Spine dynamics of PSD-95-deficient neurons in the visual cortex link silent synapses to structural cortical plasticity

Critical periods (CPs) are time windows of heightened brain plasticity during which experience refines synaptic connections to achieve mature functionality. At glutamatergic synapses on dendritic spines of principal cortical neurons, the maturation is largely governed by postsynaptic density protein-95 (PSD-95)-dependent synaptic incorporation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors into nascent AMPA-receptor silent synapses. Consequently, in mouse primary visual cortex (V1), impaired silent synapse maturation in PSD-95-deficient neurons prevents the closure of the CP for juvenile ocular dominance plasticity (jODP). A structural hallmark of jODP is increased spine elimination, induced by brief monocular deprivation (MD). However, it is unknown whether impaired silent synapse maturation facilitates spine elimination and also preserves juvenile structural plasticity. Using two-photon microscopy, we assessed spine dynamics in apical dendrites of layer 2/3 pyramidal neurons (PNs) in binocular V1 during ODP in awake adult mice. Under basal conditions, spine formation and elimination ratios were similar between PSD-95 knockout (KO) and wild-type (WT) mice. However, a brief MD affected spine dynamics only in KO mice, where MD doubled spine elimination, primarily affecting newly formed spines, and caused a net reduction in spine density similar to what has been observed during jODP in WT mice. A similar increase in spine elimination after MD occurred if PSD-95 was knocked down in single PNs of layer 2/3. Thus, structural plasticity is dictated cell autonomously by PSD-95 in vivo in awake mice. Loss of PSD-95 preserves hallmark features of spine dynamics in jODP into adulthood, revealing a functional link of PSD-95 for experience-dependent synapse maturation and stabilization during CPs.

Early life of an animal is characterized by time windows of functionally and structurally enhanced brain plasticity known as critical periods (CPs), which have been described initially in the primary visual cortex (V1) of kittens (1). During CPs, experience refines the connectivity of principal excitatory neurons to establish the mature functionality of neural networks. This refinement is governed by the constant generation and elimination of nascent synapses on dendritic spines that sample favorable connections to be consolidated and unfavorable ones to be eliminated (2–5). A fraction of nascent synapses is or becomes α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-receptor silent, expressing N-methyl-D-aspartate (NMDA) receptors only (6–8). At eye opening, silent synapses are abundant in the primary visual cortex (V1) (9, 10) and mature during CPs by stable AMPA receptor incorporation (11–14). The pace of silent synapse maturation is governed by the opposing yet cooperative function of postsynaptic density protein of 95 kDa (PSD-95) and its paralog PSD-93, two signaling scaffolds of the postsynaptic density of excitatory synapses (12, 13). However, whether silent synapses are preferential substrates for spine elimination during CPs remains to be investigated.In juvenile mice (postnatal days [P] 20 to 35), a brief monocular deprivation (MD) of the dominant contralateral eye results in a shift of the ocular dominance (OD) of binocular neurons in V1 toward the open eye, mediated by a reduction of responses to visual stimulation of the deprived eye (15–17). Structurally, MD induces an increase in spine elimination in apical dendrites of layer (L) 2/3 and L5 pyramidal neurons (PNs) which is only observed during the CP and constitutes a hallmark of juvenile OD plasticity (jODP) (18–20). After CP closure, cortical plasticity declines progressively, and in standard cage-raised mice beyond P40, a 4-d MD no longer induces the functional nor anatomical changes associated with jODP (21–24).At least three different mechanisms involved in experience-dependent maturation of cortical neural networks have been described, but the molecular and cellular mechanisms that cause CP closure remain highly debated (18, 25, 26). First, plasticity of local inhibitory neurons, such as increased inhibitory tone or a reduction of release probability by experience-dependent endocannabinoid receptor 1 (CB1R) activation was reported to close the critical period in rodent V1 (27–29). Second, the expression of so-called “plasticity brakes,” such as extracellular matrix (ECM), Nogo receptor 1 (NgR1), paired immunoglobulin-like receptor B (PirB), and Lynx1 were correlated with the end of critical periods (30–33). Experimentally decreasing the inhibitory tone or absence of plasticity brakes enhanced ODP expression in various knockout (KO) mouse models (32, 34, 35), among which only Lynx1 KO mice were shown to exhibit functional hallmarks of jODP, such as selective deprived eye depression after a short MD (36). Structurally, Lynx1 KO mice exhibited elevated spine dynamics at baseline; however, MD induced a reduction in spine elimination in apical dendrites of L5 PNs, whereas in L2/3 PNs there was no change (37). Thus, the effects of removing plasticity brakes on structural plasticity are variable, and it remains unclear to what extend manipulating the plasticity brakes can reinstate cellular signatures of CP plasticity in the adult wild-type (WT) brain (38). Third, the progressive maturation of AMPAR-silent synapses was correlated with the closure of the CP for jODP (12, 13). Consequently, in PSD-95 KO mice, the maturation of silent synapses is impaired; their fraction remains at the eye opening level, and jODP is preserved lifelong (13). Furthermore, visual cortex-specific knockdown (KD) of PSD-95 in the adult brain reinstated jODP. In contrast, in PSD-93 KO mice, silent synapses mature precociously and the CP for jODP closes precociously (12), correlating the presence of silent synapses with functional plasticity during CPs.While these three mechanisms of CP closure are not mutually exclusive in regulating cortical plasticity (26), it remains elusive whether CP-like structural plasticity can be expressed in the adult brain and whether silent synapses might be substrates for it. Here, we performed chronic two-photon imaging of dendrites of L2/3 pyramidal neurons in binocular V1 of PSD-95 KO (and KD) and WT mice, tracking the same dendritic spines longitudinally before, during, and after a 4-d period of MD. As previous studies have reported anesthesia effects on spine dynamics (39–41), we performed our experiments in awake mice, thoroughly trained for head fixation under the two-photon microscope. Our chronic spine imaging experiments revealed that in adult PSD-95 KO and KD mice, a brief MD indeed increased spine elimination about twofold, while adult WT mice did not display experience-dependent changes in spine elimination or spine formation. Thus, the loss of PSD-95 led to a high number of AMPAR-silent synapses which were correlated with jODP after MD, and with juvenile-like structural plasticity even in the adult brain, underscoring the importance of silent synapses for CP-timing and network maturation and stabilization.     

Long-term inhibitory plasticity in visual cortical layer 4 switches sign at the opening of the critical period

Sensory microcircuits are refined by experience during windows of heightened plasticity termed “critical periods” (CPs). In visual cortex the effects of visual deprivation change dramatically at the transition from the pre-CP to the CP, but the cellular plasticity mechanisms that underlie this change are poorly understood. Here we show that plasticity at unitary connections between GABAergic Fast Spiking (FS) cells and Star Pyramidal (SP) neurons within layer 4 flips sign at the transition between the pre-CP and the CP. During the pre-CP, coupling FS firing with SP depolarization induces long-term depression of inhibition at this synapse, whereas the same protocol induces long-term potentiation of inhibition at the opening of the CP. Despite being of opposite sign, both forms of plasticity share expression characteristics—a change in coefficient of variation with no change in paired-pulse ratio—and depend on GABA_B receptor signaling. Finally, we show that the reciprocal SP→FS synapse also acquires the ability to undergo long-term potentiation at the pre-CP to CP transition. Thus, at the opening of the CP, there are coordinated changes in plasticity that allow specific patterns of activity within layer 4 to potentiate feedback inhibition by boosting the strength of FS↔SP connections.Sensory microcircuits are refined by experience during windows of heightened plasticity termed “critical periods” (CPs). In visual cortex the classical CP was defined based on when visual deprivation (VD) induces ocular dominance (OD) shifts, between approximately postnatal days (P) 20–33 (1–3). However, visual cortex is also plastic during a pre-CP between eye opening (∼P14) and the onset of the classical CP (4–6). Although both developmental windows are characterized by sensitivity to visual experience, the effects of VD change dramatically at the transition between these two developmental stages (7–10).The cellular changes that underlie the transition from pre-CP to CP plasticity remain incompletely understood, but recent work has implicated a specific inhibitory network involving parvalbumin-positive fast-spiking (FS) basket cells in this process (8, 11, 12). FS cells provide strong somatic inhibition onto cortical pyramidal neurons, and this inhibition matures significantly between eye opening and the opening of the classical CP (13–15). Further, reducing or enhancing this inhibition can prevent or prematurely trigger the transition from pre-CP to CP plasticity (11, 16–18). Thus, maturation of FS inhibition is thought to be causally involved in triggering CP plasticity, but exactly what aspect of this maturation drives these changes is unknown. One characteristic of this maturation is a change in the response of FS synapses to VD. Brief monocular VD during the pre-CP decreases inhibitory synaptic strength from FS to star pyramidal (SP) neurons in layer 4 (L4) of the monocular primary visual cortex [V1m (19)] but increases inhibition at the same synapse when performed during the CP (20). There is evidence that long-term potentiation of inhibition (LTPi) is the cellular mechanism behind the VD-driven inhibitory potentiation during the CP (20), but why VD weakens this synapse during the pre-CP has not been determined.To ask whether a change in the cellular plasticity mechanisms present at FS→SP synapses might underlie this developmental shift in the effects of VD, we used paired recordings to analyze transmission and plasticity at unitary connections between FS cells and SP neurons within V1m. We found only subtle changes in the basal properties of this connection between P15–P17 (the pre-CP) and P21–P23 (the opening of the CP). In contrast, plasticity at this synapse changed dramatically. Coupling presynaptic FS firing with postsynaptic SP depolarization induced long-term depression of inhibition (LTDi) during the pre-CP, whereas the same protocol induced LTPi during the CP. Both forms of plasticity were accompanied by changes in the coefficient of variation (CV) of unitary inhibitory postsynaptic current (uIPSC) amplitude without significant changes in paired-pulse ratio (PPR) and were blocked by GABA_B receptor (GABA_BR) antagonists. Finally, we found that during the CP (but not the pre-CP) the same induction protocol at reciprocally connected FS↔SP pairs induced LTP of both connections, suggesting that during the CP both components of this feedback inhibitory loop within L4 can be potentiated as a unit.     

Syngap1 regulates experience-dependent cortical ensemble plasticity by promoting in vivo excitatory synapse strengthening

A significant proportion of autism risk genes regulate synapse function, including plasticity, which is believed to contribute to behavioral abnormalities. However, it remains unclear how impaired synapse plasticity contributes to network-level processes linked to adaptive behaviors, such as experience-dependent ensemble plasticity. We found that Syngap1, a major autism risk gene, promoted measures of experience-dependent excitatory synapse strengthening in the mouse cortex, including spike-timing–dependent glutamatergic synaptic potentiation and presynaptic bouton formation. Synaptic depression and bouton elimination were normal in Syngap1 mice. Within cortical networks, Syngap1 promoted experience-dependent increases in somatic neural activity in weakly active neurons. In contrast, plastic changes to highly active neurons from the same ensemble that paradoxically weaken with experience were unaffected. Thus, experience-dependent excitatory synapse strengthening mediated by Syngap1 shapes neuron-specific plasticity within cortical ensembles. We propose that other genes regulate neuron-specific weakening within ensembles, and together, these processes function to redistribute activity within cortical networks during experience.

Autism risk genes converge on several neurobiological functions, including the regulation of synapse biology (1–3). Synapse processes directly controlled by autism spectrum disorder (ASD) risk genes include de novo synapse formation, synapse maturation, and activity-driven changes in synapse function (i.e., synapse plasticity). Synapse plasticity, especially in cortical excitatory neurons, is a process enabling neural circuits to store new information, which is essential for experience-dependent modifications of behavior to promote survival (4, 5). Thus, risk genes are thought to contribute to ASD etiology by disrupting how neural circuits change in response to novel experiences, which in turn contributes to maladaptive behaviors. However, the study of risk gene biology and their relationship to neural plasticity is largely restricted to reduced biological preparations that focus on isolated changes to a small subset of synapses. Therefore, it is unclear how risk gene–driven regulation of synapse plasticity contributes to changes in neural dynamics within intact functional networks known to drive adaptive behaviors.Neuronal ensembles, or groups of coactivated neurons, are thought to be the direct neural substrate of cognitive processes and behavior (6). In cortex, ensemble plasticity is a multidimensional process that reflects the distribution of distinct cellular plasticity mechanisms across individual neuronal components within the assembly. For example, neurons within the same sensory-evoked cortical ensemble can undergo either increases or decreases in activity in response to the same sensory experience (7–9). While this general phenomenon has been observed in multiple contexts, it is unclear how neurons within the same functional network can have opposing changes to enduring neuronal activity in response to the same sensory experience. One way that this may occur is through the simultaneous activation of distinct forms of experience-dependent plasticity that are differentially distributed throughout neurons that comprise a functional network. Indeed, sensory experience drives the induction of Hebbian-type synaptic plasticity that can strengthen or weaken excitatory synaptic input onto sensory-responsive neurons (10). Experience-dependent circuit plasticity is not limited to changes in excitatory synaptic strength. Robust changes to the function and connectivity of GABAergic interneurons within cortical microcircuits also occurs in response to novel experience, which in turn regulates the output of pyramidal neurons (11–13). Moreover, intrinsic changes to neuronal excitability have also been observed, and in combination with changes to GABAergic function, these collective processes are thought to maintain a set firing rate within networks even as activity is redistributed among individual neurons (8, 14, 15).We propose that experience induces heterogenous changes in activity within neurons of a cortical assembly through cellular processes controlled, at least in part, by genetic mechanisms linked to ASD risk. This hypothesis originates from the clear overrepresentation of ASD risk genes that regulate the neurobiology of synapses and synapse plasticity (1–3). However, because of the multidimensional nature of cortical network plasticity, one cannot infer how a gene influences experience-dependent changes in distributed network dynamics when the function of the gene has only been studied in isolated subcellular structures, such as synapses. It is therefore important to study major ASD risk genes in the context of intact functional networks. Doing so will help to elucidate how their influence over molecular and cellular functions contribute to intermediate network-level processes more directly linked to behaviors, such as cortical ensemble plasticity.In this study, we investigated how a major ASD risk gene, SYNGAP1/Syngap1 (HUMAN/mouse–mouse only from now on), regulates specific aspects of cellular plasticity in vivo and how this process shapes experience-dependent ensemble plasticity with sensory-responsive cortical networks. The Syngap1 gene, which is a major autism risk factor (16), is a robust regulator of various forms of long-term potentiation (LTP) (17), a cellular model of Hebbian plasticity. It regulates LTP through control of excitatory synapse structure and function by gating NMDA receptor-dependent regulation of AMPA receptor trafficking and dendritic spine size (18–20). The role of Syngap1 in regulating synapse plasticity has been observed by various researchers across distinct neuronal subtypes in a variety of in vitro and ex vivo preparations (21–24). Based on this past work in reduced preparations, we hypothesized that Syngap1 regulates experience-dependent ensemble plasticity by promoting the strengthening of excitatory synapses within functional cortical networks. We found that Syngap1 was required for spike-timing-dependent (STD) synaptic potentiation and experience-mediated synapse bouton formation in layer (L) 2/3 of somatosensory cortex (SSC) but not synaptic depression or synapse bouton elimination. Syngap1 heterozygosity in mice disrupted experience-dependent potentiation of neuronal activity within a subpopulation of L2/3 SSC neurons. Syngap1 loss of function had no effect on plasticity of neurons within the same ensemble that weakens with experience. These findings indicate that disruptions to synapse-level strengthening mechanisms in Syngap1 mice contribute to imbalanced cortical ensemble plasticity driven by novel sensory experience. We propose that a key function of Syngap1 is to promote complex network-level plasticity through the strengthening of excitatory connections within cortical circuits.     

Global impairment and therapeutic restoration of visual plasticity mechanisms after a localized cortical stroke

We tested the influence of a photothrombotic lesion in somatosensory cortex on plasticity in the mouse visual system and the efficacy of anti-inflammatory treatment to rescue compromised learning. To challenge plasticity mechanisms, we induced monocular deprivation (MD) in 3-mo-old mice. In control animals, MD induced an increase of visual acuity of the open eye and an ocular dominance (OD) shift towards this eye. In contrast, after photothrombosis, there was neither an enhancement of visual acuity nor an OD-shift. However, OD-plasticity was present in the hemisphere contralateral to the lesion. Anti-inflammatory treatment restored sensory learning but not OD-plasticity, as did a 2-wk delay between photothrombosis and MD. We conclude that (i) both sensory learning and cortical plasticity are compromised in the surround of a cortical lesion; (ii) transient inflammation is responsible for impaired sensory learning, suggesting anti-inflammatory treatment as a useful adjuvant therapy to support rehabilitation following stroke; and (iii) OD-plasticity cannot be conceptualized solely as a local process because nonlocal influences are more important than previously assumed.     

All-or-none disconnection of pyramidal inputs onto parvalbumin-positive interneurons gates ocular dominance plasticity

Disinhibition is an obligatory initial step in the remodeling of cortical circuits by sensory experience. Our investigation on disinhibitory mechanisms in the classical model of ocular dominance plasticity uncovered an unexpected form of experience-dependent circuit plasticity. In the layer 2/3 of mouse visual cortex, monocular deprivation triggers a complete, “all-or-none,” elimination of connections from pyramidal cells onto nearby parvalbumin-positive interneurons (Pyr→PV). This binary form of circuit plasticity is unique, as it is transient, local, and discrete. It lasts only 1 d, and it does not manifest as widespread changes in synaptic strength; rather, only about half of local connections are lost, and the remaining ones are not affected in strength. Mechanistically, the deprivation-induced loss of Pyr→PV is contingent on a reduction of the protein neuropentraxin2. Functionally, the loss of Pyr→PV is absolutely necessary for ocular dominance plasticity, a canonical model of deprivation-induced model of cortical remodeling. We surmise, therefore, that this all-or-none loss of local Pyr→PV circuitry gates experience-dependent cortical plasticity.

Experience during a postnatal, critical period is essential to properly shape the functional connectivity of cortical circuits. A canonical model of cortical plasticity is the shift in ocular dominance following monocular deprivation (MD), which biases responses toward the nondeprived (ND) eye. Prior research established that MD-induced changes result from the reorganization of excitatory glutamatergic synapses onto excitatory pyramidal neurons (Pyr), which is, in turn, regulated by an inhibitory GABAergic network composed of parvalbumin-positive inhibitory interneurons (PVs). The current consensus is that a reduced, permissive level of inhibition from PV circuits in cortical layer 2/3 is required for plasticity at downstream excitatory synapses and that inhibition above or below the permissive range constrains the response to MD (1–3). Although the notion that rapid cortical disinhibition precedes and initiates the plasticity of glutamatergic connectivity is well established (4, 5), and decades old (6–8), the underlying cellular mechanisms remain unclear.Disinhibition of excitatory cortical neurons could be achieved indirectly, for example by suppressing PV activity via enhancing inhibition from other interneurons through cholinergic neuromodulation (9, 10) but more directly, and likely more effectively, by reducing the excitatory input onto PVs (4, 11–13). Our current investigation uncovered a unique form of experience-dependent plasticity that regulates the connectivity between pyramidal neurons and PVs. We found that the initial response to MD is the functional and structural elimination of ∼50% of these connections. In contrast to the outcome of known mechanisms of synaptic plasticity that manifest in widespread graded changes in synaptic strength, the loss of pyramidal–PV connectivity occurs in a discrete, “all-or-none,” fashion: whereas a subset of connections become completely eliminated, the persistent connections have normal strength. This disconnection is not only rapid but it is transient, affects only very local pyramidal–PV pairs, and, importantly, manipulations that promote/prevent this disconnection also promote/prevent shifts in ocular dominance. We surmise, therefore, that the rapid and transient disconnection of discrete subsets of PV circuits enables the subsequent Hebbian and homeostatic modification of glutamatergic circuitry.     

Kalirin regulates cortical spine morphogenesis and disease-related behavioral phenotypes

Dendritic spine morphogenesis contributes to brain function, cognition, and behavior, and is altered in psychiatric disorders. Kalirin is a brain-specific guanine-nucleotide exchange factor (GEF) for Rac-like GTPases and is a key regulator of spine morphogenesis. Here, we show that KALRN-knockout mice have specific reductions in cortical, but not hippocampal, Rac1 signaling and spine density, and exhibit reduced cortical glutamatergic transmission. These mice exhibit robust deficits in working memory, sociability, and prepulse inhibition, paralleled by locomotor hyperactivity reversible by clozapine in a kalirin-dependent manner. Several of these deficits are delayed and age-dependent. Our study thus links spine morphogenic signaling with age-dependent, delayed, disease-related phenotypes, including cognitive dysfunction.     

Experience-dependent functional plasticity and visual response selectivity of surviving subplate neurons in the mouse visual cortex

Subplate neurons are early-born cortical neurons that transiently form neural circuits during perinatal development and guide cortical maturation. Thereafter, most subplate neurons undergo cell death, while some survive and renew their target areas for synaptic connections. However, the functional properties of the surviving subplate neurons remain largely unknown. This study aimed to characterize the visual responses and experience-dependent functional plasticity of layer 6b (L6b) neurons, the remnants of subplate neurons, in the primary visual cortex (V1). Two-photon Ca²⁺ imaging was performed in V1 of awake juvenile mice. L6b neurons showed broader tunings for orientation, direction, and spatial frequency than did layer 2/3 (L2/3) and L6a neurons. In addition, L6b neurons showed lower matching of preferred orientation between the left and right eyes compared with other layers. Post hoc 3D immunohistochemistry confirmed that the majority of recorded L6b neurons expressed connective tissue growth factor (CTGF), a subplate neuron marker. Moreover, chronic two-photon imaging showed that L6b neurons exhibited ocular dominance (OD) plasticity by monocular deprivation during critical periods. The OD shift to the open eye depended on the response strength to the stimulation of the eye to be deprived before starting monocular deprivation. There were no significant differences in visual response selectivity prior to monocular deprivation between the OD changed and unchanged neuron groups, suggesting that OD plasticity can occur in L6b neurons showing any response features. In conclusion, our results provide strong evidence that surviving subplate neurons exhibit sensory responses and experience-dependent plasticity at a relatively late stage of cortical development.

The mammalian cerebral cortex consists of six layers, with distinct roles in information processing (1, 2). At the bottom of the neocortex, on the boundary between the gray matter and white matter, there is a thin sheet of neurons called layer 6b (L6b) (3). Layer 6b neurons are thought to be remnants of subplate neurons based on their location and cell-type marker expression (4). During prenatal and early postnatal periods, subplate neurons form transient neuronal circuits that play key roles in cortical maturation (5–7). In the embryonic cortex, subplate neurons form short-lived synapses with early immature neurons to regulate radial migration (8). During perinatal development, subplate neurons transiently receive inputs from ingrowing thalamic axons and innervate layer 4 (L4) to guide thalamic inputs to the eventual target, L4 (5, 6). Thus, the circuits formed by subplate neurons at the perinatal developmental stage are essential to establish basic neuronal circuits before starting experience-dependent refinements (5–7). Subsequently, subplate neurons largely disappear due to programmed cell death, but some survive and reside in L6b (5, 6). In the adult cortex, L6b neurons form neuronal circuits with local and long-distance neurons, which are different from those formed during early development (9–12). Therefore, surviving subplate neurons may acquire a role in information processing after remodeling of neuronal connections. A recent study using three-photon Ca²⁺ imaging demonstrated that L6b neurons show visual responses with broad orientation/direction tuning in the adult mouse primary visual cortex (V1) (13). However, comparable evidence for L6b response properties with other layer neurons in V1 is lacking (14–20). Moreover, L6b neurons have diverse morphology and molecular expression (21–24). Neurons born during subplate neurogenesis show the different expression patterns of subplate markers in postnatal L6b (4). However, the response properties in each subtype of L6b neurons remain unknown.The sensory responsiveness of cortical neurons is considerably refined by sensory experience relatively late in development, referred to as the critical period (25, 26). Previous studies have demonstrated that sensory activities before the onset of the critical period affect the arrangement of subplate neuron neurites in the barrel cortex and local subplate circuits in the auditory cortex (27, 28). However, there is no direct evidence that the sensory responses of surviving subplate neurons are modified by sensory experience during the critical period. If experience-dependent plasticity occurs in subplate neuron responses, they will contribute to the experience-dependent development of sensory functions and possibly to the functions in the mature cortex. Ocular dominance (OD) plasticity in V1 is a canonical model used to examine experience-dependent refinement of sensory responses (25, 26, 29, 30). If one eye is occluded for several days during the critical period, neurons in V1 lose their response to the deprived eye. OD plasticity is robustly preserved across species and cell types. Therefore, OD plasticity is suitable for evaluating experience-dependent plasticity in L6b neurons.This study aimed to characterize the visual responses and OD plasticity of L6b neurons in V1. Toward this goal, two-photon Ca²⁺ imaging was performed in awake juvenile mice, followed by 3D immunohistochemistry with a subplate neuronal marker, connective tissue growth factor (CTGF) (4, 31). L6b neurons showed broader tuning to visual stimuli and lower binocular matching of orientation preference than did layer 2/3 (L2/3) and L6a neurons. Chronic two-photon imaging revealed significant OD plasticity in individual L6b neurons during the critical period. Our results provide strong evidence that L6b neurons, presumed to be subplate neuron remnants, exhibit sensory responses and experience-dependent functional plasticity at a relatively late stage of cortical development.     

Essential role for a long-term depression mechanism in ocular dominance plasticity

The classic example of experience-dependent cortical plasticity is the ocular dominance (OD) shift in visual cortex after monocular deprivation (MD). The experimental model of homosynaptic long-term depression (LTD) was originally introduced to study the mechanisms that could account for deprivation-induced loss of visual responsiveness. One established LTD mechanism is a loss of sensitivity to the neurotransmitter glutamate caused by internalization of postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs). Although it has been shown that MD similarly causes a loss of AMPARs from visual cortical synapses, the contribution of this change to the OD shift has not been established. Using an herpes simplex virus (HSV) vector, we expressed in visual cortical neurons a peptide (G2CT) designed to block AMPAR internalization by hindering the association of the C-terminal tail of the AMPAR GluR2 subunit with the AP2 clathrin adaptor complex. We found that G2CT expression interferes with NMDA receptor (NMDAR)-dependent AMPAR endocytosis and LTD, without affecting baseline synaptic transmission. When expressed in vivo, G2CT completely blocked the OD shift and depression of deprived-eye responses after MD without affecting baseline visual responsiveness or experience-dependent response potentiation in layer 4 of visual cortex. These data suggest that AMPAR internalization is essential for the loss of synaptic strength caused by sensory deprivation in visual cortex.     

Selective control of cortical axonal spikes by a slowly inactivating K+ current

Neurons are flexible electrophysiological entities in which the distribution and properties of ionic channels control their behaviors. Through simultaneous somatic and axonal whole-cell recording of layer 5 pyramidal cells, we demonstrate a remarkable differential expression of slowly inactivating K(+) currents. Depolarizing the axon, but not the soma, rapidly activated a low-threshold, slowly inactivating, outward current that was potently blocked by low doses of 4-aminopyridine, alpha-dendrotoxin, and rTityustoxin-K alpha. Block of this slowly inactivating current caused a large increase in spike duration in the axon but only a small increase in the soma and could result in distal axons generating repetitive discharge in response to local current injection. Importantly, this current was also responsible for slow changes in the axonal spike duration that are observed after somatic membrane potential change. These data indicate that low-threshold, slowly inactivating K(+) currents, containing Kv1.2 alpha subunits, play a key role in the flexible properties of intracortical axons and may contribute significantly to intracortical processing.     

From the Cover: Seasonal plasticity in the adult somatosensory cortex

Seasonal cycles govern life on earth, from setting the time for the mating season to influencing migrations and governing physiological conditions like hibernation. The effect of such changing conditions on behavior is well-appreciated, but their impact on the brain remains virtually unknown. We investigate long-term seasonal changes in the mammalian brain, known as Dehnel’s effect, where animals exhibit plasticity in body and brain sizes to counter metabolic demands in winter. We find large seasonal variation in cellular architecture and neuronal activity in the smallest terrestrial mammal, the Etruscan shrew, Suncus etruscus. Their brain, and specifically their neocortex, shrinks in winter. Shrews are tactile hunters, and information from whiskers first reaches the somatosensory cortex layer 4, which exhibits a reduced width (−28%) in winter. Layer 4 width (+29%) and neuron number (+42%) increase the following summer. Activity patterns in the somatosensory cortex show a prominent reduction of touch-suppressed neurons in layer 4 (−55%), the most metabolically active layer. Loss of inhibitory gating occurs with a reduction in parvalbumin-positive interneurons, one of the most active neuronal subtypes and the main regulators of inhibition in layer 4. Thus, a reduction in neurons in layer 4 and particularly parvalbumin-positive interneurons may incur direct metabolic benefits. However, changes in cortical balance can also affect the threshold for detecting sensory stimuli and impact prey choice, as observed in wild shrews. Thus, seasonal neural adaptation can offer synergistic metabolic and behavioral benefits to the organism and offer insights on how neural systems show adaptive plasticity in response to ecological demands.

Animals have evolved to display extraordinary ethological adaptations in response to the ecological variations they face. Monarch butterflies perform annual migration cycles, each of which is completed over several generations (1), while squirrels can hibernate for several months (2). The primary organ responsible for regulating behaviors, the brain, also exhibits the ability to change: In response to environmental changes, behavioral needs, injury, or to form new memories (3–6). While the consequences of such neural plasticity have been studied at the synaptic level, minute changes in neuronal and synaptic activity over short time-scales, the impact of longer-term behavioral variations on neural structure and activity is largely unknown. Notable exceptions are songbirds that display seasonal variation in song repertoire and correlated anatomical changes in song nuclei (7). Although seasonal brain plasticity has mostly been studied in birds, mammalian brains, including humans (8), also display such effects. However, the evolutionary relations between key bird and mammalian brain regions are disputed (9, 10). Some of the most drastic yet largely unexplored seasonal changes in brain structure have been observed in small mammals, like shrews and weasels (11, 12). This phenomenon is known as Dehnel’s effect and entails a reduction in body weight, skull, and brain size during autumn and winter (11–15). We explore this effect in Etruscan shrews and find that individual shrews exhibit seasonal changes in brain size, with the cerebral cortex shrinking in winter. We then determine the microanatomical substrate of such cortical volume changes and report evidence of seasonal changes in neural activity in the cerebral cortex.     

The RNA binding protein CPEB regulates dendrite morphogenesis and neuronal circuit assembly in vivo

Visual system development requires experience-dependent mechanisms that regulate neuronal structure and function, including dendritic arbor growth, synapse formation, and stabilization. Although RNA binding proteins have been shown to affect some forms of synaptic plasticity in adult animals, their role in the development of neuronal structure and functional circuitry is not clear. Using two-photon time-lapse in vivo imaging and electrophysiology combined with morpholino-mediated knockdown and expression of functional deletion mutants, we demonstrate that the mRNA binding protein, cytoplasmic polyadenylation element binding protein1 (CPEB1), affects experience-dependent neuronal development and circuit formation in the visual system of Xenopus laevis. These data indicate that sensory experience controls circuit development by regulating translational activity of mRNAs.     

Overexpression of calcium-activated potassium channels underlies cortical dysfunction in a model of PTEN-associated autism

De novo phosphatase and tensin homolog on chromosome ten (PTEN) mutations are a cause of sporadic autism. How single-copy loss of PTEN alters neural function is not understood. Here we report that Pten haploinsufficiency increases the expression of small-conductance calcium-activated potassium channels. The resultant augmentation of this conductance increases the amplitude of the afterspike hyperpolarization, causing a decrease in intrinsic excitability. In vivo, this change in intrinsic excitability reduces evoked firing rates of cortical pyramidal neurons but does not alter receptive field tuning. The decreased in vivo firing rate is not associated with deficits in the dendritic integration of synaptic input or with changes in dendritic complexity. These findings identify calcium-activated potassium channelopathy as a cause of cortical dysfunction in the PTEN model of autism and provide potential molecular therapeutic targets.Children with inherited or de novo mutations in a single copy of phosphatase and tensin homolog on chromosome ten (PTEN) show symptoms of autism, macrocephaly, mental retardation, and epilepsy (1–3), and de novo PTEN mutations are one of the most validated causes of autism (4). This interest has led to the development of a number of lines of mutant mice with conditional homozygous Pten deletion in forebrain neurons. These mice are macrocephalic and epileptic and display abnormal social interactions and exaggerated responses to sensory stimuli (5, 6). Because these mice display behavioral deficits that are also seen in autistic children, a number of studies have examined cortical connectivity and synaptic strength following Pten gene deletion and found enlarged neural somata, overgrown dendrites, and hyperconnected neural networks (5–8). These studies and studies in other mouse lines bearing mutations found in autistic individuals (9, 10) identify deficits in synaptic strength and number as the major etiology of abnormal circuit function in autism.Alternatively, or in addition, cortical dysfunction in autism may be driven by changes in the normal expression of voltage-sensitive and/or calcium-activated ion conductances, which would alter neural excitability. Supporting this view, calcium channelopathy causes Timothy syndrome where 80% of individuals surviving past the age of three are autistic (11). Common and rare mutations in CNTNAP2, the protein product of which clusters voltage-sensitive potassium channels at the juxtaparanode of axons, are also strongly associated with autism (12–14), as are mutations in SCN2A (15), which codes the α-subunit of a voltage-gated sodium channel.The AKT/mTOR-signaling pathway that is regulated by PTEN regulates the translation Kv1.1 voltage-gated potassium channels (16). Whether Pten mutation impacts the expression of other ion channels that regulate neural excitability is not known. Nor is it understood how Pten mutation impacts the processing of sensory information in vivo. Sensory-processing deficits are found in 80–90% of autistic individuals and may underlie some of the behavioral problems associated with autism.Here we focus our studies on mice lacking one copy of Pten because heterozygous mutation more accurately models the human disease, whereas double-copy loss is lethal. Using two-photon imaging to target cell-attached and whole-cell recordings to layer 2/3 (L2/3) pyramidal neurons in the primary visual cortex, we find that single-copy loss reduces stimulus-evoked firing rates by half of their control values but has no impact on the selectivity of evoked firing to stimulus orientation and direction. Notably, the amplitude of subthreshold voltage responses to optimal visual stimuli was not different from controls. The reduction in neural firing is caused by increased expression of small-conductance calcium-activated potassium (SK_Ca) channels, which limit the firing frequency of neurons by regulating the action potential after hyperpolarization.These results identify an unsuspected mechanism by which PTEN regulates cortical gain control in vivo, thereby altering sensory processing.     

Metabotropic glutamate receptor signaling is required for NMDA receptor-dependent ocular dominance plasticity and LTD in visual cortex

A feature of early postnatal neocortical development is a transient peak in signaling via metabotropic glutamate receptor 5 (mGluR5). In visual cortex, this change coincides with increased sensitivity of excitatory synapses to monocular deprivation (MD). However, loss of visual responsiveness after MD occurs via mechanisms revealed by the study of long-term depression (LTD) of synaptic transmission, which in layer 4 is induced by acute activation of NMDA receptors (NMDARs) rather than mGluR5. Here we report that chronic postnatal down-regulation of mGluR5 signaling produces coordinated impairments in both NMDAR-dependent LTD in vitro and ocular dominance plasticity in vivo. The data suggest that ongoing mGluR5 signaling during a critical period of postnatal development establishes the biochemical conditions that are permissive for activity-dependent sculpting of excitatory synapses via the mechanism of NMDAR-dependent LTD.Temporary monocular deprivation (MD) sets in motion synaptic changes in visual cortex that result in impaired vision through the deprived eye. The primary cause of visual impairment is depression of excitatory thalamocortical synaptic transmission in layer 4 of visual cortex (1–3). The study of long-term depression (LTD) of synapses, elicited in vitro by electrical or chemical stimulation, has revealed many of the mechanisms involved in deprived-eye depression (4). In slices of visual cortex, LTD in layer 4 is induced by NMDA receptor (NMDAR) activation and expressed by posttranslational modification and internalization of AMPA receptors (AMPARs) (5, 6). MD induces identical NMDAR-dependent changes in AMPARs, and synaptic depression induced by deprivation in vivo occludes LTD in visual cortex ex vivo (6–8). Manipulations of NMDARs and AMPAR trafficking that interfere with LTD also prevent the effects of MD (7, 9–11).Although NMDAR-dependent LTD is widely expressed in the brain (12, 13), it is now understood that different circuits use different mechanisms for long-term homosynaptic depression (14). For example, in the CA1 region of hippocampus, synaptic activation of either NMDARs or metabotropic glutamate receptor 5 (mGluR5) induces LTD. In both cases, depression is expressed postsynaptically as a reduction in AMPARs, but these forms of LTD are not mutually occluding and have distinct signaling requirements (15). A defining feature of mGluR5-dependent postsynaptic LTD in CA1 is a requirement for the immediate translation of synaptic mRNAs (16). In visual cortex, there is evidence that induction of LTD in layers 2–4 requires NMDAR activation, whereas induction of LTD in layer 6 requires activation of mGluR5 (17, 18).The hypothesis that mGluRs, in addition to NMDARs, play a key role in visual cortical plasticity can be traced back more than 25 y to observations that glutamate-stimulated phosphoinositide turnover, mediated in visual cortex by mGluR5 coupled to phospholipase C, is elevated during the postnatal period of heightened sensitivity to MD (19). Early attempts to test this hypothesis were inconclusive owing to the use of weak and nonselective orthosteric compounds (20–22); however, subsequent experiments did confirm that NMDAR-dependent LTD occurs normally in layers 2/3 of visual cortex in Grm5 knockout mice (23).The idea that mGluR5 is critically involved in visual cortical plasticity in vivo was rekindled with the finding that deprived-eye depression fails to occur in layer 4 of Grm5^+/− mutant mice (24). This finding was unexpected because, as reviewed above, a considerable body of evidence has implicated the mechanism of NMDAR-dependent LTD in deprived-eye depression. In the present study, we reexamined the role of mGluR5 in LTD and ocular dominance plasticity in layer 4, using the Grm5^+/− mouse and a highly specific negative allosteric modulator, 2-chloro-4-((2,5-dimethyl-1-(4-(trifluoromethoxy)phenyl)-1H-imidazol-4-yl)ethynyl)pyridine (CTEP), that has proven suitable for chronic inhibition of mGluR5 (25, 26). Our data show that NMDAR-dependent LTD and deprived-eye depression in layer 4 require mGluR5 signaling during postnatal development.     

Prelimbic cortical BDNF is required for memory of learned fear but not extinction or innate fear

In the medial prefrontal cortex, the prelimbic area is emerging as a major modulator of fear behavior, but the mechanisms remain unclear. Using a selective neocortical knockout mouse, virally mediated prelimbic cortical-specific gene deletion, and pharmacological rescue with a TrkB agonist, we examined the role of a primary candidate mechanism, BDNF, in conditioned fear. We found consistently robust deficits in consolidation of cued fear but no effects on acquisition, expression of unlearned fear, sensorimotor function, and spatial learning. This deficit in learned fear in the BDNF knockout mice was rescued with systemic administration of a TrkB receptor agonist, 7,8-dihydroxyflavone. These data indicate that prelimbic BDNF is critical for consolidation of learned fear memories, but it is not required for innate fear or extinction of fear. Moreover, use of site-specific, inducible BDNF deletions shows a powerful mechanism that may further our understanding of the pathophysiology of fear-related disorders.     

Progressive maturation of silent synapses governs the duration of a critical period

During critical periods, all cortical neural circuits are refined to optimize their functional properties. The prevailing notion is that the balance between excitation and inhibition determines the onset and closure of critical periods. In contrast, we show that maturation of silent glutamatergic synapses onto principal neurons was sufficient to govern the duration of the critical period for ocular dominance plasticity in the visual cortex of mice. Specifically, postsynaptic density protein-95 (PSD-95) was absolutely required for experience-dependent maturation of silent synapses, and its absence before the onset of critical periods resulted in lifelong juvenile ocular dominance plasticity. Loss of PSD-95 in the visual cortex after the closure of the critical period reinstated silent synapses, resulting in reopening of juvenile-like ocular dominance plasticity. Additionally, silent synapse-based ocular dominance plasticity was largely independent of the inhibitory tone, whose developmental maturation was independent of PSD-95. Moreover, glutamatergic synaptic transmission onto parvalbumin-positive interneurons was unaltered in PSD-95 KO mice. These findings reveal not only that PSD-95–dependent silent synapse maturation in visual cortical principal neurons terminates the critical period for ocular dominance plasticity but also indicate that, in general, once silent synapses are consolidated in any neural circuit, initial experience-dependent functional optimization and critical periods end.Immature cortical neural networks, which are formed primarily under genetic control (1), require experience and training to shape and optimize their functional properties. This experience-dependent refinement is considered to be a general developmental process for all functional cortical domains and typically peaks during their respective critical periods (CPs) (2, 3). Known examples for CPs span functional domains as diverse as filial imprinting and courtship song learning in birds (4, 5); cognitive functions, such as linguistic or musical skills in humans (6, 7); and likely best studied, the different features of sensory modalities (3). CPs are characterized by the absolute requirement for experience in a restricted time window for neural network optimization. Lack of visual experience during the CP for visual cortex refinements can, for example, cause irreversible visual impairment (8). Refinements during the CP play an essential role (9). Although some functions can be substantially ameliorated after the CP, they are rarely optimally restored.It is believed that the neural network refinement is based on synapse stabilization and elimination (10–12) and includes forms of long-term synaptic plasticity to remodel excitatory synapses of principal neurons (13, 14). Although long-term plasticity at these excitatory synapses is instructive for shaping neural networks for functional output and their expression coincides with CPs, it is not known whether the remodeling itself governs the duration of CPs. In contrast, only permissive mechanisms have been shown to terminate CPs. Among these, the developmental increase of local inhibition appears to be the dominating mechanism to regulate cortical plasticity and CPs (15–17). Additionally, extracellular matrix remodeling is involved, as well as receptors of immune signaling, such as paired Ig-like receptor B (PirB), or axon pathfinding, such as Nogo (18–21). However, a specific function to directly regulate synapse remodeling during initial neural network optimization is not known and a potential instructive function of PirB was described for adult cortical plasticity but not plasticity of the initial synapse remodeling during CPs (22).AMPA receptor-silent synapses have been proposed to be efficient plasticity substrates during early cortical network refinements (13, 23, 24). Silent synapses are thought to be immature, still-developing excitatory synapses containing only NMDA receptors (NMDARs) but lacking AMPA receptors (AMPARs) (23, 24). They are functionally dormant but can evolve into fully transmitting synapses by experience-dependent insertion of AMPARs, a plasticity process thought to occur frequently in developing cortices (10). Although they appear as the ideal synaptic substrate for CP plasticity and their maturation correlates with sensory experience (10, 25), it has not been experimentally tested whether maturation of silent synapses indeed causes the termination of critical periods. This conceptual model contrasts with the current view that increased local inhibition and the expression of plasticity brakes ends critical periods (18–20, 26). We hypothesize that experience-dependent unsilencing of silent synapses, which results in strengthening and maturation of excitatory synapses, governs network stabilization and refinement during critical periods, and that the progressive decrease of silent synapses leads to the closure of critical periods.Experience-dependent cortical plasticity is classically tested with ocular dominance (OD) plasticity (ODP) in the primary visual cortex (V1), induced by monocular deprivation (MD). In the binocular region of mouse V1, neurons respond to sensory inputs from both eyes, but activity is dominated by afferents from the contralateral eye. During the critical period, a brief MD induces an OD shift of visually evoked responses in V1 toward the open eye (27–29). This juvenile ODP is mediated by a reduction of deprived eye responses in V1 and is temporally confined to a critical period (30, 31).A molecular candidate regulating the cellular basis of critical period plasticity is postsynaptic density protein-95 (PSD-95), whose expression in the visual cortex increases on eye opening and thus the onset of visual experience (32). PSD-95 promotes the maturation of AMPA receptor-silent excitatory synapses in hippocampal neurons and is required for activity-driven synapse stabilization (33–35). In juvenile PSD-95 KO mice, ODP displays the same features as in WT mice (36). However, as adult PSD-95 KO mice have not yet been analyzed, it is unknown whether PSD-95 is essential for the closure of critical periods. Thus, PSD-95 appeared to be the ideal molecular candidate to test our conceptual model that progressive silent synapse maturation marks the closure of critical periods.     

From the Cover: Successive-signal biasing for a learned sound sequence

Adult rats were trained to detect the occurrence of a two-element sound sequence in a background of nine other nontarget sound pairs. Training resulted in a modest, enduring, static expansion of the cortical areas of representation of both target stimulus sounds. More importantly, once the initial stimulus A in the target A-B sequence was presented, the cortical “map” changed dynamically, specifically to exaggerate further the representation of the “anticipated” stimulus B. If B occurred, it was represented over a larger cortical area by more strongly excited, more coordinated, and more selectively responding neurons. This biasing peaked at the expected time of B onset with respect to A onset. No dynamic biasing of responses was recorded for any sound presented in a nontarget pair. Responses to nontarget frequencies flanking the representation of B were reduced in area and in response strength only after the presentation of A at the expected time of B onset. This study shows that cortical areas are not representationally static but, to the contrary, can be biased moment by moment in time as a function of behavioral context.