The nonclassical MHC class I Qa-1 expressed in layer 6 neurons regulates activity-dependent plasticity via microglial CD94/NKG2 in the cortex.

During developmental critical periods, circuits are sculpted by a process of activity-dependent competition. The molecular machinery involved in regulating the complex process of responding to different levels of activity is now beginning to be identified. Here, we show that the nonclassical major histocompatibility class I (MHCI) molecule Qa-1 is expressed in the healthy brain in layer 6 corticothalamic neurons. In the visual cortex, Qa-1 expression begins during the critical period for ocular dominance (OD) plasticity and is regulated by neuronal activity, suggesting a role in regulating activity-dependent competition. Indeed, in mice lacking Qa-1, OD plasticity is perturbed. Moreover, signaling through CD94/NKG2, a known cognate Qa-1 heterodimeric receptor in the immune system, is implicated: selectively targeting this interaction phenocopies the plasticity perturbation observed in Qa-1 knockouts. In the cortex, CD94/NKG2 is expressed by microglial cells, which undergo activity-dependent changes in their morphology in a Qa-1­dependent manner. Our study thus reveals a neuron­microglial interaction dependent upon a nonclassical MHCI molecule expressed in L6 neurons, which regulates plasticity in the visual cortex. These results also point to an unexpected function for the Qa-1/HLA-E (ligand) and CD94/NKG2 (receptor) interaction in the nervous system, in addition to that described in the immune system.

Activity-dependent modulation of hippocampal synaptic plasticity via PirB and endocannabinoids.

The threshold for Hebbian synaptic plasticity in the CNS is modulated by prior synaptic activity. At adult CA3-CA1 synapses, endocannabinoids play a role in this process, but how activity engages and maintains this retrograde signaling system is not well understood. Here we show that conditional deletion of Paired Immunoglobulin-like receptor B (PirB) from pyramidal neurons in adult mouse hippocampus results in deficient LTD at CA3-CA1 synapses over a range of stimulation frequencies, accompanied by an increase in LTP. This finding can be fully explained by the disengagement of retrograde endocannabinoid signaling selectively at excitatory synapses. In the absence of PirB, the NMDAR-dependent regulation of endocannabinoid signaling is lost, while CB1R-dependent and group I mGluR-dependent regulation are intact. Moreover, mEPSC frequency in mutant CA1 pyramidal cells is elevated, consistent with a higher density of excitatory synapses and altered synapse pruning. Mice lacking PirB also perform better than WT in learning and memory tasks. These observations suggest that PirB is an integral part of an NMDA receptor-mediated synaptic mechanism that maintains bidirectional Hebbian plasticity and learning via activity-dependent endocannabinoid signaling.

Synapse elimination and learning rules co-regulated by MHC class I H2-Db.

The formation of precise connections between retina and lateral geniculate nucleus (LGN) involves the activity-dependent elimination of some synapses, with strengthening and retention of others. Here we show that the major histocompatibility complex (MHC) class I molecule H2-D(b) is necessary and sufficient for synapse elimination in the retinogeniculate system. In mice lacking both H2-K(b) and H2-D(b) (K(b)D(b)(-/-)), despite intact retinal activity and basal synaptic transmission, the developmentally regulated decrease in functional convergence of retinal ganglion cell synaptic inputs to LGN neurons fails and eye-specific layers do not form. Neuronal expression of just H2-D(b) in K(b)D(b)(-/-) mice rescues both synapse elimination and eye-specific segregation despite a compromised immune system. When patterns of stimulation mimicking endogenous retinal waves are used to probe synaptic learning rules at retinogeniculate synapses, long-term potentiation (LTP) is intact but long-term depression (LTD) is impaired in K(b)D(b)(-/-) mice. This change is due to an increase in Ca(2+)-permeable AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors. Restoring H2-D(b) to K(b)D(b)(-/-) neurons renders AMPA receptors Ca(2+) impermeable and rescues LTD. These observations reveal an MHC-class-I-mediated link between developmental synapse pruning and balanced synaptic learning rules enabling both LTD and LTP, and demonstrate a direct requirement for H2-D(b) in functional and structural synapse pruning in CNS neurons.

Developmental Sculpting of Intracortical Circuits by MHC Class I H2-Db and H2-Kb.

Synapse pruning is an activity-regulated process needed for proper circuit sculpting in the developing brain. Major histocompatibility class I (MHCI) molecules are regulated by activity, but little is known about their role in the development of connectivity in cortex. Here we show that protein for 2 MHCI molecules H2-Kb and H2-Db is associated with synapses in the visual cortex. Pyramidal neurons in mice lacking H2-Kb and H2-Db (KbDb KO) have more extensive cortical connectivity than normal. Modified rabies virus tracing was used to monitor the extent of pyramidal cell connectivity: Horizontal connectivity is greater in the visual cortex of KbDb KO mice. Basal dendrites of L2/3 pyramids, where many horizontal connections terminate, are more highly branched and have elevated spine density in the KO. Furthermore, the density of axonal boutons is elevated within L2/3 of mutant mice. These increases are accompanied by elevated miniature excitatory postsynaptic current frequency, consistent with an increase in functional synapses. This functional and anatomical increase in intracortical connectivity is also associated with enhanced ocular dominance plasticity that persists into adulthood. Thus, these MHCI proteins regulate sculpting of local cortical circuits and in their absence, the excess connectivity can function as a substrate for cortical plasticity throughout life.

PirB regulates a structural substrate for cortical plasticity.

Experience-driven circuit changes underlie learning and memory. Monocular deprivation (MD) engages synaptic mechanisms of ocular dominance (OD) plasticity and generates robust increases in dendritic spine density on L5 pyramidal neurons. Here we show that the paired immunoglobulin-like receptor B (PirB) negatively regulates spine density, as well as the threshold for adult OD plasticity. In PirB(-/-) mice, spine density and stability are significantly greater than WT, associated with higher-frequency miniature synaptic currents, larger long-term potentiation, and deficient long-term depression. Although MD generates the expected increase in spine density in WT, in PirB(-/-) this increase is occluded. In adult PirB(-/-), OD plasticity is larger and more rapid than in WT, consistent with the maintenance of elevated spine density. Thus, PirB normally regulates spine and excitatory synapse density and consequently the threshold for new learning throughout life.

Synaptic plasticity defect following visual deprivation in Alzheimer's disease model transgenic mice.

Amyloid-ß (Aß)-induced changes in synaptic function in experimental models of Alzheimer's disease (AD) suggest that Aß generation and accumulation may affect fundamental mechanisms of synaptic plasticity. To test this hypothesis, we examined the effect of APP overexpression on a well characterized, in vivo, developmental model of systems-level plasticity, ocular dominance plasticity. Following monocular visual deprivation during the critical period, mice that express mutant alleles of amyloid precursor protein (APPswe) and Presenilin1 (PS1dE9), as well as mice that express APPswe alone, lack ocular dominance plasticity in visual cortex. Defects in the spatial extent and magnitude of the plastic response are evident using two complementary approaches, Arc induction and optical imaging of intrinsic signals in awake mice. This defect in a classic paradigm of systems level synaptic plasticity shows that Aß overexpression, even early in postnatal life, can perturb plasticity in cerebral cortex, and supports the idea that decreased synaptic plasticity due to elevated Aß exposure contributes to cognitive impairment in AD.

H2-K(b) and H2-D(b) regulate cerebellar long-term depression and limit motor learning.

There are more than 50 class I MHC (MHCI) molecules in the mouse genome, some of which are now known to be expressed in neurons; however, the role of classical MHCI molecules in synaptic plasticity is unknown. We report that the classical MHCI molecules, H2-K(b) and H2-D(b), are co-expressed by Purkinje cells (PCs). In the cerebellum of mice deficient for both H2-K(b) and H2-D(b) (K(b)D(b-/-)), there is a lower threshold for induction of long-term depression (LTD) at parallel fiber to PC synapses. This change may be a result of additional glutamate release observed at K(b)D(b-/-) CF to PC synapses, which are thought to "train" the cerebellar circuit. A behavioral correlate of cerebellar LTD is motor learning; acquisition and retention of a Rotarod behavioral task is significantly better in K(b)D(b-/-) mice than in WT cohorts. These physiological and behavioral phenotypes in K(b)D(b-/-) mice reveal a surprising role for classical MHCI molecules in synaptic plasticity and motor learning.

Enhancing motor learning by increasing the stability of newly formed dendritic spines in the motor cortex.

Dendritic spine dynamics are thought to be substrates for motor learning and memory, and altered spine dynamics often lead to impaired performance. Here, we describe an exception to this rule by studying mice lacking paired immunoglobulin receptor B (PirB-/-). Pyramidal neuron dendrites in PirB-/- mice have increased spine formation rates and density. Surprisingly, PirB-/- mice learn a skilled reaching task faster than wild-type (WT) littermates. Furthermore, stabilization of learning-induced spines is elevated in PirB-/- mice. Mechanistically, single-spine uncaging experiments suggest that PirB is required for NMDA receptor (NMDAR)-dependent spine shrinkage. The degree of survival of newly formed spines correlates with performance, suggesting that increased spine stability is advantageous for learning. Acute inhibition of PirB function in M1 of adult WT mice increases the survival of learning-induced spines and enhances motor learning. These results demonstrate that there are limits on motor learning that can be lifted by manipulating PirB, even in adulthood.

Subplate neurons regulate maturation of cortical inhibition and outcome of ocular dominance plasticity.

Synaptic plasticity during critical periods of development requires intact inhibitory circuitry. We report that subplate neurons are needed both for maturation of inhibition and for the proper sign of ocular dominance (OD) plasticity. Removal of subplate neurons prevents the developmental upregulation of genes involved in mature, fast GABAergic transmission in cortical layer 4, including GABA receptor subunits and KCC2, and thus prevents the switch to a hyperpolarizing effect of GABA. To understand the implications of these changes, a realistic circuit model was formulated. Simulations predicted that without subplate neurons, monocular deprivation (MD) paradoxically favors LGN axons representing the deprived (less active) eye, exactly what was then observed experimentally. Simulations also account for published results showing that OD plasticity requires mature inhibition. Thus, subplate neurons regulate molecular machinery required to establish an adult balance of excitation and inhibition in layer 4, and thereby influence the outcome of OD plasticity.

A burst-based "Hebbian" learning rule at retinogeniculate synapses links retinal waves to activity-dependent refinement.

Patterned spontaneous activity in the developing retina is necessary to drive synaptic refinement in the lateral geniculate nucleus (LGN). Using perforated patch recordings from neurons in LGN slices during the period of eye segregation, we examine how such burst-based activity can instruct this refinement. Retinogeniculate synapses have a novel learning rule that depends on the latencies between pre- and postsynaptic bursts on the order of one second: coincident bursts produce long-lasting synaptic enhancement, whereas non-overlapping bursts produce mild synaptic weakening. It is consistent with "Hebbian" development thought to exist at this synapse, and we demonstrate computationally that such a rule can robustly use retinal waves to drive eye segregation and retinotopic refinement. Thus, by measuring plasticity induced by natural activity patterns, synaptic learning rules can be linked directly to their larger role in instructing the patterning of neural connectivity.

Synaptogenesis in purified cortical subplate neurons.

An ideal preparation for investigating events during synaptogenesis would be one in which synapses are sparse, but can be induced at will using a rapid, exogenous trigger. We describe a culture system of immunopurified subplate neurons in which synaptogenesis can be triggered, providing the first homogeneous culture of neocortical neurons for the investigation of synapse development. Synapses in immunopurified rat subplate neurons are sparse, and can be induced by a 48-h exposure to feeder layers of neurons and glia, an induction more rapid than any previously reported. Induced synapses are electrophysiologically functional and ultrastructurally normal. Microarray and real-time PCR experiments reveal a new program of gene expression accompanying synaptogenesis. Surprisingly few known synaptic genes are upregulated during the first 24 h of synaptogenesis; Gene Ontology annotation reveals a preferential upregulation of synaptic genes only at a later time. In situ hybridization confirms that some of the genes regulated in cultures are also expressed in the developing cortex. This culture system provides both a means of studying synapse formation in a homogeneous population of cortical neurons, and better synchronization of synaptogenesis, permitting the investigation of neuron-wide events following the triggering of synapse formation.

Effects of visual experience on activity-dependent gene regulation in cortex.

There are critical periods in development when sensory experience directs the maturation of synapses and circuits within neocortex. We report that the critical period in mouse visual cortex has a specific molecular logic of gene regulation. Four days of visual deprivation regulated one set of genes during the critical period, and different sets before or after. Dark rearing perturbed the regulation of these age-specific gene sets. In addition, a 'common gene set', comprised of target genes belonging to a mitogen-activated protein (MAP) kinase signaling pathway, was regulated by vision at all ages but was impervious to prior history of sensory experience. Together, our results demonstrate that vision has dual effects on gene regulation in visual cortex and that sensory experience is needed for the sequential acquisition of age-specific, but not common, gene sets. Thus, a dynamic interplay between experience and gene expression drives activity-dependent circuit maturation.

Co-regulation of ocular dominance plasticity and NMDA receptor subunit expression in glutamic acid decarboxylase-65 knock-out mice.

Experience can shape cortical circuits, especially during critical periods for plasticity. In visual cortex, imbalance of activity from the two eyes during the critical period shifts ocular dominance (OD) towards the more active eye. Inhibitory circuits are crucial in this process: OD plasticity is absent in GAD65KO mice that show diminished inhibition. This defect can be rescued by application of benzodiazepines, which increase GABAergic signalling. However, it is unknown how such changes in inhibition might disrupt and then restore OD plasticity. Since NMDA dependent synaptic plasticity mechanisms are also known to contribute to OD plasticity, we investigated whether NMDA receptor levels and function are also altered in GAD65KO. There are reduced NR2A levels and slower NMDA currents in visual cortex of GAD65KO mice. Application of benzodiazepines, which rescues OD plasticity, also increases NR2A levels. Thus it appears as if OD plasticity can be restored by adding a critical amount of excitatory transmission through NR2A-containing NMDA receptors. Together, these observations can unify competing ideas of how OD plasticity is regulated: changes in either inhibition or excitation would engage homeostatic mechanisms that converge to regulate NMDA receptors, thereby enabling plasticity mechanisms and also ensuring circuit stability.

Multiple periods of functional ocular dominance plasticity in mouse visual cortex.

The precise period when experience shapes neural circuits in the mouse visual system is unknown. We used Arc induction to monitor the functional pattern of ipsilateral eye representation in cortex during normal development and after visual deprivation. After monocular deprivation during the critical period, Arc induction reflects ocular dominance (OD) shifts within the binocular zone. Arc induction also reports faithfully expected OD shifts in cat. Shifts towards the open eye and weakening of the deprived eye were seen in layer 4 after the critical period ends and also before it begins. These shifts include an unexpected spatial expansion of Arc induction into the monocular zone. However, this plasticity is not present in adult layer 6. Thus, functionally assessed OD can be altered in cortex by ocular imbalances substantially earlier and far later than expected.

Automated dendritic spine detection using convolutional neural networks on maximum intensity projected microscopic volumes.

BACKGROUND: Dendritic spines are structural correlates of excitatory synapses in the brain. Their density and structure are shaped by experience, pointing to their role in memory encoding. Dendritic spine imaging, followed by manual analysis, is a primary way to study spines. However, an approach that analyses dendritic spines images in an automated and unbiased manner is needed to fully capture how spines change with normal experience, as well as in disease. NEW METHOD: We propose an approach based on fully convolutional neural networks (FCNs) to detect dendritic spines in two-dimensional maximum-intensity projected images from confocal fluorescent micrographs. We experiment on both fractionally strided convolution and efficient sub-pixel convolutions. Dendritic spines far from the dendritic shaft are pruned by extraction of the shaft to reduce false positives. Performance of the proposed method is evaluated by comparing predicted spine positions to those manually marked by experts. RESULTS: The averaged distance between predicted and manually annotated spines is 2.81 ± 2.63 pixels (0.082 ± 0.076 microns) and 2.87 ± 2.33 pixels (0.084 ± 0.068 microns) based on two different experts. FCN-based detection achieves F scores > 0.80 for both sets of expert annotations. COMPARISON WITH EXISTING METHODS: Our method significantly outperforms two well-known software, NeuronStudio and Neurolucida (p-value < 0.02). CONCLUSIONS: FCN architectures used in this work allow for automated dendritic spine detection. Superior outcomes are possible even with small training data-sets. The proposed method may generalize to other datasets on larger scales.

A saturation hypothesis to explain both enhanced and impaired learning with enhanced plasticity.

Across many studies, animals with enhanced synaptic plasticity exhibit either enhanced or impaired learning, raising a conceptual puzzle: how enhanced plasticity can yield opposite learning outcomes? Here, we show that the recent history of experience can determine whether mice with enhanced plasticity exhibit enhanced or impaired learning in response to the same training. Mice with enhanced cerebellar LTD, due to double knockout (DKO) of MHCI H2-Kb/H2-Db (KbDb-/-), exhibited oculomotor learning deficits. However, the same mice exhibited enhanced learning after appropriate pre-training. Theoretical analysis revealed that synapses with history-dependent learning rules could recapitulate the data, and suggested that saturation may be a key factor limiting the ability of enhanced plasticity to enhance learning. Optogenetic stimulation designed to saturate LTD produced the same impairment in WT as observed in DKO mice. Overall, our results suggest that the recent history of activity and the threshold for synaptic plasticity conspire to effect divergent learning outcomes.

Cell-Autonomous Regulation of Dendritic Spine Density by PirB.

Synapse density on cortical pyramidal neurons is modulated by experience. This process is highest during developmental critical periods, when mechanisms of synaptic plasticity are fully engaged. In mouse visual cortex, the critical period for ocular dominance (OD) plasticity coincides with the developmental pruning of synapses. At this time, mice lacking paired Ig-like receptor B (PirB) have excess numbers of dendritic spines on L5 neurons; these spines persist and are thought to underlie the juvenile-like OD plasticity observed in adulthood. Here we examine whether PirB is required specifically in excitatory neurons to exert its effect on dendritic spine and synapse density during the critical period. In mice with a conditional allele of PirB (PirBfl/fl), PirB was deleted only from L2/3 cortical pyramidal neurons in vivo by timed in utero electroporation of Cre recombinase. Sparse mosaic expression of Cre produced neurons lacking PirB in a sea of wild-type neurons and glia. These neurons had significantly elevated dendritic spine density, as well as increased frequency of miniature EPSCs, suggesting that they receive a greater number of synaptic inputs relative to Cre- neighbors. The effect of cell-specific PirB deletion on dendritic spine density was not accompanied by changes in dendritic branching complexity or axonal bouton density. Together, results imply a neuron-specific, cell-autonomous action of PirB on synaptic density in L2/3 pyramidal cells of visual cortex. Moreover, they are consistent with the idea that PirB functions normally to corepress spine density and synaptic plasticity, thereby maintaining headroom for cells to encode ongoing experience-dependent structural change throughout life.

A novel role for p75NTR in subplate growth cone complexity and visual thalamocortical innervation.

In cortical development, subplate axons pioneer the pathway from neocortex to the internal capsule, leading to the proposal that they are required for subsequent area-specific innervation of cortex by thalamic axons. A role for p75 neutrophin receptor (NTR) in area-specific thalamic innervation of cortex is suggested by the observation that p75NTR expression is restricted to subplate neurons in a low-rostral to high-caudal gradient throughout the period of thalamocortical innervation. In vitro, neurotrophin 3 binding to p75NTR increases neurite length and filopodial formation of immunopurified subplate neurons, suggesting a role for p75NTR in subplate growth cone morphology and function in vivo. Consistent with this idea, subplate growth cones have markedly fewer filopodia in mice lacking p75NTR than in wild type mice. Despite this gross morphologic defect, many subplate axons in knock-out mice pioneer the projection to the internal capsule as they do in wild-type mice. However a few subplate axons in the knock-out mice make ectopic projections rostral in the intermediate zone and frontal cortex. Concomitant with the altered morphology of subplate growth cones, mice lacking p75NTR have diminished innervation of visual cortex from the lateral geniculate nucleus, with markedly reduced or absent connections in 48% of knock-out mice. Thalamic projections to auditory and somatosensory cortex are normal, consistent with the gradient of p75NTR expression. Our present results are unusual in that they argue that p75NTR functions in a novel way in subplate neurons, that is, in growth cone morphology and function rather than in axon extension or neuronal survival.