronal activity (14–18). Stimuli inducing synaptic plasticity such as long-term potentiation (LTP) drive GluR1-containing AMPA receptors into synapses in vitro (19, 20). In vivo, whisker experience drives GluR1-containing AMPA receptors into synapses in the developing barrel cortex (12, 21, 22). Synaptic addition of GluR1-containing AMPA receptors contributes to behavioral modification in several systems (1, 23–28).

A number of studies have revealed that behavioral stress, including social isolation, affects LTP (29, 30). While some stress protocols (e.g., inescapable restraint-tail shock, exposure to brightly lit unfamiliar chambers) attenuate LTP in the hippocampus (31–33), other protocols, such as brief exposure to a predator, enhance LTP (1). Therefore, the effect of stress on synaptic plasticity may be dependent on the stress protocol and show an inverted U-shape dose-response relation (relating the “dose” of stress and plasticity response). Recently, we have shown that brief arousal facilitates synaptic AMPA receptor delivery via norepinephrine-mediated (NE-mediated) signaling (1). However, the molecular mechanism of how behavioral stress attenuates synaptic plasticity is still unclear.

Rodents and humans may display similar effects of neonatal maltreatment on the nervous system (34–36). Here we examine the effects of early social isolation on synaptic function during cortical development. We focus on the synapses formed from layer 4 to layer 2/3 in the developing rat barrel cortex (BC-L4, 2/3). We examine effects between P12 and P14, a time when GluR1-containing AMPA receptors are delivered into synapses by whisker experience and when the fine mapping in the barrel field is formed (12, 37). We find that brief periods of neonatal social isolation (but not merely whisker sensory deprivation) disrupt this process as well as LTP and result in the attenuation of whisker sensitivity at layer 2/3 of the barrel cortex. This leads to disruption of whisker-dependent behavior. Furthermore, we find that these effects of neonatal social isolation are mediated by a glucocorticoid stress hormone. These results indicate that neonatal social isolation alters the formation of proper neuronal circuitry by a glucocorticoid signaling–mediated blockade of experience-dependent synaptic AMPA receptor insertion and leads to long-lasting behavioral perturbations.

Social isolation disrupts whisker experience–dependent synaptic delivery of GluR1-containing AMPA receptors. In order to study the effect of social isolation on receptor trafficking, we isolated rat pups from their mothers and other siblings for 6 hours per day from P7 to P11 (38) (see Supplemental Chart of experimental procedures; supplemental material available online with this article; doi: 10.1172/JCI63060DS1). At P12, we microinjected Sindbis virus expressing GFP-GluR1 in the barrel cortex of either isolated or nonisolated siblings from the same litter. We used low-titer virus to avoid toxic effects and infected about 1 of 1,000 cells in the injected area. Viral-mediated overexpression of GFP-GluR1 leads to about 4-fold increase in cell bodies and 35% increase in dendrites of GluR1-containing AMPA receptors, indicating near physiological levels of AMPA receptor expression in infected dendrites (39). The expression level of GFP-GluR1 was comparable between isolated and nonisolated animals (data not shown). Furthermore, the basic electrophysiological properties such as input resistance and firing properties were indistinguishable between infected and noninfected neurons (Supplemental Table 1). These profiles were comparable between isolated and nonisolated animals (Supplemental Table 1). Recombinantly expressed GluR1 formed homomeric receptors which, in contrast with endogenous receptors, showed little outward current at positive potential, and thus synaptic incorporation of recombinant AMPA receptors was detected as an increased rectification (ratio of response at –60 mV to +40 mV) of AMPA receptor–mediated synaptic transmission. From P12 to P14, litters were maintained under normal conditions, and acute coronal brain slices were prepared at P14. Animals were maintained with whiskers intact throughout the experiment. We performed whole-cell recordings of layer 2/3–infected pyramidal neurons and nearby noninfected neurons and evoked transmission by stimulating layer 4. GFP-GluR1 infected neurons from nonisolated control siblings showed increased rectification compared with nearby noninfected neurons (Figure 1A). However, no increase of rectification was detected from GFP-GluR1–expressing neurons in isolated animals (Figure 1A). Therefore, experience-driven synaptic delivery of GluR1 was blocked by social isolation at this early postnatal period. While isolation for 1 hour per day during P7–P11 or for 6 hours only at P7 still prevented experience-driven synaptic GluR1 delivery during P12–P14, 1 hour isolation for just 1 day at P7 did not (Supplemental Figure 1A). Thus, the effect of social isolation on GluR1 delivery depends on the duration of isolation. In order to determine whether synaptic delivery of endogenous AMPA receptors was prevented by social isolation, we measured the ratio of AMPA to NMDA responses (A/N). In brain slices from P14 animals isolated between P7 and P11 for 6 hours per day, the A/N ratio for transmission at BC-L4, 2/3 synapses was significantly lower than that observed in nonisolated animals of the same age (Figure 1D), indicating that social isolation from P7 to P11 disrupted synaptic delivery of endogenous AMPA receptors. The kinetics of NMDA receptor–mediated synaptic responses was not different between isolated and nonisolated animals, indicating that NMDA receptor–mediated synaptic currents are relatively stable (Supplemental Figure 1B). Consistent with these results, synaptosome fraction of isolated animals (6 hours per day during P7–P11 and assayed at P11) contained a reduced amount of GluR1 compared with that of nonisolated animals (Supplemental Figure 1C).

Figure 1

Social isolation disrupts experience-dependent synaptic delivery of AMPA receptors in the developing rat barrel cortex. (A–C) Top insets: time line of experiments. R1, GFP-GluR1–expressing neurons; non, nonexpressing neurons. (A) Isolation during P7–P11 disrupted GluR1 delivery at P12–P14 (*P < 0.05, R1 versus non; n = 8 nonisolated control, n = 9 isolated). (B) Removal of mother during P7–P11 (mother removed) did not disrupt GluR1 delivery at P12–P14 (*P < 0.05, R1 versus non; n = 8) (left). Blockade of whisker input during removal of mother (whisker mask) did not prevent GluR1 delivery (*P < 0.05, R1 versus non; n = 5) (right). (C) Isolation (P4–P7) disrupted GluR1 delivery during P12–P14 (*P < 0.05, R1 versus non; n = 6). (D) A/N ratio at P14 was lower with rats isolated than nonisolated rats (*P < 0.05, isolated vs, nonisolated; n = 18). (E) A/N ratio at P14 of pups, either mother removed or whisker masked, was comparable with nonisolated rats, but larger than isolated animals (n = 16) (*P < 0.05, compared with nonisolated [n = 15], mother removed [n = 17], whisker mask [n = 21] animals; F_(3,65) = 4.022). (F) A/N ratio at P14 was lower with rats isolated at P4–P7 than nonisolated rats (*P < 0.05, isolated vs. nonisolated; n = 15 nonisolated control animals, n = 18 isolated animals). Scale bars: 20 pA/20 ms (A, D); 10 pA/20 ms (B); 10 pA/15 ms (E, F). Data were analyzed by paired, 2-tailed t test (A–D, F) or 1-way factorial ANOVA (E).

Since weaning occurs at a later stage, pups from P7 to P11 depend on nutrition provided by the dam. Therefore, pups were deprived of nutrition during social isolation. Although we did not detect any difference of body weights between isolated and nonisolated animals (Supplemental Table 2), we wished to rule out the possibility that the lack of nutrition during social isolation was responsible for the disruption of synaptic delivery of GluR1. We separated pups from the dam, but kept 10 pups together for 6 hours per day from P7 to P11 (mother removed). Thus, these pups were deprived of nutrition from the dam, but retained social interaction with other pups. In these pups, GFP-GluR1–infected neurons showed increased rectification, indicating synaptic incorporation of GFP-GluR1 (Figure 1B). The A/N ratio for transmission at BC-L4, 2/3 synapses of pups deprived of the dam was not different from that of control pups, but was significantly larger than that of isolated animals (Figure 1E), indicating that mother removal from P7 to P11 did not disrupt synaptic delivery of endogenous AMPA receptors. This shows that experience-driven synaptic delivery of GluR1 in the development was intact in pups separated from the mother if they retained social interaction with other pups. Thus, disruptions in nursing do not appear responsible for the observed deficits.

During 6 hours per day of social isolation, pups did not interact with other siblings or the mother, and thus they may have had reduced whisker experience. A reduction of whisker experience during social isolation procedure could be responsible for the disruption of synaptic delivery of GluR1 by social isolation. To rule out this possibility, we deprived whisker experience for 6 hours per day from P7 to P11 by masking whiskers (whisker mask). This method is effective in depriving experience, as continuous masking for 2 days from P12 to P14 blocks experience-dependent delivery of GluR1 into BC-L4, 2/3 synapses (infected, 102% ± 15%; noninfected, 100% ± 17%; P > 0.5). In order to prevent their mother from removing the mask, we separated a masked animal from the dam, but kept it with several other siblings to retain social interaction with other pups. We expressed GFP-GluR1 in the barrel cortex from P12 to P14 in these masked animals. We found that GFP-GluR1–expressing cells showed increased rectification (Figure 1B). The A/N ratio at these synapses of pups with masks was not different from that of control pups, but was significantly larger than that in isolated animals (Figure 1E), indicating that whisker masking for 6 hours per day from P7 to P11 did not disrupt synaptic delivery of endogenous AMPA receptors. This indicates that deprivation of whisker experience for 6 hours per day from P7 to P11 did not have any effect on experience-driven synaptic incorporation of GluR1 from P12 to P14.

Taken together, social isolation from P7 to P11 disrupts whisker experience–dependent synaptic delivery of GluR1 at P12 to P14. This effect is neither due to lack of nutrition from the dam nor possible reduction of sensory experience during social isolation.

Social isolation at early postnatal stage disrupts experience-driven synaptic delivery of GluR1 later in development. We examined longer-term effects of social isolation on experience-driven synaptic delivery of AMPA receptors. Pups were isolated from the mother and other siblings for 6 hours per day from P4 to P7 and then maintained under normal conditions from P8 to P14; GFP-GluR1 was expressed from P12 to P14 by viral-mediated gene transfer. Whole-cell recording of slices prepared at P14 showed no increase of rectification of GFP-GluR1–expressing cells, while normal GFP-GluR1 delivery into synapses was observed in nonisolated control siblings (Figure 1C). Consistent with this, the A/N ratio for transmission at BC-L4, 2/3 synapses of isolated animals was significantly lower than that observed in nonisolated animals of the same age (Figure 1F). This indicates that social deprivation early in development disturbs synaptic delivery of GluR1 1 week later in development.

Social isolation disrupts LTP. Above, we show that social isolation disrupts experience-dependent delivery of AMPA receptors in vivo. This could be due to a disruption of biochemical pathways necessary for the trafficking of AMPA receptors. Since similar biochemical pathways are used between LTP and experience-driven synaptic delivery of AMPA receptors (14, 16, 17, 19), disturbance of molecular machinery for the delivery of AMPA receptors should result in the disruption of LTP. Alternatively, rats exposed to social isolation could develop aberrant social behavior that could lead to inappropriate sensory stimuli (e.g., reduction of contact through whiskers) during the period when receptor trafficking requires whisker activity; this aberrant experience could prevent synaptic delivery of AMPA receptors. In this case, neurons of isolated rats could retain normal endogenous biochemical pathways that would permit LTP in vitro. In order to distinguish these 2 possibilities, we examined the effect of social isolation on LTP tested in vitro.

Acute slices were prepared from P14 rats that had been isolated either from P4 to P7 or from P7 to P11 for 6 hours per day. Whole-cell recordings were obtained from layer 2/3 pyramidal neurons, and transmission was evoked by stimulating layer 4. LTP was induced by pairing synaptic stimulation with postsynaptic depolarization (3 Hz, +20 mV, 90 seconds). While nonisolated rats showed intact LTP, paired-induced LTP was significantly reduced in siblings exposed to social isolation (Figure 2, A and B). We examined the effect of early social isolation on LTP at a later developmental age (P27). Although we did not detect attenuation of LTP induced by the same protocol as used in young animals in isolated rats (data not shown), social isolation during P4–P7 partially attenuated LTP induced by the weaker condition (1 Hz, 0 mV, 90 seconds), indicating that the milder but persistent effect of neonatal social isolation continues until juvenile age (Figure 2C). These data indicate that biochemical pathways necessary for the trafficking of AMPA receptors were perturbed by this social isolation protocol.

Figure 2

Social isolation disrupts LTP in the developing rat barrel cortex. LTP was induced by pairing 3-Hz stimulation with depolarization of the postsynaptic neuron (+20 mV) for 90 seconds. Recordings were maintained for at least 40 minutes after pairing. Mean amplitude between 30 minutes and 40 minutes after LTP induction was normalized to baseline amplitude (right). Social isolation during P7–P11 (A: *P < 0.05, nonisolated induced pathway vs. isolated induced pathway; n = 6 per each group) and P4–P7 (B: *P < 0.05, nonisolated induced pathway vs. isolated induced pathway; n = 5 per each group) prevented LTP at P14 in the barrel cortex. (C) Social isolation during P4–P7 partially blocked LTP in the barrel cortex at P27 (*P < 0.05, nonisolated induced pathway vs. isolated induced pathway; n = 6 nonisolated control animals, n = 8 isolated animals). Ind, LTP-inducing pathway; cont, control pathway; noniso, nonisolated animals; iso, isolated animals. Data were analyzed by paired, 2-tailed t test.

Glucocorticoid mediates social isolation–induced disruption of AMPA receptor delivery into synapses. What mediates the disruption of experience-driven synaptic delivery of AMPA receptors by social isolation? Given that plasticity was globally affected, we considered corticosterone, a major stress hormone in rodents. Since the active form of corticosterone is free corticosterone (rather than that bound to corticosterone-binding globulin), we measured the free blood corticosterone level (see Methods). During social isolation experiments, prolonged rise of free blood corticosterone levels was observed (Figure 3, A and B).

Figure 3

Increased glucocorticoid mediates synaptic plasticity disruption by isolation. (A and B) Isolation increased free corticosterone levels from blood samples collected at 1 hour and 6 hours after the initiation of isolation (*P < 0.05 compared with control animals, A: isolated at P4–P7, assayed at P4 and P7, B: isolated at P7–P11, assayed at P7 and P11; n = 10). (C) Administration of RU486 prevented the disruption of GluR1 delivery by isolation at P7–P11 (*P < 0.05, R1 versus non; n = 5), while vehicle showed no effects (n = 5). (D) A/N ratio at P14 of isolated pups with RU486 was comparable with nonisolated, but larger than isolated animals without RU486 (F_[2,41] = 3.481, *P < 0.05, isolated vs. nonisolated, isolated versus RU486; n = 15). (E) Administration of corticosterone (Cort) disrupted synaptic GluR1 delivery of animals housed in a normal environment (n = 8). (F) A/N ratio at P14 of nonisolated pups treated with corticosterone (n = 12) was lower than nonisolated (F_(2,35) = 3.395, *P < 0.05, nonisolated vs. isolated, nonisolated vs. Cort, n = 15), but comparable with isolated rats (n = 16). (G) Attenuated LTP at P14 of animals treated with corticosterone (*P < 0.05, control vs. corticosterone-injected animals; n = 7 nonisolated control animals, n = 8 nonisolated animals with corticosterone). Ind, LTP-inducing pathway; cont, control pathway. Scale bars: 10 pA/20 ms (C, E); 10 pA/15 ms (D, F). Data were analyzed by paired, 2-tailed t test (A–C, E, G) or 1-way factorial ANOVA (D, F).

Next, we injected RU486, an antagonist of glucocorticoid receptor (GR), during 6 hours per day social isolation from P7 to P11. As described above, GFP-GluR1–expressing Sindbis virus was injected into layer 2/3 of the barrel cortex at P12, and acute brain slices were prepared at P14. Whole-cell recording of slices prepared from isolated rats injected with RU486 (8 μg/g of body weight) revealed increased rectification of infected neurons compared with noninfected neurons (but there was no statistical difference between the rectification of infected neurons from isolated animals with RU486 and those from control nonisolated animals; control infected, 138% ± 7%; noninfected, 100% ± 3%, n = 27, P < 0.05; isolation with RU486 infected, 143% ± 12%; noninfected, 100% ± 5%, n = 7, P < 0.05, no statistical difference between “control infected” and “isolation with RU486 infected”, P > 0.5), indicating normal rescue of synaptic delivery of GluR1 (Figure 3C and Supplemental Figure 2A). Injection of vehicle (DMSO) did not block the effect of social isolation in both cases (Figure 3C). The A/N ratio for transmission at BC-L4, 2/3 synapses of pups isolated for 6 hours per day in the presence of RU486 (8 μg/g of body weight) was not different from that of control nonisolated pups, but was significantly larger than that of isolated animals (Figure 3D), indicating that blockade of GR with RU486 during social isolation rescued isolation-induced disruption of synaptic delivery of endogenous AMPA receptors. The effect of RU486 was dose dependent, since isolated animals injected with reduced RU486 (1.6 μg/g of body weight) were not rescued (Supplemental Figure 2A).

In order to elucidate further the role of increased glucocorticoid on experience-dependent synaptic delivery of AMPA receptors, we determined whether injection of glucocorticoid mimics the effect of social isolation. Animals injected with corticosterone (4.17 μg per day) from P7 to P11 were housed in a normal environment (see Supplemental Chart). Then a GFP-GluR1–expressing virus was injected into rat barrel cortex at P12, and acute slices were prepared at P14. Whole-cell recording showed no difference of rectification between GFP-GluR1–expressing and nonexpressing neurons, indicating no synaptic delivery of GFP-GluR1 (Figure 3E). Furthermore, the A/N ratio for transmission at BC-L4, 2/3 synapses of nonisolated animals injected with corticosterone was significantly lower than that observed in nonisolated animals without injection and comparable to that of isolated pups, indicating that endogenous AMPA receptor delivery was prevented by the injection of corticosterone (Figure 3F). Consistent with these results, injection of this amount of corticosterone attenuated LTP at P14 (Figure 3G). Injection of reduced corticosterone (0.83 μg per day) did not block GFP-GluR1 delivery, showing a dose-dependent effect of corticosterone (Supplemental Figure 2B). These results indicate that elevated glucocorticoid levels are sufficient to prevent experience-driven AMPA receptor trafficking to synapses, as is seen with social isolation.

To determine whether social isolation activates glucocorticoid signaling at the barrel cortex and prevents experience-dependent synaptic AMPA receptor delivery, we first examined whether social isolation induces nuclear translocation of GR in the barrel cortex, a mechanism for activation of glucocorticoid signaling. Immunostaining with anti-GR antibody exhibited the expression of GR in the layer 2/3 neurons of the barrel cortex at P11 (Figure 4A). Three hours of social isolation at P11 increased the amount of GR in the nuclear fraction (Figure 4B). These data indicate that social isolation activates glucocorticoid signaling at the barrel cortex. If social isolation–induced prevention of AMPA receptor delivery is mediated by the activation of GR in the barrel cortex, local injection of RU486 into the barrel cortex should block the effect of social isolation. To test this, we injected RU486 together with virus expressing GFP-GluR1 at P12 (see Methods). One hour later, we isolated rat pups for 6 hours. Pups were returned to the home cage, and whole-cell recordings were performed 27 hours later (Figure 4C). GFP-GluR1–expressing neurons showed increased rectification in the presence of RU486, while no increase of rectification from infected neurons was detected in the absence of RU486, indicating that local injection of RU486 prevented the effect of social isolation (Figure 4C). We found no statistical difference in the rectification index (RI) between infected neurons from control nonisolated animals (measured 34 hours after virus injection without social isolation) and those from isolated animals with RU486 (control animals: infected, 131% ± 9%; noninfected, 100% ± 7%, n = 9; isolated animals with RU486: infected, 120 ± 7%, noninfected, 100% ± 5%, n = 8, P > 0.2 between infected neurons from control animals and isolated animals with RU486), indicating normal rescue of synaptic delivery of GluR1. These results suggest that blockade of synaptic AMPA receptor delivery with social isolation in the barrel cortex was mediated by the local activation of glucocorticoid signaling.

Figure 4

Mechanisms underlying isolation-induced disruption of synaptic AMPA receptor delivery. (A) Immunohistostaining of the barrel cortex of animals at P11 with anti-GR antibody. (B) Nuclear fraction contained larger amount of GR at 3 hours after social isolation than nonisolated animals. The amount of GR in the nuclear fraction of isolated animals was normalized to nonisolated rats (*P < 0.05, isolated vs. nonisolated; n = 6 per each group). (C) Local injection of RU486 during social isolation prevented the disruption of synaptic GluR1 delivery by social isolation. Top inset showed time line of experimental manipulations (see text) (*P < 0.05, R1 versus non; n = 9 isolated with RU486, n = 7 isolated with vehicle). (D) Social isolation decreased phosphorylation of CaMKII at Thr286 compared with nonisolated rats. Phosphorylation level (calculated as the ratio to total CaMKII) of rats with social isolation in the presence of RU486 was comparable to nonisolated rats (F_(2,11) = 8.163, *P < 0.05; n = 5 per each group). (E) Social isolation decreased phosphorylation of Ser831 of GluR1 compared with nonisolated rats. Phosphorylation level of rats with social isolation in the presence of RU486 was comparable to nonisolated rats but larger than isolated animals without RU486 (F_(2,45) = 6.950, *P < 0.05; n = 12 per each group). (F) No delivery of 831A (R1 831A) at layer 4-2/3 synapses in the barrel cortex of intact rats at P12–P14 (n = 16). Scale bars: 200 μm (A); 100 μm (A, inset); 10 pA/20 ms. (B, D, and E); 20 pA/20 ms (C). Lanes were run on the same gel but are noncontiguous. Data were analyzed by paired, 2-tailed t test (B, C, F) or 1-way factorial ANOVA (D, E).

Social isolation decreases CaMKII activity and phosphorylation of GluR1 at a site critical for its synaptic delivery. Activation of calcium/calmodulin-dependent protein kinase type II (CaMKII) is critical for synaptic AMPA receptor delivery, LTP, and behavioral plasticity (40). To elucidate the signaling mechanisms that mediate the connection between social isolation and synaptic GluR1 insertion, we first examined the effect of social isolation on CaMKII activity. Synaptosome fractions from the barrel cortex of isolated animals (6 hours per day during P7–P11) with or without RU486 and nonisolated animals were prepared at P11. Then phosphorylation of Thr286 of CaMKIIα, an indicator of CaMKII activation, was assessed. We found that social isolation decreased phosphorylation at Thr286, indicating downregulation of CaMKII activity (Figure 4D). This effect of social isolation was prevented in the presence of RU486 (8 μg/g of body weight) (Figure 4D). Downregulation of CaMKII activity by neonatal social isolation persisted until a later developmental age (P27) (nonisolated, 100% ± 15%; isolated, 61% ± 10%, n = 12, P < 0.05; data not illustrated), consistent with the attenuation of LTP at P27 by early social isolation (Figure 2C).

Ser831 of GluR1 is a target of CaMKII and known to be phos­phorylated during LTP (41). We next examined the effect of social isolation on phosphorylation at Ser831 of GluR1. Synaptosome fraction from the barrel cortex of isolated animals exhibited decreased phosphorylation at Ser831 compared with that of nonisolated control animals (Figure 4E). Phosphorylation levels at Ser831 of rats with social isolation in the presence of RU486 (8 μg/g of body weight) were comparable to those of nonisolated control rats, but significantly larger than those of isolated animals without RU486 (Figure 4E). Consistent with the persistent downregulation of CaMKII activity until juvenile age in isolated animals, we found that phosphorylation of Ser831 was still reduced at P27 in isolated animals (nonisolated, 100% ± 5%; isolated, 69% ± 9%, n = 12, P < 0.05). We did not observe the change of the phosphorylation at Ser845 with social isolation (data not shown).

Previous studies indicate that mutation of GluR1 at Ser831 to alanine does not prevent its synaptic incorporation in neurons expressing constitutively active CaMKII, indicating that increase of phosphorylation at Ser831 was not required for this form of plasticity (19). To determine whether this is the case with experience-dependent synaptic delivery of GluR1 in vivo, we expressed GluR1 mutant (Ser831 to alanine: 831A) in the barrel cortex at P12 and examined whisker experience–driven delivery of 831A at layer 2/3-4 synapses. We detected no difference of rectification between 831A-expressing and nonexpressing neurons at P14, indicating no synaptic delivery of 831A (Figure 4F). Thus, phosphorylation of Ser831 is required for experience-dependent synaptic GluR1 delivery. Expression of constitutively active CaMKII may activate mechanisms other than those produced by input from sensory experience in vivo and could overcome lack of phosphorylation at Ser831.

These results suggested that social isolation downregulates CaMKII activity via glucocorticoid, decreases phosphorylation at Ser831, and results in attenuation of synaptic GluR1 delivery.

NE does not mediate social isolation–induced disruption of synaptic AMPA receptor delivery. NE is also a crucial stress hormone in mammals. In order to elucidate the role of NE in the effect of social isolation, we injected propranolol (3.0 mg/kg), a blocker of β-adrenergic receptors, during social isolation from P7 to P11. We used this dose of propranolol, since we found that increase of phosphorylation at Ser845 of GluR1 induced by the systemic injection of epinephrine (0.5 mg/kg i.p.) was blocked in the presence of propranolol (3.0 mg/kg i.p.), suggesting the effectiveness of propranolol at this dose (data not shown). As was described above, synaptic delivery of recombinant GFP-GluR1 was assessed by whole-cell recording. Isolated animals showed no difference of rectification between GFP-GluR1–expressing and nonexpressing neurons even in the presence of propranolol (Supplemental Figure 3A), indicating no synaptic delivery of GFP-GluR1 receptors. Synaptic delivery of endogenous AMPA receptors was also prevented by social isolation in the presence of propranolol, since the A/N ratio of isolated rats with propranolol at P14 was significantly lower than that of nonisolated rats and comparable to that of isolated animals without propranolol (Supplemental Figure 3B). These results indicate that social isolation–induced disruption of experience-dependent synaptic delivery of AMPA receptors in the developing rat barrel cortex requires glucocorticoid signaling, but not NE signaling.

Whisker-barrel function is attenuated by social isolation. Disruption of AMPA receptor trafficking to BC-L4, 2/3 synapses could lead to an aberrant functional relationship between whiskers and layer 2/3 barrel columns. Since the effect of neonatal social isolation during P4–P7 on LTP persisted until juvenile age (Figure 2C), we chose juvenile age to assess the effect of early isolation (P4–P7) on whisker-barrel function.

The decrease of synaptic transmission of BC-L4, 2/3 synapses by the disruption of synaptic GluR1 delivery could result in an attenuation of the sensitivity of each barrel to the corresponding whiskers. To examine this, we measured the slope of field excitatory postsynaptic potential (EPSP) and the discharge frequency of individual neurons in response to the deflection of whiskers with in vivo tetrode recordings from a single barrel. Whiskers with the highest response were defined as principal whiskers. We collected data from recording electrodes placed in layer 2/3 of the barrel field and at the almost identical tangential location within the barrel column (Figure 5C). Single-unit activities were semiautomatically isolated based on the spike waveform using the ND Manager software suite (ref. 42 and Supplemental Figure 4, A–C). As we excluded fast-spiking interneurons from the analysis, the majority (>90%) of the isolated unit activities originated from pyramidal cells, from which we detected synaptic dysfunction (see above).

Figure 5

Social isolation attenuates the sensitivity of whiskers. Responses (field EPSP slopes and the frequency of spikes) in layer 2/3 neurons of the barrel column to the deflection of principal whiskers. (A and B) The sensitivity of whiskers was attenuated by social isolation. (A) Rats with social isolation exhibited lower-field EPSP slopes than control animals, indicating attenuated sensitivity of whiskers (*P < 0.05, nonisolated versus isolated; n = 10 per each group). (B) Rats with social isolation exhibited lower spike frequency (spike number during 5–50 ms from whisker stimulation) than control animals, indicating decreased sensitivity of whiskers (*P < 0.05, nonisolated versus isolated; n = 10 per each group). (C) Wire electrode was implanted into almost identical tangential location of barrel column (arrowhead indicates the location of the implanted electrode) throughout experiments (left). Example of implanted electrode into the layer 2/3 of the barrel cortex (right). Coronal cortical slices infected with GFP-GluR1ct–expressing virus. The electrode was implanted into layer 2/3 of the barrel cortex (layer numbers are indicated). Scale bar: 500 μm. (D) Rats with GFP-GluR1ct (C tail) expression exhibited lower-field EPSP slopes than GFP-expressing animals, indicating decreased sensitivity of whiskers by the expression of GFP-GluR1ct (*P < 0.05, GFP versus C tail; C tail, n = 7, GFP, n = 4). Data were analyzed by paired, 2-tailed t test.

First, we examined the response to principal whisker deflections of rats either with or without social isolation. Rats were either isolated for 6 hours per day from P4 to P7 or kept under normal conditions. We measured the slope of field EPSP and the frequency of spikes in response to the deflection of principal whiskers at P23. We found that the slope of field EPSP and the discharge frequency by the deflection of principal whiskers of isolated animals were significantly smaller than those of control animals, indicating that the sensitivity of the barrel to its principal whisker was attenuated (Figure 5, A and B). There was no difference in spike latency by the deflection of the principal whisker between animals with social isolation during P4–P7 and control nonisolated rats at P23 (Supplemental Figure 4D). This suggests that social isolation deteriorates the sensitivity of the barrel column to its principal whisker at layer 2/3. Absolute values of spike frequency and field EPSP at layer 4 of the barrel cortex from isolated animals displayed no difference with those of nonisolated control rats (Supplemental Figure 4, E and F), suggesting that the decrease of the response to the whisker deflection at layer 2/3 was not inherited from dysfunction at layer 4.

Next, we wished to relate the attenuation of whisker-barrel function to the disruption of experience-dependent synaptic delivery of GluR1. We expressed the cytoplasmic portion of GluR1 tagged with GFP (GFP-GluR1ct) in layer 2/3 of the barrel cortex, beginning at P4, using lentivirus-mediated in vivo gene transfer. GFP-GluR1ct prevents experience-driven synaptic delivery of endogenous GluR1 (12, 23). We found that the slope of field EPSP in response to a deflection of the principal whisker was significantly smaller in GFP-GluR1ct–expressing rats than in control GFP-expressing rats, indicating that the sensitivity of the barrel to its principal whisker was attenuated in GFP-GluR1ct–expressing rats (Figure 5D). These results suggest that social isolation attenuates the sensitivity of the barrel column to its principal whisker by the disruption of GluR1 delivery to BC-L4, 2/3 synapses.

Social isolation attenuates whisker-dependent behavior. We next examined the effects of the attenuated whisker sensitivity on behavior. We assessed the disruption of whisker sensitivity with the ability of an animal to detect distances using a modified version of the gap-crossing task (see Methods), which is known to be dependent on the detection ability of whiskers (refs. 43, 44, and Figure 6A). In this task, rats were initially trained to find a reward at the opposite end of a runway. After this training phase, a gap was inserted in the runway and was widened at 0.5-cm intervals. The gap was widened until rats would no longer step across it. This gap size was defined as the upper threshold (SU). Subsequently, the gap was narrowed at 0.5-cm intervals until rats crossed it (Figure 6A). This gap size was defined as the lower threshold (SL). The difference between SU and SL was calculated (defined as ΔS). Rats touched the target platform even on noncrossing trials (data not shown), suggesting that rats utilize their whiskers to perform this task.

Figure 6

Social isolation disrupts behavior dependent on whisker-barrel function. (A) Schematic of gap-crossing test. The gap was widened until rats could no longer cross (middle, SU), and then the gap was narrowed until they could cross (right: SL). Difference between SU and SL is indicated by ΔS (see text and Methods for details). (B) Rats with GFP expression in the barrel cortex showed lower ΔS than GFP-GluR1ct–expressing (C tail) animals, indicating that GFP-expressing animals have better distance detection ability than GFP-GluR1ct–expressing animals (*P < 0.05, GFP versus C tail; n = 8 per each group). (C) Isolated rats showed higher ΔS than nonisolated rats, indicating worse distance detection. Isolated rats treated with RU486 showed lower ΔS than untreated isolated rats, indicating RU486 rescued distance detection of isolated rats (F_(5,45) = 8.668, *P < 0.05 compared with nonisolated [n = 9] and isolated postnatal P4–P7 with RU486 [n = 9] animals, ^#P < 0.05 compared with nonisolated and isolated P7–P11 with RU486 [n = 8] animals). RU486 treatment alone had no effect on distance detection (n = 8). Data were analyzed by paired, 2-tailed t test (B) or 1-way factorial ANOVA (C).

Since the effect of neonatal social isolation on synaptic plasticity and whisker-barrel function lasts until juvenile age (Figure 2C and Figure 5), we performed whisker-barrel function–dependent behavior task at a juvenile age.

We first used this test on P21 animals that had been expressing either GFP-GluR1ct or GFP in layer 2/3 of the barrel cortex (delivered at P4 by lentivirus-mediated in vivo gene transfer). GFP-GluR1ct–expressing animals showed larger distance discrimination values (ΔS) than GFP-expressing animals, indicating that expression of GFP-GluR1ct impaired the distance-detection ability (Figure 6B). Therefore, this gap-crossing test is dependent on AMPA receptor trafficking and proper whisker-barrel function.

Next, animals were exposed to social isolation for 6 hours per day either during P4–P7 or P7–P11. At P21, rats were tested in the gap-crossing task. Figure 6C shows distance discrimination values (ΔS) of rats housed in control nonisolated environments and of isolated rats treated with RU486 (8.0 μg/g of body weight) or without RU486 during social isolation. ΔS was significantly different among the groups. Post hoc analyses revealed that control rats showed lower ΔS than rats with social isolation, indicating a better distance detection ability in control rats (Figure 6C). Moreover, isolated rats treated with RU486 showed lower ΔS than isolated rats without RU486, but no difference was observed between control and RU486-treated isolated rats, indicating that RU486 rescued the effect of social isolation on the ability of distance discrimination (Figure 6C). Application of RU486 did not affect this behavior task, since nonisolated rats treated with RU486 exhibited the same level of distance discrimination ability as control animals (Figure 6C). It is unlikely that these effects of social isolation on this task were due to general anxiety, since we observed no difference of indicators of anxiety on the open field apparatus and the device used for the gap-crossing task between isolated and nonisolated rats (Supplemental Figure 5, A and B). Moreover, there was no significant difference in food consumption, suggesting no effect of social isolation on the motivation in this task (Supplemental Figure 5C). Furthermore, we did not detect any difference in the length of whiskers between isolated and nonisolated animals (isolated, 22.4 ± 0.4 mm; nonisolated, 23.3 ± 0.7 mm, n = 7, P > 0.3, D2 whisker), suggesting that this stress protocol did not have any effect on whisker growth. These results indicate that neonatal social isolation impaired the distance discrimination ability of animals and that this effect was mediated by the glucocorticoid pathway.

Since social isolation during P4–P7 induced synaptic defect at early developmental age (P14), we determined whether disruption of whisker-dependent behavior appeared at P14. To test this, we used the forelimb-placing test (45), which can be used on animals of this age. In this test, a pup’s body was supported by the experimenter so that the limbs of the pup were free. Then, whiskers were brushed. If barrel function is normal, a pup extends the forelimb contralateral to deflected whiskers, but not if it has aberrant barrel function. We counted the number of normal limb responses per 10 trials. We found significantly lower scores of normal forelimb response in animals isolated either during P4–P7 or P7–P11 than in pups housed under normal conditions (Supplemental Figure 6). This indicates that impairments in whisker-dependent behavior also occurred at the same age at which synaptic dysfunction was detected.

While numerous studies have revealed profound effects of stress on neural function, few have comprehensively related molecular, synaptic, and circuit deficits to behavioral aberrations (30, 46, 47). In this study we found that the stress produced by neonatal social isolation disrupts synaptic delivery of AMPA receptors, circuit formation in the cortex, and behaviors related to somatosensory function. We focused on plasticity in the barrel cortex, since it is likely to play an important role in the integration of social information. This region is the primary sensory cortical area receiving input from whiskers, which are important for social interaction (10, 11). Although we analyzed only the barrel cortex in this study, we elucidated the molecular mechanism underlying the effect of neonatal isolation on synaptic plasticity, which may play an important role in the pathophysiology of many CNS diseases.

We examined the potential signaling mechanisms underlying the effects of isolation. We found that a prolonged rise of corticosterone level was induced by neonatal social isolation. This downregulates CaMKII activity and phosphorylation at Ser831 of GluR1, and this could contribute to the interruption of experience-driven synaptic delivery of AMPA receptors, while other posttranslational modifications with social isolation could be involved in the disruption of synaptic AMPA receptor delivery. NE, another important stress hormone, does not mediate the effect of social isolation on AMPA receptor trafficking, since propranolol did not prevent social isolation–induced disruption of AMPA receptor delivery. A recent in vitro study showed that brief treatment with corticosterone increases synaptic AMPA receptors (48). In contrast with the effects of chronic elevation of corticosterone observed with social isolation, a lower brief rise of glucocorticoid might enhance synaptic delivery of AMPA receptors, suggesting an inverted U dose-response effect of glucocorticoid signaling on AMPA receptor trafficking. By what molecular mechanism glucocoritcoid signaling alters CaMKII activity remains to be elucidated. One possible mechanism is an increase in a protein phosphatase such as calcineurin and attenuation of CaMKII activity through dephosphorylation of phosphorylated active CaMKII. Indeed, chronic stress was shown to increase the calcineurin level in neurons (49). Downregulation of CaMKII activity in isolated animals persisted until juvenile age (P27), when attenuation of LTP was still observed in isolated animals, suggesting that neonatal chronic stress could induce permanent changes in signaling machinery required for synaptic plasticity. Although PKC could also be involved in the phosphorylation of Ser831, social isolation did not decrease PKC activity, suggesting that decrease in the phosphorylation of Ser831 was not due to the downregulation of PKC activity (data not shown). Social isolation induced the reduction of CaMKII activity as well as the decrease of the phosphorylation of Ser831 on GluR1. Social isolation–induced decrease of the phosphorylation of Ser831 could be due to the reduction of CaMKII activity or to mechanisms other than CaMKII action. Thus, our results delineate molecular mechanism underlying long-lasting effects of neonatal chronic stress on synaptic function.

Sensory processing is crucial for cognitive functions such as attention, emotion, memory, and social behavior. Alterations in sensory processing constitute prominent symptoms in various psychiatric disorders (e.g., autism, schizophrenia, and personality disorders) (50–53). While genetic alterations are considered to be important as etiologies of these mental illnesses, postnatal environment could also contribute to the onset of these mental disorders. In human studies, it has been shown that traumatic events such as child abuse could lead to misprocessing of sensory information (54–58). Our findings show that neonatal social isolation, which could be a traumatic experience, can cause disruption of experience-dependent synaptic delivery of AMPA receptors and aberrant circuit formation in the rodent somatosensory cortex and leads to behavioral abnormalities. Since rodents and humans may display similar effects of neonatal maltreatment on the nervous system (36), elucidation of molecular and cellular events in the somatosensory cortex of animals with postnatal maltreatment could lead to the discovery of novel biomarkers of mental disorders induced by postnatal severe stress such as child abuse.

View Supplemental data

This project was supported by Grant-in-Aid for Young Scientists (Start-up) (18800037), Grant-in-Aid for Scientific Research (20300131) (to T. Takahashi), JST, CREST (to T. Takahashi), Special Coordination Funds for Promoting Science and Technology (to T. Takahashi), the Sumitomo Foundation (to T. Takahashi), the KANAE Foundation (to T. Takahashi), the Takeda Science Foundation (to T. Takahashi), NARSAD (to T. Takahashi), the grant for 2007 Strategic Research Project (K19025) of Yokohama City University (to T. Takahashi), “Development of biomarker candidates for social behavior” carried out under the Strategic Research Program for Brain Sciences by the Ministry of Education, Culture, Sports, Science and Technology of Japan (to T. Takahashi), and the NIH (MH049159 to R. Malinow). We also thank Yoshiko Kannno for excellent technical assistance and Helmut Kessels for the critical reading of the manuscript.

Address correspondence to: Takuya Takahashi, Yokohama City University, Graduate School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan. Phone: 81.0.45.787.2579; Fax: 81.0.45.787.2578; E-mail: takahast@yokohama-cu.ac.jp.

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J Clin Invest. 2012;122(7):2690–2701. doi:10.1172/JCI63060.

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