Thalamus Controls Development and Expression of Arousal States in Visual Cortex.

Two major checkpoints of development in cerebral cortex are the acquisition of continuous spontaneous activity and the modulation of this activity by behavioral state. Despite the critical importance of these functions, the circuit mechanisms of their development remain unknown. Here we use the rodent visual system as a model to test the hypothesis that the locus of circuit change responsible for the developmental acquisition of continuity and state dependence measured in sensory cortex is relay thalamus, rather than the local cortical circuitry or the interconnectivity of the two structures. We conducted simultaneous recordings in the dorsal lateral geniculate nucleus (dLGN) and primary visual cortex (VC) of awake, head-fixed male and female rats using linear multielectrode arrays throughout early development. We find that activity in dLGN becomes continuous and positively correlated with movement (a measure of state dependence) on P13, the same day as VC, and that these properties are not dependent on VC activity. By contrast, silencing dLGN after P13 causes activity in VC to become discontinuous and movement to suppress, rather than augment, cortical firing, effectively reversing development. Thalamic bursting, a core characteristic of non-aroused states, emerged later, on P16, suggesting these processes are developmentally independent. Together our results indicate that cellular or circuit changes in relay thalamus are critical drivers for the maturation of background activity, which occurs around term in humans.SIGNIFICANCE STATEMENT The developing brain acquires two crucial features, continuous spontaneous activity and its modulation by arousal state, around term in humans and before the onset of sensory experience in rodents. This developmental transition in cortical activity, as measured by electroencephalogram (EEG), is an important milestone for normal brain development and indicates a good prognosis for babies born preterm and/or suffering brain damage such as hypoxic-ischemic encephalopathy. By using the awake rodent visual system as a model, we identify changes occurring at the level of relay thalamus, the major input to cortex, as the critical driver of EEG maturation. These results could help understand the circuit basis of human EEG development to improve diagnosis and treatment of infants in vulnerable situations.

Independent optical excitation of distinct neural populations.

Optogenetic tools enable examination of how specific cell types contribute to brain circuit functions. A long-standing question is whether it is possible to independently activate two distinct neural populations in mammalian brain tissue. Such a capability would enable the study of how different synapses or pathways interact to encode information in the brain. Here we describe two channelrhodopsins, Chronos and Chrimson, discovered through sequencing and physiological characterization of opsins from over 100 species of alga. Chrimson's excitation spectrum is red shifted by 45 nm relative to previous channelrhodopsins and can enable experiments in which red light is preferred. We show minimal visual system-mediated behavioral interference when using Chrimson in neurobehavioral studies in Drosophila melanogaster. Chronos has faster kinetics than previous channelrhodopsins yet is effectively more light sensitive. Together these two reagents enable two-color activation of neural spiking and downstream synaptic transmission in independent neural populations without detectable cross-talk in mouse brain slice.

Eye opening and PSD95 are required for long-term potentiation in developing superior colliculus.

The only major glutamate receptor membrane-associated guanylate kinase scaffolds expressed in the young superficial superior colliculus (SC) are synapse-associated protein 102 (SAP102) and postsynaptic density protein 95 (PSD95). In this, as in all visual brain regions examined, synaptic PSD95 increases rapidly following simultaneous eyelid opening (EO). We show that EO and PSD95 are necessary for SC NMDA receptor (NMDAR)-dependent long-term potentiation (LTP) and this LTP is eliminated or reinstated by manipulating EO. PSD95 knockdown (KD) in vivo blocks this LTP, but not long-term depression, and reduces frequencies of miniature AMPA receptor and NMDAR currents with no change in presynaptic release. Furthermore, miniature NMDAR currents after PSD95 KD show an activity-triggered calcineurin sensitivity that is normally only found in the pre-EO period when SAP102 binds mixed GluN2A/GluN2B NMDARs. These data indicate that young SC LTP arises from PSD95 unsilencing of silent synapses, that unsilencing is labile in young brain, and that even though SAP102 and PSD95 can bind the same NMDARs, only PSD95 enables SC synaptic maturation.

An intracellular domain with a novel sequence regulates cell surface expression and synaptic clustering of leucine-rich repeat transmembrane proteins in hippocampal neurons.

Leucine-rich repeat transmembrane proteins (LRRTMs) are single-spanning transmembrane proteins that belong to the family of synaptically localized adhesion molecules that play various roles in the formation, maturation, and function of synapses. LRRTMs are highly localized in the post-synaptic density; however, the mechanisms and significance of LRRTM synaptic clustering remain unclear. Here, we focus on the intracellular domain of LRRTMs and investigate its role in cell surface expression and synaptic clustering. The deletion of 55-56 residues in the cytoplasmic tail caused significantly reduced synaptic clustering of LRRTM1-4 in rat hippocampal neurons, whereas it simultaneously resulted in augmented LRRTM1-2 cell surface expression. A series of deletions and further single amino acid substitutions in the intracellular domain of LRRTM2 demonstrated that a previously uncharacterized sequence at the region of -16 to -13 from the C-terminus was responsible for efficient synaptic clustering and proper cell surface trafficking of LRRTMs. Furthermore, the clustering-deficient LRRTM2 mutant lost the ability to promote the accumulation of post-synaptic density protein-95 (PSD-95). These results suggest that trafficking to the cell surface and synaptic clustering of LRRTMs are regulated by a specific mechanism through this novel sequence in the intracellular domain that underlies post-synaptic molecular assembly and maturation. Leucine-rich repeat transmembrane proteins (LRRTMs) are synaptic cell adhesion molecules promoting synapse formation. LRRTMs are highly localized in the postsynaptic density. We report amino acid sequence YxxC in the intracellular domain of LRRTMs is responsible for the postsynaptic localization of LRRTMs. This novel amino acid sequence of LRRTMs facilitates synapse maturation. We propose this regulated synaptic clustering of LRRTMs by the intracellular domain presents a novel molecular mechanism of synapse maturation.

Postsynaptic density scaffold SAP102 regulates cortical synapse development through EphB and PAK signaling pathway.

Membrane-associated guanylate kinases (MAGUKs), including SAP102, PSD-95, PSD-93, and SAP97, are scaffolding proteins for ionotropic glutamate receptors at excitatory synapses. MAGUKs play critical roles in synaptic plasticity; however, details of signaling roles for each MAGUK remain largely unknown. Here we report that SAP102 regulates cortical synapse development through the EphB and PAK signaling pathways. Using lentivirus-delivered shRNAs, we found that SAP102 and PSD-95, but not PSD-93, are necessary for excitatory synapse formation and synaptic AMPA receptor (AMPAR) localization in developing mouse cortical neurons. SAP102 knockdown (KD) increased numbers of elongated dendritic filopodia, which is often observed in mouse models and human patients with mental retardation. Further analysis revealed that SAP102 coimmunoprecipitated the receptor tyrosine kinase EphB2 and RacGEF Kalirin-7 in neonatal cortex, and SAP102 KD reduced surface expression and dendritic localization of EphB. Moreover, SAP102 KD prevented reorganization of actin filaments, synapse formation, and synaptic AMPAR trafficking in response to EphB activation triggered by its ligand ephrinB. Last, p21-activated kinases (PAKs) were downregulated in SAP102 KD neurons. These results demonstrate that SAP102 has unique roles in cortical synapse development by mediating EphB and its downstream PAK signaling pathway. Both SAP102 and PAKs are associated with X-linked mental retardation in humans; thus, synapse formation mediated by EphB/SAP102/PAK signaling in the early postnatal brain may be crucial for cognitive development.

TrkB and protein kinase Mζ regulate synaptic localization of PSD-95 in developing cortex.

Postsynaptic density 95 (PSD-95), the major scaffold at excitatory synapses, is critical for synapse maturation and learning. In rodents, eye opening, the onset of pattern vision, triggers a rapid movement of PSD-95 from visual neuron somata to synapses. We showed previously that the PI3 kinase-Akt pathway downstream of BDNF/TrkB signaling stimulates synaptic delivery of PSD-95 via vesicular transport. However, vesicular transport requires PSD-95 palmitoylation to attach it to a lipid membrane. Also, PSD-95 insertion at synapses is known to require this lipid modification. Here, we show that BDNF/TrkB signaling is also necessary for PSD-95 palmitoylation and its transport to synapses in mouse visual cortical layer 2/3 neurons. However, palmitoylation of PSD-95 requires the activation of another pathway downstream of BDNF/TrkB, namely, signaling through phospholipase Cγ and the brain-specific PKC variant protein kinase M ζ (PKMζ). We find that PKMζ selectively regulates phosphorylation of the palmitoylation enzyme ZDHHC8. Inhibition of PKMζ results in a reduction of synaptic PSD-95 accumulation in vivo, which can be rescued by overexpressing ZDHHC8. Therefore, TrkB and PKMζ, two critical regulators of synaptic plasticity, facilitate PSD-95 targeting to synapses. These results also indicate that palmitoylation can be regulated by a trophic factor. Our findings have implications for neurodevelopmental disorders as well as aging brains.

A new role for visual experience in top-down cortical development.

In this issue of Neuron, Ibrahim et al. (2021) examine the rules by which top-down connections are made on visual cortical layer 1 interneurons, discovering activity-dependent cooperative interactions with visual input that are specific to neurogliaform cells and anterior cingulate cortex.

GABAergic interneurons excite neonatal hippocampus in vivo.

GABAergic interneurons are proposed to be critical for early activity and synapse formation by directly exciting, rather than inhibiting, neurons in developing hippocampus and neocortex. However, the role of GABAergic neurons in the generation of neonatal network activity has not been tested in vivo, and recent studies have challenged the excitatory nature of early GABA. By locally manipulating interneuron activity in unanesthetized neonatal mice, we show that GABAergic neurons are excitatory in CA1 hippocampus at postnatal day 3 (P3) and are responsible for most of the spontaneous firing of pyramidal cells at that age. Hippocampal interneurons become inhibitory by P7, whereas visual cortex interneurons are already inhibitory by P3 and remain so throughout development. These regional and age-specific differences are the result of a change in chloride reversal potential, because direct activation of light-gated anion channels in glutamatergic neurons drives CA1 firing at P3, but silences it at P7 in CA1, and at all ages in visual cortex. This study in the intact brain reveals that GABAergic interneuron excitation is essential for network activity in neonatal hippocampus and confirms that visual cortical interneurons are inhibitory throughout early postnatal development.

Myosin Va Brain-Specific Mutation Alters Mouse Behavior and Disrupts Hippocampal Synapses.

Myosin Va (MyoVa) is a plus-end filamentous-actin motor protein that is highly and broadly expressed in the vertebrate body, including in the nervous system. In excitatory neurons, MyoVa transports cargo toward the tip of the dendritic spine, where the postsynaptic density (PSD) is formed and maintained. MyoVa mutations in humans cause neurologic dysfunction, intellectual disability, hypomelanation, and death in infancy or childhood. Here, we characterize the Flailer (Flr) mutant mouse, which is homozygous for a myo5a mutation that drives high levels of mutant MyoVa (Flr protein) specifically in the CNS. Flr protein functions as a dominant-negative MyoVa, sequestering cargo and blocking its transport to the PSD. Flr mice have early seizures and mild ataxia but mature and breed normally. Flr mice display several abnormal behaviors known to be associated with brain regions that show high expression of Flr protein. Flr mice are defective in the transport of synaptic components to the PSD and in mGluR-dependent long-term depression (LTD) and have a reduced number of mature dendritic spines. The synaptic and behavioral abnormalities of Flr mice result in anxiety and memory deficits similar to that of other mouse mutants with obsessive-compulsive disorder and autism spectrum disorder (ASD). Because of the dominant-negative nature of the Flr protein, the Flr mouse offers a powerful system for the analysis of how the disruption of synaptic transport and lack of LTD can alter synaptic function, development and wiring of the brain and result in symptoms that characterize many neuropsychiatric disorders.

Thalamic inhibitory circuits and network activity development.

Inhibitory circuits in thalamus and cortex shape the major activity patterns observed by electroencephalogram (EEG) in the adult brain. Their delayed maturation and circuit integration, relative to excitatory neurons, suggest inhibitory neuronal development could be responsible for the onset of mature thalamocortical activity. Indeed, the immature brain lacks many inhibition-dependent activity patterns, such as slow-waves, delta oscillations and sleep-spindles, and instead expresses other unique oscillatory activities in multiple species including humans. Thalamus contributes significantly to the generation of these early oscillations. Compared to the abundance of studies on the development of inhibition in cortex, however, the maturation of thalamic inhibition is poorly understood. Here we review developmental changes in the neuronal and circuit properties of the thalamic relay and its interconnected inhibitory thalamic reticular nucleus (TRN) both in vitro and in vivo, and discuss their potential contribution to early network activity and its maturation. While much is unknown, we argue that weak inhibitory function in the developing thalamus allows for amplification of thalamocortical activity that supports the generation of early oscillations. The available evidence suggests that the developmental acquisition of critical thalamic oscillations such as slow-waves and sleep-spindles is driven by maturation of the TRN. Further studies to elucidate thalamic GABAergic circuit formation in relation to thalamocortical network function would help us better understand normal as well as pathological brain development.

Uncorrelated Neural Firing in Mouse Visual Cortex during Spontaneous Retinal Waves.

Synchronous firing among the elements of forming circuits is critical for stabilization of synapses. Understanding the nature of these local network interactions during development can inform models of circuit formation. Within cortex, spontaneous activity changes throughout development. Unlike the adult, early spontaneous activity occurs in discontinuous population bursts separated by long silent periods, suggesting a high degree of local synchrony. However, whether the micro-patterning of activity within early bursts is unique to this early age and specifically tuned for early development is poorly understood, particularly within the column. To study this we used single-shank multi-electrode array recordings of spontaneous activity in the visual cortex of non-anesthetized neonatal mice to quantify single-unit firing rates, and applied multiple measures of network interaction and synchrony throughout the period of map formation and immediately after eye-opening. We find that despite co-modulation of firing rates on a slow time scale (hundreds of ms), the number of coactive neurons, as well as pair-wise neural spike-rate correlations, are both lower before eye-opening. In fact, on post-natal days (P)6-9 correlated activity was lower than expected by chance, suggesting active decorrelation of activity during early bursts. Neurons in lateral geniculate nucleus developed in an opposite manner, becoming less correlated after eye-opening. Population coupling, a measure of integration in the local network, revealed a population of neurons with particularly strong local coupling present at P6-11, but also an adult-like diversity of coupling at all ages, suggesting that a neuron's identity as locally or distally coupled is determined early. The occurrence probabilities of unique neuronal "words" were largely similar at all ages suggesting that retinal waves drive adult-like patterns of co-activation. These findings suggest that the bursts of spontaneous activity during early visual development do not drive hyper-synchronous activity within columns. Rather, retinal waves provide windows of potential activation during which neurons are active but poorly correlated, adult-like patterns of correlation are achieved soon after eye-opening.

A leucine-rich repeat membrane protein, 5T4, is expressed by a subtype of granule cells with dendritic arbors in specific strata of the mouse olfactory bulb.

Segregation of neuron-type-specific synaptic connections in different strata is a characteristic feature shared by the olfactory bulb (OB) and retina. In the mammalian OB, mitral cells form dendrodendritic synapses with granule cells (GCs) in the deep stratum of the external plexiform layer (EPL), whereas tufted cells form dendrodendritic synapses in the superficial stratum. In the search for membrane proteins with strata-specific expression patterns, we found that a leucine-rich repeat membrane protein (5T4 oncofetal trophoblast glycoprotein) was expressed selectively by a subset of superficial GCs. The somata of 5T4-positive GCs were localized in or near the mitral cell layer, and their apical dendrites ramified preferentially in the superficial stratum of the EPL, where tufted cell dendrites ramified. Strata-specific expression of 5T4 was found also in the retina: 5T4 was expressed selectively by rod-bipolar cells and a subset of amacrine cells whose dendrites ramified in a specific sublamina of the inner plexiform layer. During the perinatal and postnatal development of the OB, 5T4 expression paralleled in time the formation of dendrodendritic synapses in the EPL. Odor deprivation during the first postnatal month selectively reduced the thickness of the superficial stratum of the EPL and the number of 5T4-positive GCs. Because 5T4 is known to interact with actin cytoskeleton, these observations suggest that 5T4 is involved in the formation or maintenance of strata-specific dendritic ramification or synaptic connection of subsets of local interneurons.

An excitatory cortical feedback loop gates retinal wave transmission in rodent thalamus.

Spontaneous retinal waves are critical for the development of receptive fields in visual thalamus (LGN) and cortex (VC). Despite a detailed understanding of the circuit specializations in retina that generate waves, whether central circuit specializations also exist to control their propagation through visual pathways of the brain is unknown. Here we identify a developmentally transient, corticothalamic amplification of retinal drive to thalamus as a mechanism for retinal wave transmission in the infant rat brain. During the period of retinal waves, corticothalamic connections excite LGN, rather than driving feedforward inhibition as observed in the adult. This creates an excitatory feedback loop that gates retinal wave transmission through the LGN. This cortical multiplication of retinal wave input ends just prior to eye-opening, as cortex begins to inhibit LGN. Our results show that the early retino-thalamo-cortical circuit uses developmentally specialized feedback amplification to ensure powerful, high-fidelity transmission of retinal activity despite immature connectivity.

The Conserved VPS-50 Protein Functions in Dense-Core Vesicle Maturation and Acidification and Controls Animal Behavior.

The modification of behavior in response to experience is crucial for animals to adapt to environmental changes. Although factors such as neuropeptides and hormones are known to function in the switch between alternative behavioral states, the mechanisms by which these factors transduce, store, retrieve, and integrate environmental signals to regulate behavior are poorly understood. The rate of locomotion of the nematode Caenorhabditis elegans depends on both current and past food availability. Specifically, C. elegans slows its locomotion when it encounters food, and animals in a food-deprived state slow even more than animals in a well-fed state. The slowing responses of well-fed and food-deprived animals in the presence of food represent distinct behavioral states, as they are controlled by different sets of genes, neurotransmitters, and neurons. Here we describe an evolutionarily conserved C. elegans protein, VPS-50, that is required for animals to assume the well-fed behavioral state. Both VPS-50 and its murine homolog mVPS50 are expressed in neurons, are associated with synaptic and dense-core vesicles, and control vesicle acidification and hence synaptic function, likely through regulation of the assembly of the V-ATPase complex. We propose that dense-core vesicle acidification controlled by the evolutionarily conserved protein VPS-50/mVPS50 affects behavioral state by modulating neuropeptide levels and presynaptic neuronal function in both C. elegans and mammals.

A Diencephalic Dopamine Source Provides Input to the Superior Colliculus, where D1 and D2 Receptors Segregate to Distinct Functional Zones.

Modulation of neural responses is frequently observed in the superior colliculus (SC), a retinorecipient midbrain structure that controls orienting and the localization of attention. Although behavioral contingencies that influence SC responses are well documented, the neural pathways and molecular mechanisms responsible for this modulation are not completely understood. Here, we illustrate a dopaminergic system that strongly impacts neural responses in the SC. After using RNA sequencing (RNA-seq) to detail the transcriptome of dopamine-related genes in the SC, we show that D1 receptors are enriched in the superficial visual SC, while D2 receptors segregate to the intermediate multimodal/motor SC. Retrograde injections into the SC consistently label A13, a small dopamine cell group located in the zona incerta. We surmise that A13 mimics dopaminergic effects that we observed in SC slices, which suggests that dopamine in the SC may reduce the tendency of an animal to orient or attend to salient stimuli.

Pentraxins coordinate excitatory synapse maturation and circuit integration of parvalbumin interneurons.

Circuit computation requires precision in the timing, extent, and synchrony of principal cell (PC) firing that is largely enforced by parvalbumin-expressing, fast-spiking interneurons (PVFSIs). To reliably coordinate network activity, PVFSIs exhibit specialized synaptic and membrane properties that promote efficient afferent recruitment such as expression of high-conductance, rapidly gating, GluA4-containing AMPA receptors (AMPARs). We found that PVFSIs upregulate GluA4 during the second postnatal week coincident with increases in the AMPAR clustering proteins NPTX2 and NPTXR. Moreover, GluA4 is dramatically reduced in NPTX2(-/-)/NPTXR(-/-) mice with consequent reductions in PVFSI AMPAR function. Early postnatal NPTX2(-/-)/NPTXR(-/-) mice exhibit delayed circuit maturation with a prolonged critical period permissive for giant depolarizing potentials. Juvenile NPTX2(-/-)/NPTXR(-/-) mice display reduced feedforward inhibition yielding a circuit deficient in rhythmogenesis and prone to epileptiform discharges. Our findings demonstrate an essential role for NPTXs in controlling network dynamics highlighting potential therapeutic targets for disorders with inhibition/excitation imbalances such as schizophrenia.

CFBP is a novel tyrosine-phosphorylated protein that might function as a regulator of CIN85/CD2AP.

To decipher the global network of the epidermal growth factor (EGF) receptor-mediated signaling pathway, a large scale proteomic analysis of tyrosine-phosphorylated proteins was conducted. Here, we focus on characterizing a novel protein, CFBP (CIN85/CD2AP family binding protein), identified in the study. CFBP was found to be phosphorylated at tyrosine 204 upon EGF stimulation, and the CIN85/CD2AP family was identified as a binding partner. A proline-rich motif of CFBP is recognized by one of the three Src-homology 3 domains of CIN85/CD2AP, and the affinity of the interaction is regulated by the tyrosine phosphorylation of CFBP. They co-localize in actinenriched structures, and overexpression of CFBP induced morphological changes with actin reorganization. Furthermore, CFBP accelerated the EGF receptor's down-regulation by facilitating the recruitment of Cbl to the CD2AP/CIN85 complex. Two spliced variants of CFBP lacking either exon 5 or 8 are also expressed, and the variant lacking exon 5 without the proline-rich motif lacks the ability to bind to the CIN85/CD2AP family. The CFBP protein seems to play a key role in the ligand-mediated internalization and down-regulation of the EGF receptor.

Proteomic analysis revealed a novel synaptic proline-rich membrane protein (PRR7) associated with PSD-95 and NMDA receptor.

Proteomic analyses have revealed a novel synaptic proline-rich membrane protein: PRR7 (proline rich 7), in the postsynaptic density (PSD) fraction of rat forebrain. PRR7 is 269 amino acid residues long, and displays a unique architecture, composed of a very short N-terminal extracellular region, a single membrane spanning domain, and a cytoplasmic domain possessing a proline-rich sequence and a C-terminal type-1 PDZ binding motif. A fraction of PRR7 accumulates in spines along with synapse maturation, and colocalizes with PSD-95 in a punctate pattern in rat hippocampal neural cultures. Immunoprecipitation and GST pull-down assays demonstrated that PRR7 binds to the third PDZ domain of PSD-95. In addition, the NMDA receptor subunits, NR1 and NR2B, specifically co-immunoprecipitated with PRR7. These results suggest that PRR7 is involved in modulating neural activities via interactions with the NMDA receptor and PSD-95, and PSD core formation.