Fast-spiking cells (FS cells) are a prominent subtype of neocortical GABAergic interneurons with important functional roles. Multiple FS cell properties are coordinated for rapid response. Here, we describe an FS cell feature that serves to gate the powerful inhibition produced by FS cell activity. We show that FS cells in layer 2/3 barrel cortex possess a dampening mechanism mediated by Kv1.1-containing potassium channels localized to the axon initial segment. These channels powerfully regulate action potential threshold and allow FS cells to respond preferentially to large inputs that are fast enough to "outrun" Kv1 activation. In addition, Kv1.1 channel blockade converts the delay-type discharge pattern of FS cells to one of continuous fast spiking without influencing the high-frequency firing that defines FS cells. Thus, Kv1 channels provide a key counterbalance to the established rapid-response characteristics of FS cells, regulating excitability through a unique combination of electrophysiological properties and discrete subcellular localization.

A vesicular glutamate transporter, VGluT2, has been suggested to be the transporter utilized in the thalamocortical pathway. We examined the reliability of this marker in identifying and discriminating thalamic terminals in adult and developing ferret visual cortex. We studied brain sections stained for the transporter protein and/or anterogradely filled thalamocortical or intracortical axons, by using light, confocal, and electron microscopy. Under light microscopy, VGluT2 immunoreactivity (ir) in adult animals [past postnatal day (P)90] and in neonatal animals as early as P27 formed a dense band in layer 4 and appeared as scattered puncta in layers 6 and 1. Confocal dual-labeling analyses of P46 and adult striate cortices indicated that VGluT2 was present in thalamocortical axons, suggesting that thalamic projections utilize this transporter during postnatal development as well as adulthood. In contrast, extracellularly filled intracortical axons failed to colocalize with VGluT2-ir, suggesting that no significant terminal population originating in cortex contained VGluT2 in layer 4. Electron microscopic analysis revealed that, in adult layer 4, VGluT2-ir was present in large terminals, forming asymmetric synapses. Similar to anterogradely labeled thalamocortical terminals, VGluT2-ir synaptic terminals were different from their unlabeled counterparts in terms of terminal area (0.6 vs. 0.3 microm), synaptic length (486 vs. 353 nm), and preference for synapsing on spines (77% vs. 59%). Moreover, no significant differences were found between VGluT2-ir and anterogradely labeled thalamocortical terminals. Comparable similarities were also demonstrated at P46. These results indicate that thalamocortical terminals in layer 4 of visual cortex utilize VGluT2 and suggest that this marker can be used to identify thalamic axons specifically in adult and developing animals.

Monocular lid closure (MC) causes a profound shift in the ocular dominance (OD) of neurons in primary visual cortex (V1). Anatomical studies in both cat and mouse V1 suggest that large-scale structural rearrangements of eye-specific thalamocortical (TC) axons in response to MC occur much more slowly than the shift in OD. Consequently, there has been considerable debate as to whether the plasticity of TC synapses, which transmit competing visual information from each eye to V1, contributes to the early functional consequences of MC or is simply a feature of long-term deprivation. Here, we used quantitative immuno-electron microscopy to examine the possibility that alterations of TC synapses occur rapidly enough to impact OD after brief MC. The effect of short-term deprivation on TC synaptic structure was examined in male C57BL/6 mice that underwent 3 and 7 d of MC or monocular retinal inactivation (MI) with tetrodotoxin. The data show that 3 d of MC is sufficient to induce substantial remodeling of TC synapses. In contrast, 3 d of MI, which alters TC activity but does not shift OD, does not significantly affect the structure of TC synapses. Our results support the hypothesis that the rapid plasticity of TC synapses is a key step in the sequence of events that shift OD in visual cortex.

Synaptic scaling is a form of homeostatic plasticity that stabilizes neuronal firing in response to changes in synapse number and strength. Scaling up in response to action-potential blockade is accomplished through increased synaptic accumulation of GluA2-containing AMPA receptors (AMPAR), but the receptor trafficking steps that drive this process remain largely obscure. Here, we show that the AMPAR-binding protein glutamate receptor-interacting protein-1 (GRIP1) is essential for regulated synaptic AMPAR accumulation during scaling up. Synaptic abundance of GRIP1 was enhanced by activity deprivation, directly increasing synaptic GRIP1 abundance through overexpression increased the amplitude of AMPA miniature excitatory postsynaptic currents (mEPSCs), and shRNA-mediated GRIP1 knockdown prevented scaling up of AMPA mEPSCs. Furthermore, knockdown and replace experiments targeting either GRIP1 or GluA2 revealed that scaling up requires the interaction between GRIP1 and GluA2. Finally, GRIP1 synaptic accumulation during scaling up did not require GluA2 binding. Taken together, our data support a model in which activity-dependent trafficking of GRIP1 to synaptic sites drives the forward trafficking and enhanced synaptic accumulation of GluA2-containing AMPAR during synaptic scaling up.P roper development of neuronal circuits, as well as efficient information storage during learning and memory, are thought to depend upon the presence of homeostatic mechanisms that stabilize neuronal excitability (1-3). One such mechanism is synaptic scaling, which compensates for perturbations in average firing by scaling up or down the postsynaptic strength of all of a neuron's excitatory synapses (4). Synaptic scaling is a cell-autonomous process in which neurons detect changes in their own firing through a set of calcium-dependent sensors that then regulate receptor trafficking to increase or decrease the accumulation of AMPA receptors (AMPAR) at synaptic sites and thus increase or decrease synaptic strength (4-6). Despite great recent interest, the AMPA receptor-trafficking events that underlie synaptic scaling remain largely obscure. Defects in synaptic scaling have been postulated to contribute to disorders as diverse as Alzheimer's disease (7) and epilepsy (8), so illuminating the underlying AMPAR trafficking steps could shed light into the genesis of a wide range of neurological disorders.Most neocortical AMPAR are heteromeric receptors composed of both GluA1 and GluA2 subunits, which have unique phosphorylation sites and interact with distinct trafficking proteins (9). During synaptic scaling up in response to action potential blockade, synaptic strength is increased through enhanced synaptic accumulation of GluA1 and GluA2-containing AMPAR (5, 10-13) and requires the C-terminal domain of the GluA2 subunit (12), but which subunit-specific interactions underlie synaptic scaling remain controversial (12,14). Several trafficking proteins are known to interact with the GluA2, but not the GluA1, C-tail, including glutamate recepto...

Summary In visual cortex monocular deprivation (MD) during a critical period (CP) reduces the ability of the deprived eye to activate cortex, but the underlying cellular plasticity mechanisms are incompletely understood. Here we show that MD reduces the intrinsic excitability of layer 5 (L5) pyramidal neurons and enhances long-term potentiation of intrinsic excitability (LTP-IE). Further, MD and LTP-IE induce reciprocal changes in Kv2.1 current, and LTP-IE reverses the effects of MD on intrinsic excitability. Taken together these data suggest that MD reduces intrinsic excitability by preventing sensory-drive induced LTP-IE. The effects of MD on excitability were correlated with the classical visual system CP, and (like the functional effects of MD) could be rapidly reversed when vision was restored. These data establish LTP-IE as a novel candidate mechanism mediating loss of visual responsiveness within L5, and suggest that intrinsic plasticity plays an important role in experience-dependent refinement of visual cortical circuits.

A primary goal of research on developmental critical periods is the recapitulation of a juvenile-like state of malleability in the adult brain that might enable recovery from injury. These ambitions are often framed in terms of the simple reinstatement of enhanced plasticity in the growth-restricted milieu of an injured adult brain. Here, we provide an analysis of the similarities and differences between deprivation-induced and injury-induced cortical plasticity, to provide for a nuanced comparison of these remarkably similar processes. As a first step, we review the factors that drive ocular dominance plasticity in the primary visual cortex of the uninjured brain during the critical period (CP) and in adults, to highlight processes that might confer adaptive advantage. In addition, we directly compare deprivation-induced cortical plasticity during the CP and plasticity following acute injury or ischemia in mature brain. We find that these two processes display a biphasic response profile following deprivation or injury: an initial decrease in GABAergic inhibition and synapse loss transitions into a period of neurite expansion and synaptic gain. This biphasic response profile emphasizes the transition from a period of cortical healing to one of reconnection and recovery of function. Yet while injury-induced plasticity in adult shares several salient characteristics with deprivation-induced plasticity during the CP, the degree to which the adult injured brain is able to functionally rewire, and the time required to do so, present major limitations for recovery. Attempts to recapitulate a measure of CP plasticity in an adult injury context will need to carefully dissect the circuit alterations and plasticity mechanisms involved while measuring functional behavioral output to assess their ultimate success.

No abstract

Superresolution microscopy has fundamentally altered our ability to resolve subcellular proteins, but improving on these techniques to study dense structures composed of single-molecule-sized elements has been a challenge. One possible approach to enhance superresolution precision is to use cryogenic fluorescent imaging, reported to reduce fluorescent protein bleaching rates, thereby increasing the precision of superresolution imaging. Here, we describe an approach to cryogenic photoactivated localization microscopy (cPALM) that permits the use of a room-temperature high-numerical-aperture objective lens to image frozen samples in their native state. We find that cPALM increases photon yields and show that this approach can be used to enhance the effective resolution of two photoactivatable/switchable fluorophore-labeled structures in the same frozen sample. This higher resolution, two-color extension of the cPALM technique will expand the accessibility of this approach to a range of laboratories interested in more precise reconstructions of complex subcellular targets.