Melanopsin-driven increases in maintained activity enhance thalamic visual response reliability across a simulated dawn

Twice a day, at dawn and dusk, we experience gradual but very high amplitude changes in background light intensity (irradiance). Although we perceive the associated change in environmental brightness, the representation of such very slow alterations in irradiance by the early visual system has been little studied. Here, we addressed this deficit by recording electrophysiological activity in the mouse dorsal lateral geniculate nucleus under exposure to a simulated dawn. As irradiance increased we found a widespread enhancement in baseline firing that extended to units with ON as well as OFF responses to fast luminance increments. This change in baseline firing was equally apparent when the slow irradiance ramp appeared alone or when a variety of higher-frequency artificial or natural visual stimuli were superimposed upon it. Using a combination of conventional knockout, chemogenetic, and receptor-silent substitution manipulations, we continued to show that, over higher irradiances, this increase in firing originates with inner-retinal melanopsin photoreception. At the single-unit level, irradiance-dependent increases in baseline firing were strongly correlated with improvements in the amplitude of responses to higher-frequency visual stimuli. This in turn results in an up to threefold increase in single-trial reliability of fast visual responses. In this way, our data indicate that melanopsin drives a generalized increase in dorsal lateral geniculate nucleus excitability as dawn progresses that both conveys information about changing background light intensity and increases the signal:noise for fast visual responses.The rotation of the earth imposes slow but very high amplitude changes in background light intensity (irradiance) across dawn and dusk. An array of light adaptation mechanisms acts to buffer the visual code against this substantial variation in its physical origins (1–8). Nonetheless, we certainly perceive the change in brightness of our environment, raising the question of how the visual system represents such gradual changes in irradiance.The question of how the early visual system encodes background light levels has previously been addressed by extended exposure to spatially uniform stimuli. Under these conditions, many neurons in the retina and visual thalamus scale their maintained firing rate according to stimulus irradiance over many decades (9–14). This behavior, sometimes termed “luxotonic,” implies that natural slow changes in irradiance would be represented by gradual modulations in firing rate across at least a fraction of the visual projection. However, we are unaware of a direct test of that prediction. Moreover, the extent to which luxotonic activity survives in the presence of higher-frequency visual stimuli (as would be the case in any natural scene) remains unknown. We start here by addressing these deficits and showing that naturalistic dawn transitions do indeed induce a diffuse increase in firing across the mouse dorsal lateral geniculate nucleus (dLGN).Although the luxotonic capacity of the mammalian visual system has been appreciated for >50 y, both the retinal origins and functional significance of this mode of action remain controversial. An important question is how luxotonic changes in maintained firing impact the ability of neurons to encode other visual features. There is the obvious potential for increases in baseline firing to occlude other responses; it has also been suggested that luxotonic behavior could improve signal:noise for fast visual responses by regularizing firing patterns (9, 15; although see refs. 16 and 17). A second aim of this study was to resolve this controversy, by directly measuring responses to high-frequency visual stimuli across naturalistic ramps in irradiance.Our final objective was to address the retinal circuitry responsible for luxotonic activity. Under mesopic and photopic conditions, cone photoreceptors track fast modulations in local luminance, with rapid alterations in membrane potential. However, they also can encode background light intensity with changes in steady-state voltage, and this signal can be propagated through the retinal circuitry to support luxotonic activity in ganglion cells and beyond (18–21). At brighter backgrounds, however, the photoreceptor steady state response approaches saturation (6, 22, 23). Under these conditions, further changes in maintained firing could be driven by intrinsically photosensitive retinal ganglion cells (ipRGCs), which can rely upon their intrinsic melanopsin-dependent light response to accurately encode higher irradiances (24). Here we used a combination of conventional knockout, receptor-silent substitution and chemogenetic manipulations to test the hypothesis that ipRGCs support responses to naturalistic changes in irradiance under these conditions.     

Epileptic baboons have lower numbers of neurons in specific areas of cortex

Epilepsy is characterized by recurrent seizure activity that can induce pathological reorganization and alter normal function in neocortical networks. In the present study, we determined the numbers of cells and neurons across the complete extent of the cortex for two epileptic baboons with naturally occurring seizures and two baboons without epilepsy. Overall, the two epileptic baboons had a 37% average reduction in the number of cortical neurons compared with the two nonepileptic baboons. The loss of neurons was variable across cortical areas, with the most pronounced loss in the primary motor cortex, especially in lateral primary motor cortex, representing the hand and face. Less-pronounced reductions of neurons were found in other parts of the frontal cortex and in somatosensory cortex, but no reduction was apparent in the primary visual cortex and little in other visual areas. The results provide clear evidence that epilepsy in the baboon is associated with considerable reduction in the numbers of cortical neurons, especially in frontal areas of the cortex related to motor functions. Whether or not the reduction of neurons is a cause or an effect of seizures needs further investigation.Epilepsy is associated with structural changes in the cerebral cortex (e.g., refs. 1–6), and partial epilepsies (i.e., seizures originating from a brain region) may lead to loss of neurons (7) and altered connectivity (8). The cerebral cortex is a heterogeneous structure comprised of multiple sensory and motor information-processing systems (e.g., refs. 9 and 10) that vary according to their processing demands, connectivity (e.g., refs. 11 and 12), and intrinsic numbers of cells and neurons (13–16). Chronic seizures have been associated with progressive changes in the region of the epileptic focus and in remote but functionally connected cortical or subcortical structures (3, 17). Because areas of the cortex are functionally and structurally different, they may also differ in susceptibility to pathological changes resulting from epilepsy.The relationship between seizure activity and neuron damage can be difficult to study in humans. Seizure-induced neuronal damage can be convincingly demonstrated in animals using electrically or chemically induced status epilepticus (one continuous seizure episode longer than 5 min) to reveal morphometric (e.g., refs. 18 and 19) or histological changes (e.g., refs. 20 and 21). Subcortical brain regions are often studied for vulnerability to seizure-induced injury (21–27); however, a recent study by Karbowski et al. (28) observed reduction of neurons in cortical layers 5 and 6 in the frontal lobes of rats with seizures. Seizure-induced neuronal damage in the cortex has also been previously demonstrated in baboons with convulsive status epilepticus (29).The goal of the present study was to determine if there is a specific pattern of cell or neuron reduction across the functionally divided areas of the neocortex in baboons with epilepsy. Selected strains of baboons have been studied as a natural primate model of generalized epilepsy (30–36) that is analogous to juvenile myoclonic epilepsy in humans. The baboons demonstrate generalized myoclonic and tonic-clonic seizures, and they have generalized interictal and ictal epileptic discharges on scalp EEG. Because of their phylogenetic proximity to humans, baboons and other Old World monkeys share many cortical areas and other features of cortical organization with humans (e.g., refs. 9 and 10). Cortical cell and neuron numbers were determined using the flow fractionator method (37, 38) in epileptic baboon tissue obtained from the Texas Biomedical Research Institute, where a number of individuals develop generalized epilepsy within a pedigreed baboon colony (31–36). Our results reveal a regionally specific neuron reduction in the cortex of baboons with naturally occurring, generalized seizures.     

Elevated intraocular pressure decreases response sensitivity of inner retinal neurons in experimental glaucoma mice

Glaucoma is the second leading cause of blindness in the United States and the world, characterized by progressive degeneration of the optic nerve and retinal ganglion cells (RGCs). Glaucoma patients exhibit an early diffuse loss of retinal sensitivity followed by focal loss of RGCs in sectored patterns. Recent evidence has suggested that this early sensitivity loss may be associated with dysfunctions in the inner retina, but detailed cellular and synaptic mechanisms underlying such sensitivity changes are largely unknown. In this study, we use whole-cell voltage-clamp techniques to analyze light responses of individual bipolar cells (BCs), AII amacrine cells (AIIACs), and ON and sustained OFF alpha-ganglion cells (ONαGCs and sOFFαGCs) in dark-adapted mouse retinas with elevated intraocular pressure (IOP). We present evidence showing that elevated IOP suppresses the rod ON BC inputs to AIIACs, resulting in less sensitive AIIACs, which alter AIIAC inputs to ONαGCs via the AIIAC→cone ON BC→ONαGC pathway, resulting in lower ONαGC sensitivity. The altered AIIAC response also reduces sOFFαGC sensitivity via the AIIAC→sOFFαGC chemical synapses. These sensitivity decreases in αGCs and AIIACs were found in mice with elevated IOP for 3–7 wk, a stage when little RGC or optic nerve degeneration was observed. Our finding that elevated IOP alters neuronal function in the inner retina before irreversible structural damage occurs provides useful information for developing new diagnostic tools and treatments for glaucoma in human patients.Glaucoma is a leading cause of irreversible blindness in the United States and the world (1, 2), and is characterized by optic nerve cupping (thinning of the neuroretinal rim at the optic nerve head) and progressive optic nerve and retinal ganglion cell (RGC) degeneration as well as functional deficit revealed by psychophysical tests (3, 4). Although factors causing the eventual RGC death and blindness remain controversial (1, 5–8), increasing evidence from human patients and animal models has shown that the disease is associated with an early mild diffuse loss of retinal sensitivity or inner retinal response decrease (9–14). Although it is unclear whether these functional changes are a prelude or even causal to RGC death and blindness, elucidating the underlying synaptic and cellular mechanisms for such sensitivity/response decline will nevertheless provide novel insights pertaining to early detection and treatment of human glaucoma.Multiple risk factors are associated with glaucomatous diseases, among which elevated intraocular pressure (IOP) is widely accepted as the most significant for both disease onset and progression (2, 15). Because high IOP (H-IOP) is an important risk factor, many experimental animal models of elevated IOP have been developed in multiple species including monkeys, rats, and mice (16–22). Most experiments performed in animal models have focused on anatomical and histopathological analyses of RGC death, axon loss, and changes to axonal projections to higher visual centers in the brain (23–25). Only a few studies have attempted to address whether function and light sensitivity of retinal neurons are affected. Some reports have suggested a possible but inconclusive involvement of amacrine cells (26, 27). A recent study examining the scotopic threshold responses (STRs) in an elevated IOP mouse model generated by the microbead occlusion method (28) has suggested that the voltage gains (ratio of post/presynaptic signals) of the negative STR [possibly representing AII amacrine cell (AIIAC) responses (29)] and positive STR [possibly representing ON GC responses (30)] are both reduced at stages before morphological changes or RGC death occurs (12). However, no changes in single RGC or their presynaptic bipolar cell (BC)/AIIAC responses have been reported in experimental glaucoma models. Studies using electroretinogram, STR, and optic nerve recording techniques lack the power to identify or establish cellular and synaptic sites of retinal dysfunction (27, 31, 32), leaving a disabling gap preventing us from knowing how elevated IOP affects light responses of individual retinal neurons. In this study, we fill this gap by using whole-cell voltage-clamp techniques to study light responses of individual alpha-RGCs (αGCs) and AIIACs, as well as their presynaptic BCs, in two experimental glaucoma mouse models.It has been shown that light responses of mammalian AIIACs are mediated by rod bipolar cell (DBC_R) inputs via a 6,7-dinitroquinoxaline-2,3-dione (DNQX)–sensitive glutamatergic synapse and certain types of cone depolarizing bipolar cell [DBC_R/MC; ON bipolar cells with mixed rod and M-cone inputs (33), or B6-7] input via a connexin36 (Cx36)-dependent electrical synapse (34–36). AIIACs are perhaps the most sensitive (with the lowest response threshold) neurons in the mouse retina (37, 38), and thus they send highly sensitive output signals to postsynaptic neurons such as certain types of cone hyperpolarizing bipolar cells [HBC_R/MCs; OFF bipolar cells with mixed rod and M-cone inputs (39), or B1-2] and OFF GCs (37, 40). ON and sustained OFF alpha-GCs (ONαGCs and sOFFαGCs) are two primary types of GCs in the mouse retina, and their synaptic circuitries include many major types of mammalian BCs and ACs (40–42). By studying the effects of elevated IOP on these GCs and their presynaptic neurons, we will be able to gain insights into general mechanisms underlying inner retina dysfunction in glaucoma. ONαGCs and sOFFαGCs exhibit characteristic morphology (large somas and dendritic trees) and light response signatures (40). ONαGCs have no or very little spike activity in darkness, increased spikes in light, inward light-evoked cation current (ΔI_C; mediated by DBC_R/MC inputs), and outward light-evoked chloride current (ΔI_Cl; mediated by AC inputs) (40, 43). sOFFαGCs exhibit maintained spike activity in darkness, sustained decrease of spikes in light, outward ΔI_C (mediated by HBC_R/MC inputs), and outward ΔI_Cl (mediated by AIIAC/AC inputs) (40, 44). Fig. 1 is a schematic diagram of synaptic connections between ONαGCs/sOFFαGCs and their primary presynaptic neurons: DBC_Rs, DBC_R/MCs, HBC_R/MCs, and AIIACs (key synapses are labeled 1–5 in the figure; see below). In this report, we analyzed light responses of these retinal neurons in treated mice (in which H-IOP was induced; Materials and Methods) and compared them with the corresponding responses measured in nontreated mice with normal IOP (n-IOP).Open in a separate windowFig. 1.Schematic diagram of major synaptic connections in the ON and OFF α-ganglion pathways in the mouse retina. Green, rods and rod BCs; blue, M cones and mixed rod/M-cone BCs; orange, AIIACs; gray, αGCs; arrows, chemical synapses (red, glutamatergic; blue, glycinergic; +, sign-preserving; −, sign-inverting); zigzag (red), electrical synapses. a, sublamina a; b, sublamina b; GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; OPL, outer plexiform layer; PRL, photoreceptor layer. Synapses directly relevant to this study are marked with numbers in circles: 1: DBC_R→AIIAC glutamatergic; 2: DBC_C↔AIIAC electrical; 3: DBC_R/MC/HBC_R/MC→ONαGC/sOFFαGC glutamatergic; 4: AIIAC→HBC_R/MC glycinergic; and 5: AIIAC→sOFFαGC glycinergic.     

Behavioral response inhibition and maturation of goal representation in prefrontal cortex after puberty

Executive functions including behavioral response inhibition mature after puberty, in tandem with structural changes in the prefrontal cortex. Little is known about how activity of prefrontal neurons relates to this profound cognitive development. To examine this, we tracked neuronal responses of the prefrontal cortex in monkeys as they transitioned from puberty into adulthood and compared activity at different developmental stages. Performance of the antisaccade task greatly improved in this period. Among neural mechanisms that could facilitate it, reduction of stimulus-driven activity, increased saccadic activity, or enhanced representation of the opposing goal location, only the latter was evident in adulthood. Greatly accentuated in adults, this neural correlate of vector inversion may be a prerequisite to the formation of a motor plan to look away from the stimulus. Our results suggest that the prefrontal mechanisms that underlie mature performance on the antisaccade task are more strongly associated with forming an alternative plan of action than with suppressing the neural impact of the prepotent stimulus.Behavioral response inhibition, and cognitive task performance more generally, improves substantially between the time of puberty and adulthood (1–4). Risky decision-making peaks in adolescence, the time period between puberty and adulthood that is most closely linked to delinquent behavior in humans (5–7). Performance in tasks that assay response inhibition, such as the antisaccade task, improves into adulthood, reflecting the progressive development of behavioral control (3). This period of cognitive enhancement parallels the maturation of the prefrontal cortex (8–11). Anatomical changes in the prefrontal cortex continue during adolescence, involving gray and white matter volumes and myelination of axon fibers within the prefrontal cortex and between the prefrontal cortex and other areas (8–15). Changes in prefrontal activation, including increases (12, 16–20) and decreases (21, 22), have been documented in imaging studies for tasks that require inhibition of prepotent behavioral responses and filtering of distractors.Much less is known about how the physiological properties of prefrontal neurons develop after puberty. Similar to the human pattern of development, the monkey prefrontal cortex undergoes anatomical maturation in adolescence and early adulthood (23, 24). Male monkeys enter puberty at ∼3.5 y of age and reach full sexual maturity at 5 y, approximately equivalent to the human ages of 11 y and 16 y, respectively (25, 26). By some accounts, biochemical and anatomical changes characteristic of adolescence in humans occur at an earlier, prepubertal age in the monkey prefrontal cortex (27, 28), so it is not known if cognitive maturation or neurophysiological changes occur in monkeys after puberty. The contribution of prefrontal cortex to antisaccade performance has also been a matter of debate, with contrasting views favoring mechanisms of inhibiting movement toward the visual stimulus or enhancing movement away from it (29–31). Potential maturation of behavioral response inhibition may therefore be associated with a more efficient suppression of the stimulus representation in neural activity (weaker visual responses to stimuli inside the receptive field), stronger motor responses (higher activity to saccades), or enhancement of the appropriate goal representation (stronger activity for planning a saccade away from the stimulus). To examine the mechanisms that facilitate the mature ability to resist generating a response toward a salient stimulus, we used developmental markers to track transition from puberty to adulthood in monkeys and sought to identify neural correlates of changes in antisaccade performance within the visual and saccade-related activations of prefrontal neurons.     

Visual stimuli recruit intrinsically generated cortical ensembles

The cortical microcircuit is built with recurrent excitatory connections, and it has long been suggested that the purpose of this design is to enable intrinsically driven reverberating activity. To understand the dynamics of neocortical intrinsic activity better, we performed two-photon calcium imaging of populations of neurons from the primary visual cortex of awake mice during visual stimulation and spontaneous activity. In both conditions, cortical activity is dominated by coactive groups of neurons, forming ensembles whose activation cannot be explained by the independent firing properties of their contributing neurons, considered in isolation. Moreover, individual neurons flexibly join multiple ensembles, vastly expanding the encoding potential of the circuit. Intriguingly, the same coactive ensembles can repeat spontaneously and in response to visual stimuli, indicating that stimulus-evoked responses arise from activating these intrinsic building blocks. Although the spatial properties of stimulus-driven and spontaneous ensembles are similar, spontaneous ensembles are active at random intervals, whereas visually evoked ensembles are time-locked to stimuli. We conclude that neuronal ensembles, built by the coactivation of flexible groups of neurons, are emergent functional units of cortical activity and propose that visual stimuli recruit intrinsically generated ensembles to represent visual attributes.There is a growing consensus in neuroscience that ensembles of neurons working in concert, as opposed to single neurons, are the underpinnings of cognition and behavior (1–3). At the microcircuit level, the cortex is dominated by recurrent excitatory connections (4, 5). Such densely interconnected excitatory networks are ideal for generating reverberating activity (1, 6, 7) that could link neurons into functional neuronal ensembles. Moreover, most cortical neurons are part of highly distributed synaptic circuits, receiving inputs from and projecting outputs to, thousands of other neurons (8, 9). In fact, the basic excitatory neurons of the cortex, pyramidal cells, appear to be biophysically designed to perform large-scale integration of inputs (10). All of these structural features indicate the possibility that rather than relying on the firing of individual neurons, cortical circuits may generate responses built out of the coordinated activity of groups of neurons. These postulated emergent circuit states could represent the building blocks of mental and behavioral processes (1–3, 11).In the visual cortex, there has been continuing progress in understanding functional properties and receptive fields of single neurons using single-unit electrophysiology and optical imaging (12–14). These single-neuron studies have provided a solid foundation for neuroscience. However, the focus on single neurons may provide an incomplete picture of this highly distributed neural circuit (3, 15). In fact, in recent years, the network activity patterns of the primary visual cortex (V1) in vitro and in vivo have been shown to be highly structured in spatiotemporal properties (13, 16–18). For example, using voltage-sensitive imaging, one can measure large-scale cortical dynamics with high temporal resolution, albeit without single-cell resolution (19–22). At this bird’s-eye view, wave-like spontaneous spatiotemporal patterns of activity appear similar to those patterns measured during visual stimulation (19, 21, 22). These findings imply that groups of neurons are active together in the absence of any visual input and that the same groups of neurons are also active together in response to visual stimulation. However, to test this hypothesis, one must measure the circuit activity with single-cell resolution.With calcium imaging, multineuronal activity can be visualized with single-cell resolution (16, 23), so it has become possible to discern exactly which neurons are activated under spontaneous and visually evoked conditions, cell by cell. Indeed, calcium imaging of brain slices from mouse visual cortex has revealed that groups of neurons become coactive spontaneously (24, 25) and that the same groups of neurons can be triggered by stimulation of thalamic afferents (26, 27). However, the patterns of activity found in slices may differ from the patterns of activity in vivo. Therefore, to determine the relation between spontaneous and evoked cortical activity patterns properly, it is necessary to measure them in vivo.Using two-photon calcium imaging in vivo, we have now mapped the spontaneous reverberating activity patterns in the V1 from awake mice with single-cell resolution and analyzed their relation to the activity patterns evoked by visual stimulation. We find patterns of coactive neurons that we term “ensembles,” defined as “a group of items viewed as a whole rather than individually” (28). Although the mere existence of these coactive neurons does not prove their functional importance, we provide converging lines of evidence that these ensembles are, in fact, functional units of cortical activity. This work provides a step in the progression of defining neuronal ensembles, rather than receptive fields of individual cells, as a building block of cortical microcircuits and suggests that these intrinsic neuronal ensembles are recruited when the cortex performs some of its most basic functions.     

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: PNAS Plus: Neuronal medium that supports basic synaptic functions and activity of human neurons in vitro

Human cell reprogramming technologies offer access to live human neurons from patients and provide a new alternative for modeling neurological disorders in vitro. Neural electrical activity is the essence of nervous system function in vivo. Therefore, we examined neuronal activity in media widely used to culture neurons. We found that classic basal media, as well as serum, impair action potential generation and synaptic communication. To overcome this problem, we designed a new neuronal medium (BrainPhys basal + serum-free supplements) in which we adjusted the concentrations of inorganic salts, neuroactive amino acids, and energetic substrates. We then tested that this medium adequately supports neuronal activity and survival of human neurons in culture. Long-term exposure to this physiological medium also improved the proportion of neurons that were synaptically active. The medium was designed to culture human neurons but also proved adequate for rodent neurons. The improvement in BrainPhys basal medium to support neurophysiological activity is an important step toward reducing the gap between brain physiological conditions in vivo and neuronal models in vitro.Induced pluripotent stem cell (iPSC) technology is currently being used to model human diseases in vitro and may contribute to the discovery and validation of new pharmacological treatments (1–3). In particular, neuroscientists have seized the opportunity to culture neurons from patients with neurological and psychiatric disorders and have demonstrated that phenotypes associated with particular disorders can be recapitulated in the dish (4–7). However, the basic culture conditions for growing neurons in vitro have not been updated to reflect fundamental principles of brain physiology. Currently, most human neuronal cultures are grown in media based on DMEM/F12 (4, 5, 7–24), Neurobasal (25–30), or a mixture of DMEM and Neurobasal (DN) (31–34). To promote neuronal differentiation and survival, a variety of supplements, such as serum, growth factors, hormones, proteins, and antioxidants, are typically added to these basal media. Although these media were designed and optimized to promote neuronal survival in vitro, they were not tested for their ability to support fundamental neuronal functions. Using several electrophysiological techniques such as patch clamping, calcium imaging, and multielectrode arrays, we found that widely used tissue culture media (e.g., DMEM basal, Neurobasal, serum) actually impaired neurophysiological functions.We identified several neuroactive components in these media that acutely interfered with neuronal function. To solve these issues, we designed a chemically defined basal medium: BrainPhys basal. We used human neurons in vitro to demonstrate in a series of experiments that this new medium, combined with the appropriate supplements, better supports important neuronal functions while sustaining cell survival in vitro. Notably, BrainPhys-based medium better mimics the environment present in healthy living brains, unlike previous media based on DMEM, Neurobasal or serum. Although BrainPhys basal was specifically designed for the culturing of mature human neurons, our studies also showed that BrainPhys provided a functional environment for ex vivo brain slices and for culturing rodent primary neurons.     

Drosophila seminal protein ovulin mediates ovulation through female octopamine neuronal signaling

Across animal taxa, seminal proteins are important regulators of female reproductive physiology and behavior. However, little is understood about the physiological or molecular mechanisms by which seminal proteins effect these changes. To investigate this topic, we studied the increase in Drosophila melanogaster ovulation behavior induced by mating. Ovulation requires octopamine (OA) signaling from the central nervous system to coordinate an egg’s release from the ovary and its passage into the oviduct. The seminal protein ovulin increases ovulation rates after mating. We tested whether ovulin acts through OA to increase ovulation behavior. Increasing OA neuronal excitability compensated for a lack of ovulin received during mating. Moreover, we identified a mating-dependent relaxation of oviduct musculature, for which ovulin is a necessary and sufficient male contribution. We report further that oviduct muscle relaxation can be induced by activating OA neurons, requires normal metabolic production of OA, and reflects ovulin’s increasing of OA neuronal signaling. Finally, we showed that as a result of ovulin exposure, there is subsequent growth of OA synaptic sites at the oviduct, demonstrating that seminal proteins can contribute to synaptic plasticity. Together, these results demonstrate that ovulin increases ovulation through OA neuronal signaling and, by extension, that seminal proteins can alter reproductive physiology by modulating known female pathways regulating reproduction.Throughout internally fertilizing animals, seminal proteins play important roles in regulating female fertility by altering female physiology and, in some cases, behavior after mating (reviewed in refs. 1–3). Despite this, little is understood about the physiological mechanisms by which seminal proteins induce postmating changes and how their actions are linked with known networks regulating female reproductive physiology.In Drosophila melanogaster, the suite of seminal proteins has been identified, as have many seminal protein-dependent postmating responses, including changes in egg production and laying, remating behavior, locomotion, feeding, and in ovulation rate (reviewed in refs. 2 and 3). For example, the Drosophila seminal protein ovulin elevates ovulation rate to maximal levels during the 24 h following mating (4, 5), and the seminal protein sex peptide (SP) suppresses female mating receptivity and increases egg-laying behavior for several days after mating (6–10). However, although a receptor for SP has been identified (11), along with elements of the neural circuit in which it is required (12–14), SP’s mechanism of action has not yet been linked to regulatory networks known to control postmating behaviors. Thus, a crucial question remains: how do male-derived seminal proteins interact with regulatory networks in females to trigger postmating responses?We addressed this question by examining the stimulation of Drosophila ovulation by the seminal protein ovulin. In insects, ovulation, defined here as the release of an egg from the ovary to the uterus, is among the best understood reproductive processes in terms of its physiology and neurogenetics (15–27). In D. melanogaster, ovulation requires input from neurons in the abdominal ganglia that release the catecholaminergic neuromodulators octopamine (OA) and tyramine (17, 18, 28). Drosophila ovulation also requires an OA receptor, OA receptor in mushroom bodies (OAMB) (19, 20). Moreover, it has been proposed that OA may integrate extrinsic factors to regulate ovulation rates (17). Noradrenaline, the vertebrate structural and functional equivalent to OA (29, 30), is important for mammalian ovulation, and its dysregulation has been associated with ovulation disorders (31–38). In this paper we investigate the role of neurons that release OA and tyramine in ovulin’s action. For simplicity, we refer to these neurons as “OA neurons” to reflect the well-established role of OA in ovulation behavior (16–20, 22).We investigated how action of the seminal protein ovulin relates to the conserved canonical neuromodulatory pathway that regulates ovulation physiology (39–41). We found that ovulin increases ovulation and egg laying through OA neuronal signaling. We also found that ovulin relaxes oviduct muscle tonus, a postmating process that is also mediated by OA neuronal signaling. Finally, subsequent to these effects we detected an ovulin-dependent increase in synaptic sites between OA motor neurons and oviduct muscle, suggesting that ovulin’s stimulation of OA neurons could have increased their synaptic activity. These results suggest that ovulin affects ovulation by manipulating the gain of a neuromodulatory pathway regulating ovulation physiology.     

Local generation of multineuronal spike sequences in the hippocampal CA1 region

Sequential activity of multineuronal spiking can be observed during theta and high-frequency ripple oscillations in the hippocampal CA1 region and is linked to experience, but the mechanisms underlying such sequences are unknown. We compared multineuronal spiking during theta oscillations, spontaneous ripples, and focal optically induced high-frequency oscillations (“synthetic” ripples) in freely moving mice. Firing rates and rate modulations of individual neurons, and multineuronal sequences of pyramidal cell and interneuron spiking, were correlated during theta oscillations, spontaneous ripples, and synthetic ripples. Interneuron spiking was crucial for sequence consistency. These results suggest that participation of single neurons and their sequential order in population events are not strictly determined by extrinsic inputs but also influenced by local-circuit properties, including synapses between local neurons and single-neuron biophysics.A hypothesized hallmark of cognition is self-organized sequential activation of neuronal assemblies (1). Self-organized neuronal sequences have been observed in several cortical structures (2–5) and neuronal models (6–7). In the hippocampus, sequential activity of place cells (8) may be induced by external landmarks perceived by the animal during spatial navigation (9) and conveyed to CA1 by the upstream CA3 region or layer 3 of the entorhinal cortex (10). Internally generated sequences have been also described in CA1 during theta oscillations in memory tasks (4, 11), raising the possibility that a given neuronal substrate is responsible for generating sequences at multiple time scales. The extensive recurrent excitatory collateral system of the CA3 region has been postulated to be critical in this process (4, 7, 12, 13).The sequential activity of place cells is “replayed” during sharp waves (SPW) in a temporally compressed form compared with rate modulation of place cells (14–20) and may arise from the CA3 recurrent excitatory networks during immobility and slow wave sleep. The SPW-related convergent depolarization of CA1 neurons gives rise to a local, fast oscillatory event in the CA1 region (“ripple,” 140–180 Hz; refs. 8 and 21). Selective elimination of ripples during or after learning impairs memory performance (22–24), suggesting that SPW ripple-related replay assists memory consolidation (12, 13). Although the local origin of the ripple oscillations is well demonstrated (25, 26), it has been tacitly assumed that the ripple-associated, sequentially ordered firing of CA1 neurons is synaptically driven by the upstream CA3 cell assemblies (12, 15), largely because excitatory recurrent collaterals in the CA1 region are sparse (27). However, sequential activity may also emerge by local mechanisms, patterned by the different biophysical properties of CA1 pyramidal cells and their interactions with local interneurons, which discharge at different times during a ripple (28–30). A putative function of the rich variety of interneurons is temporal organization of principal cell spiking (29–32). We tested the “local-circuit” hypothesis by comparing the probability of participation and sequential firing of CA1 neurons during theta oscillations, natural spontaneous ripple events, and “synthetic” ripples induced by local optogenetic activation of pyramidal neurons.     

Optogenetic activation of cholinergic neurons in the PPT or LDT induces REM sleep

Rapid eye movement (REM) sleep is an important component of the natural sleep/wake cycle, yet the mechanisms that regulate REM sleep remain incompletely understood. Cholinergic neurons in the mesopontine tegmentum have been implicated in REM sleep regulation, but lesions of this area have had varying effects on REM sleep. Therefore, this study aimed to clarify the role of cholinergic neurons in the pedunculopontine tegmentum (PPT) and laterodorsal tegmentum (LDT) in REM sleep generation. Selective optogenetic activation of cholinergic neurons in the PPT or LDT during non-REM (NREM) sleep increased the number of REM sleep episodes and did not change REM sleep episode duration. Activation of cholinergic neurons in the PPT or LDT during NREM sleep was sufficient to induce REM sleep.Rapid eye movement (REM) sleep is tightly regulated, yet the mechanisms that control REM sleep remain incompletely understood. Early pharmacological and unit recording studies suggested that ACh was important for REM sleep regulation (1, 2). For example, injection of cholinergic drugs into the dorsal mesopontine tegmentum reliably induced a state very similar to natural REM sleep in cats (3–6). Unit recordings from the cholinergic areas of the mesopontine tegmentum revealed cells that were active during wakefulness and REM sleep, as well as neurons active only during REM sleep (7–13). Electrical stimulation of the laterodorsal tegmentum (LDT) in cats increased the percentage of time spent in REM sleep (14), and activation of the pedunculopontine tegmentum (PPT) in rats induced wakefulness and REM sleep (15). If cholinergic PPT and LDT neurons are necessary for REM sleep to occur, as the early studies suggest, then lesioning the PPT or LDT should decrease REM sleep. In cats, lesions of the PPT and LDT do disrupt REM sleep (16, 17), but lesions in rodents have had little effect on REM sleep or increased REM sleep (18–22). Additionally, c-fos studies have found very few cholinergic cells activated under high-REM sleep conditions. When c-fos–positive cholinergic neurons in the PPT and LDT are found to correlate with the percentage of REM sleep, they still account for only a few of the total cholinergic cells in the area (23). Juxtacellular recordings of identified cholinergic neurons in the LDT found these cells had wake and REM active firing profiles, with the majority firing the highest during REM sleep (13). These discrepancies have led to alternative theories of REM sleep regulation, where cholinergic neurons do not play a key role (18, 19, 23, 24 and reviewed in 25, 26).The PPT and LDT are made up of heterogeneous populations of cells, including distinct populations of cholinergic, GABAergic, and glutamatergic neurons (27–29). Many GABAergic neurons are active during REM sleep, as indicated by c-fos (23), and both GABAergic and glutamatergic neurons have been found with maximal firing rates during REM sleep in the LDT and medial PPT (13). To distinguish the differential roles of each cell type in REM sleep regulation, a method that can modulate specific cell types in the behaving animal is needed. Optogenetics now provides this ability to target specific subpopulations of neurons and control them with millisecond temporal resolution (30). Therefore, we aimed to determine the role of cholinergic neurons in the PPT and LDT in REM sleep regulation using optogenetics.     

Distinct dopamine neurons mediate reward signals for short- and long-term memories

Drosophila melanogaster can acquire a stable appetitive olfactory memory when the presentation of a sugar reward and an odor are paired. However, the neuronal mechanisms by which a single training induces long-term memory are poorly understood. Here we show that two distinct subsets of dopamine neurons in the fly brain signal reward for short-term (STM) and long-term memories (LTM). One subset induces memory that decays within several hours, whereas the other induces memory that gradually develops after training. They convey reward signals to spatially segregated synaptic domains of the mushroom body (MB), a potential site for convergence. Furthermore, we identified a single type of dopamine neuron that conveys the reward signal to restricted subdomains of the mushroom body lobes and induces long-term memory. Constant appetitive memory retention after a single training session thus comprises two memory components triggered by distinct dopamine neurons.Memory of a momentous event persists for a long time. Whereas some forms of long-term memory (LTM) require repetitive training (1–3), a highly relevant stimulus such as food or poison is sufficient to induce LTM in a single training session (4–7). Recent studies have revealed aspects of the molecular and cellular mechanisms of LTM formation induced by repetitive training (8–11), but how a single training induces a stable LTM is poorly understood (12).Appetitive olfactory learning in fruit flies is suited to address the question, as a presentation of a sugar reward paired with odor induces robust short-term memory (STM) and LTM (6, 7). Odor is represented by a sparse ensemble of the 2,000 intrinsic neurons, the Kenyon cells (13). A current working model suggests that concomitant reward signals from sugar ingestion cause associative plasticity in Kenyon cells that might underlie memory formation (14–20). A single activation session of a specific cluster of dopamine neurons (PAM neurons) by sugar ingestion can induce appetitive memory that is stable over 24 h (19), underscoring the importance of sugar reward to the fly.The mushroom body (MB) is composed of the three different cell types, α/β, α′/β′, and γ, which have distinct roles in different phases of appetitive memories (11, 21–25). Similar to midbrain dopamine neurons in mammals (26, 27), the structure and function of PAM cluster neurons are heterogeneous, and distinct dopamine neurons intersect unique segments of the MB lobes (19, 28–34). Further circuit dissection is thus crucial to identify candidate synapses that undergo associative modulation.By activating distinct subsets of PAM neurons for reward signaling, we found that short- and long-term memories are independently formed by two complementary subsets of PAM cluster dopamine neurons. Conditioning flies with nutritious and nonnutritious sugars revealed that the two subsets could represent different reinforcing properties: sweet taste and nutritional value of sugar. Constant appetitive memory retention after a single training session thus comprises two memory components triggered by distinct reward signals.     

Cortical oscillations arise from contextual interactions that regulate sparse coding

Precise spike times carry information and are important for synaptic plasticity. Synchronizing oscillations such as gamma bursts could coordinate spike times, thus regulating information transmission in the cortex. Oscillations are driven by inhibitory neurons and are modulated by sensory stimuli and behavioral states. How their power and frequency are regulated is an open question. Using a model cortical circuit, we propose a regulatory mechanism that depends on the activity balance of monosynaptic and disynaptic pathways to inhibitory neurons: Monosynaptic input causes more powerful oscillations whereas disynaptic input increases the frequency of oscillations. The balance of stimulation to the two pathways modulates the overall distribution of spikes, with stronger disynaptic stimulation (e.g., preferred stimuli inside visual receptive fields) producing high firing rates and weak oscillations; in contrast, stronger monosynaptic stimulation (e.g., suppressive contextual stimulation from outside visual receptive fields) generates low firing rates and strong oscillatory regulation of spike timing, as observed in alert cortex processing complex natural stimuli. By accounting for otherwise paradoxical experimental findings, our results demonstrate how the frequency and power of oscillations, and hence spike times, can be modulated by both sensory input and behavioral context, with powerful oscillations signifying a cortical state under inhibitory control in which spikes are sparse and spike timing is precise.Individual neurons can precisely time their spikes when driven by temporally fluctuating synaptic inputs (1). Narrowband oscillations mediated by inhibitory neurons are thought to be a key source of coordinated fluctuating discharges from input neurons, and they vary in power and frequency during wakeful behavior and sleep. Oscillations in the gamma range (30–80 Hz), thought to be mediated by fast-spiking inhibitory neurons expressing the calcium-binding protein parvalbumin (2, 3), are modulated by the sensory environment (4–6), attention (7), and volition (8), as well as by specific memory tasks, causing changes in sensory responses (2) and information transfer (3) in the cortex. The modulation is observed both in the oscillation power, which we define as the peak of a distinct “bump” in the power spectrum of the local field potential (LFP), as well as the oscillation frequency, which is the frequency at this peak in the power spectrum (5, 6). In current models of oscillations in neuronal networks, oscillations are regulated by stimulation of inhibitory neurons such that increasing stimulation mainly increases their frequency (9–11) or power (12). In the visual cortex, both the contrast and size of visual stimuli increase the stimulation to local inhibitory neurons (13, 14), but the former increases the frequency of gamma-range oscillations (6), and the latter decreases it (5). The power of gamma oscillations increases in the somatosensory, medial temporal (15), motor (8), olfactory (16), and primary visual cortex (5) with increased stimulation to local inhibitory neurons. However, the peak power of oscillations decreases with increased stimulation of inhibitory neurons with attention (17) in some cortical areas (7). In a third scenario, whereas the broadband power in the LFP signal increases with increasing visual contrast (6, 18), peak narrowband power shows no significant trend in response to increasing contrast (8), which is thought to increase the stimulation to the local inhibitory neurons (13).We show that these diverse experimental observations can be explained by the following hypothesis: The balance of two distinct pathways that activate local inhibitory neurons mediates bidirectional regulation of oscillations (Fig. 1A). We classify these pathways as monosynaptic (MS), those that make direct excitatory synaptic connections to the inhibitory neurons, and disynaptic (DS), those that act through the local excitatory neurons.Fig. 1.Relative strength of MS and DS stimulation to inhibitory neurons determines the power and frequency of oscillations in spiking activity. (A) Schematic of local network and the monosynaptic (solid black) and disynaptic (dotted black) pathways for stimulating ...     

Dissecting neural pathways for forgetting in Drosophila olfactory aversive memory

Recent studies have identified molecular pathways driving forgetting and supported the notion that forgetting is a biologically active process. The circuit mechanisms of forgetting, however, remain largely unknown. Here we report two sets of Drosophila neurons that account for the rapid forgetting of early olfactory aversive memory. We show that inactivating these neurons inhibits memory decay without altering learning, whereas activating them promotes forgetting. These neurons, including a cluster of dopaminergic neurons (PAM-β′1) and a pair of glutamatergic neurons (MBON-γ4>γ1γ2), terminate in distinct subdomains in the mushroom body and represent parallel neural pathways for regulating forgetting. Interestingly, although activity of these neurons is required for memory decay over time, they are not required for acute forgetting during reversal learning. Our results thus not only establish the presence of multiple neural pathways for forgetting in Drosophila but also suggest the existence of diverse circuit mechanisms of forgetting in different contexts.Although forgetting commonly has a negative connotation, it is a functional process that shapes memory and cognition (1–4). Recent studies, including work in relatively simple invertebrate models, have started to reveal basic biological mechanisms underlying forgetting (5–15). In Drosophila, single-session Pavlovian conditioning by pairing an odor (conditioned stimulus, CS) with electric shock (unconditioned stimulus, US) induces aversive memories that are short-lasting (16). The memory performance of fruit flies is observed to drop to a negligible level within 24 h, decaying rapidly early after training and slowing down thereafter (17). Memory decay or forgetting requires the activation of the small G protein Rac, a signaling protein involved in actin remodeling, in the mushroom body (MB) intrinsic neurons (6). These so-called Kenyon cells (KCs) are the neurons that integrate CS–US information (18, 19) and support aversive memory formation and retrieval (20–22). In addition to Rac, forgetting also requires the DAMB dopamine receptor (7), which has highly enriched expression in the MB (23). Evidence suggests that the dopamine-mediated forgetting signal is conveyed to the MB by dopamine neurons (DANs) in the protocerebral posterior lateral 1 (PPL1) cluster (7, 24). Therefore, forgetting of olfactory aversive memory in Drosophila depends on a particular set of intracellular molecular pathways within KCs, involving Rac, DAMB, and possibly others (25), and also receives modulation from extrinsic neurons. Although important cellular evidence supporting the hypothesis that memory traces are erased under these circumstances is still lacking, these findings lend support to the notion that forgetting is an active, biologically regulated process (17, 26).Although existing studies point to the MB circuit as essential for forgetting, several questions remain to be answered. First, whereas the molecular pathways for learning and forgetting of olfactory aversive memory are distinct and separable (6, 7), the neural circuits seem to overlap. Rac-mediated forgetting has been localized to a large population of KCs (6), including the γ-subset, which is also critical for initial memory formation (21, 27). The site of action of DAMB for forgetting has yet to be established; however, the subgroups of PPL1-DANs implicated in forgetting are the same as those that signal aversive reinforcement and are required for learning (28–30). It leaves open the question of whether the brain circuitry underlying forgetting and learning is dissociable, or whether forgetting and learning share the same circuit but are driven by distinct activity patterns and molecular machinery (26). Second, shock reinforcement elicits multiple memory traces through at least three dopamine pathways to different subdomains in the MB lobes (28, 29). Functional imaging studies have also revealed Ca²⁺-based memory traces in different KC populations (31). It is poorly understood how forgetting of these memory traces differs, and it remains unknown whether there are multiple regulatory neural pathways. Notably, when PPL1-DANs are inactivated, forgetting still occurs, albeit at a lower rate (7). This incomplete block suggests the existence of an additional pathway(s) that conveys forgetting signals to the MB. Third, other than memory decay over time, forgetting is also observed through interference (32, 33), when new learning or reversal learning is introduced after training (6, 34, 35). Time-based and interference-based forgetting shares a similar dependence on Rac and DAMB (6, 7). However, it is not known whether distinct circuits underlie forgetting in these different contexts.In the current study, we focus on the diverse set of MB extrinsic neurons (MBENs) that interconnect the MB lobes with other brain regions, which include 34 MB output neurons (MBONs) of 21 types and ∼130 dopaminergic neurons of 20 types in the PPL1 and protocerebral anterior medial (PAM) clusters (36, 37). These neurons have been intensively studied in olfactory memory formation, consolidation, and retrieval in recent years (e.g., 24, 28–30, 38–48); however, their roles in forgetting have not been characterized except for the aforementioned PPL1-DANs. In a functional screen, we unexpectedly found that several Gal4 driver lines of MBENs showed significantly better 3-h memory retention when the Gal4-expressing cells were inactivated. The screen has thus led us to identify two types of MBENs that are not involved in initial learning but play important and additive roles in mediating memory decay. Furthermore, neither of these MBEN types is required for reversal learning, supporting the notion that there is a diversity of neural circuits that drive different forms of forgetting.     

Tectal microcircuit generating visual selection commands on gaze-controlling neurons

The optic tectum (called superior colliculus in mammals) is critical for eye–head gaze shifts as we navigate in the terrain and need to adapt our movements to the visual scene. The neuronal mechanisms underlying the tectal contribution to stimulus selection and gaze reorientation remains, however, unclear at the microcircuit level. To analyze this complex—yet phylogenetically conserved—sensorimotor system, we developed a novel in vitro preparation in the lamprey that maintains the eye and midbrain intact and allows for whole-cell recordings from prelabeled tectal gaze-controlling cells in the deep layer, while visual stimuli are delivered. We found that receptive field activation of these cells provide monosynaptic retinal excitation followed by local GABAergic inhibition (feedforward). The entire remaining retina, on the other hand, elicits only inhibition (surround inhibition). If two stimuli are delivered simultaneously, one inside and one outside the receptive field, the former excitatory response is suppressed. When local inhibition is pharmacologically blocked, the suppression induced by competing stimuli is canceled. We suggest that this rivalry between visual areas across the tectal map is triggered through long-range inhibitory tectal connections. Selection commands conveyed via gaze-controlling neurons in the optic tectum are, thus, formed through synaptic integration of local retinotopic excitation and global tectal inhibition. We anticipate that this mechanism not only exists in lamprey but is also conserved throughout vertebrate evolution.Visual scenes are composed of abundant stimuli, and the gaze needs continuously to be redirected toward different objects—an important task for the brain. Current models postulate that stimulus selection occurs through a process involving competitive interaction between different visual stimuli, resulting in the appropriate eye–head movement (1–5). The optic tectum (superior colliculus in mammals) has a causal role in the stimulus selection process (6–12) and not only in the control of saccades and eye–head gaze shifts (13–16). Although the collicular contribution to the selection process is of central importance, the underlying neuronal processes have remained elusive due to methodological limitations. It is our aim here to address this issue in a novel experimental model.The optic tectum is well developed in the lamprey, belonging to the oldest extant vertebrate group that evolved 560 million years ago (17), and it has remained conserved throughout vertebrate phylogeny (18–22). Afferents from retina provide a direct input to the superficial layers of the optic tectum, where a retinotopic map is formed (23–26). The intermediate and deep layers give rise to projections to brainstem areas and a motor map is formed that is responsible for the coordination of eye, head, and body movements (22, 27–29).To uncover the mechanisms underlying visual stimulus selection for gaze reorientation, detailed intracellular analysis is needed in combination with visual activation, which has not been possible in the classic mammalian preparations. To achieve this, we have developed a preparation in lamprey that maintains the eye and the midbrain intact in vitro, which has allowed us to perform whole-cell recordings from identified gaze-controlling cells in the optic tectum, while delivering natural and focal light stimuli within the visual field and monitoring and manipulating the synaptic responses. The retinotopic map in the lamprey tectum and the aligned motor map have been described in considerable detail (22, 26), and the lamprey nervous system from forebrain to spinal cord is experimentally very accessible, well described, and also conserved throughout vertebrate evolution (30–32).Although most mammalian studies have focused on the role of the retinotopic excitatory circuits that mediate signal transmission between the superficial layer and the deeper layers (33–36), only more recent studies have emphasized the role of GABAergic circuits in the collicular control of gaze (37–45). The question how collicular GABAergic systems affect stimulus selection for gaze motor action has, however, remained unanswered because most studies have relied on extracellular recordings in vivo and therefore have not allowed an analysis of the synaptic basis for these inhibitory interactions.We show here that tectal gaze-controlling cells receive intense excitatory monosynaptic retinal input in a bottom-up manner, which with a slight delay is followed by an even stronger inhibition from local GABAergic neurons. The local inhibition gives rise to visual stimulus selectivity by suppressing competing retinotopic stimuli with long-range horizontal projections across tectum. When this inhibition is blocked, stimulus selection is eliminated and gaze-controlling cells could discharge action potentials indiscriminately whether or not competing stimuli were present. The presence of a potential target will trigger local inhibitory and horizontally projecting neurons that will aim to reduce the excitability of cells in other tectal areas, hence, reducing the likelihood of executing a gaze motor response to other distracting stimuli. This stimulus suppression mechanism provides a robust solution for generating accurate attentional shifts, because the outcome of the synaptic interaction is directly integrated by the tectal output cells responsible for gaze action.     

Acuity-independent effects of visual deprivation on human visual cortex

Visual development depends on sensory input during an early developmental critical period. Deviation of the pointing direction of the two eyes (strabismus) or chronic optical blur (anisometropia) separately and together can disrupt the formation of normal binocular interactions and the development of spatial processing, leading to a loss of stereopsis and visual acuity known as amblyopia. To shed new light on how these two different forms of visual deprivation affect the development of visual cortex, we used event-related potentials (ERPs) to study the temporal evolution of visual responses in patients who had experienced either strabismus or anisometropia early in life. To make a specific statement about the locus of deprivation effects, we took advantage of a stimulation paradigm in which we could measure deprivation effects that arise either before or after a configuration-specific response to illusory contours (ICs). Extraction of ICs is known to first occur in extrastriate visual areas. Our ERP measurements indicate that deprivation via strabismus affects both the early part of the evoked response that occurs before ICs are formed as well as the later IC-selective response. Importantly, these effects are found in the normal-acuity nonamblyopic eyes of strabismic amblyopes and in both eyes of strabismic patients without amblyopia. The nonamblyopic eyes of anisometropic amblyopes, by contrast, are normal. Our results indicate that beyond the well-known effects of strabismus on the development of normal binocularity, it also affects the early stages of monocular feature processing in an acuity-independent fashion.Over 50 y of research on experimental animal models has indicated that deprivation of normal visual experience during a developmental critical period perturbs both the structure and function of primary visual cortex (1–4). The animal models were developed to understand the underlying neural mechanisms of amblyopia, a common human developmental disorder of spatial vision associated with the presence of strabismus, anisometropia, or form deprivation during early life (5). Amblyopia is classically defined on the basis of poor visual acuity, but many other visual functions are known to be affected (6–8).The earliest experimental studies of visual deprivation focused on the effects of monocular lid suture, and these studies showed devastating effects on the ability of the deprived eye to drive neural responses, retain synaptic connections, and guide visual behavior (9–11). Later work studied less extreme forms of deprivation that are common in humans, such as the effects of strabismus (deviation of the pointing direction of the two eyes) (12, 13) or anisometropia (chronic optical blur) (14, 15). More recent studies (16, 17) have found that losses in cell responses in primary visual cortex appear to be insufficient to explain the magnitude of behaviorally measured deficits. Based on these results, a hypothesis has been put forward that these forms of deprivation have their primary effects in extrastriate cortex (16).Motivated by this idea, psychophysicists have sought evidence that extrastriate cortex is particularly impaired in human amblyopia. This work has used tasks whose execution is fundamentally limited by processing resources that single-cell physiology suggests are located in extrastriate cortex. As a second step, these studies have scaled stimuli based on visual acuity and compensated for contrast sensitivity losses to equate the output of early visual cortex from the amblyopic eye to that of normal-vision participants. Despite a nominal match at the level of early visual cortex outputs, patients with amblyopia still show deficits on illusory tilt perception (18), contour integration (19–23), global motion sensitivity (8, 24–28), object enumeration (29), and object tracking (7, 30). The impairments listed above have been interpreted to indicate that amblyopia may involve abnormalities in “higher-level” (e.g., extrastriate) neural processing that occur independent of any deficits in early processing stages (e.g., in striate cortex). A limitation of the existing psychophysical approaches has been the need to make an assumption that the stimulus scaling used to equate stimuli for visibility fully equilibrates the activity of early visual cortex. It would be preferable to take an approach that allows one to measure neural responses directly from both early and later stages of visual processing. Here we use event-related potentials (ERPs) and a stimulation paradigm that allow us to record responses from both early visual cortex and higher-level, extrastriate areas.Our approach is similar in spirit to existing psychophysical approaches: We use a stimulus configuration—illusory contours (ICs)—that previous single-unit studies have shown to be first extracted in extrastriate cortex (31–34). ICs, also referred to as subjective contours, render object borders that are perceptually vivid but that are created in the absence of luminance contrast or chrominance gradients (35). ICs have been widely used to study mechanisms of scene segmentation and grouping operations that are among the most fundamental tasks the visual system has to perform (36). ICs have garnered considerable interest because of their “inferential” nature—despite the lack of luminance edges, the visual system uses implicit configural cues to infer the presence of a contour. Finally, behavioral investigations in macaque suggest that IC perception is strongly dependent on higher visual areas, including V4 (37, 38) and inferotemporal (IT) cortex (39, 40).Instead of attempting to equate the visibility of stimuli in the amblyopic eye to that of normal control eyes, as has been typical practice in the study of amblyopia, we make a close analysis of the effects of deprivation that is based on ERP responses from the nonamblyopic eyes of patients with anisometropic or strabismic amblyopia. These eyes have normal visual acuity and normal or even supernormal contrast sensitivity (41), making the stimuli nominally equivisible without the need for scaling. We then measure evoked responses at early latencies before the time that IC selectivity arises to assess the integrity of early visual cortex, and compare these responses to those measured at longer latencies after robust IC selectivity has been established. Previous single-unit studies that have used ICs of the type used in the present study indicate that they are first extracted no later than V2 (31, 42, 43) or V4 (34). Given the difference in species and stimuli, we will refer in the following to evoked responses that lack IC sensitivity as having arisen in “early” visual cortex, rather than in specific visual areas. To further specify the site of deprivation effects, we also study a group of stereo-blind patients with strabismus who do not have amblyopia (normal visual acuity in each eye).A second goal of our study is to compare the effects of deprivation from unilateral blur (anisometropia) to that caused by strabismus. The human psychophysical literature has made a distinction in the pattern of visual loss associated with strabismus versus that associated with anisometropia (44). At least some of the differences in performance between these two types of deprivation can be explained on the level of residual stereopsis, which typically differs between these two populations (41). Whenever these two types of deprivation have been compared in terms of their effects on the monocular cell properties of V1, there has been little to differentiate the effects of the two types of deprivation (16, 45, 46). Unfortunately, there are relatively few studies of the effects of critical period deprivation on the cell-tuning properties in extrastriate cortex of any species (15, 17, 47), and there has been no comparison of the effects of strabismus vs. anisometropia in extrastriate cortex. The implication of the existing animal literature is that strabismus and anisometropia have comparable effects on early visual cortex and thus the divergence in their behavioral phenotype, as well as the major effects of deprivation, will lie in extrastriate cortex. Here we show that these two types of deprivation have differential effects very early in visual cortex, possibly as early as the transfer of information from V1 to V2.     

In vivo imaging of immediate early gene expression reveals layer-specific memory traces in the mammalian brain

The dynamic processes of formatting long-term memory traces in the cortex are poorly understood. The investigation of these processes requires measurements of task-evoked neuronal activities from large numbers of neurons over many days. Here, we present a two-photon imaging-based system to track event–related neuronal activity in thousands of neurons through the quantitative measurement of EGFP proteins expressed under the control of the EGR1 gene promoter. A recognition algorithm was developed to detect GFP-positive neurons in multiple cortical volumes and thereby to allow the reproducible tracking of 4,000 neurons in each volume for 2 mo. The analysis revealed a context-specific response in sparse layer II neurons. The context-evoked response gradually increased during several days of training and was maintained 1 mo later. The formed traces were specifically activated by the training context and were linearly correlated with the behavioral response. Neuronal assemblies that responded to specific contexts were largely separated, indicating the sparse coding of memory-related traces in the layer II cortical circuit.In the mammalian brain, memory traces in cortical areas are poorly understood. In contrast to the medial temporal lobe, particularly the hippocampus, which is involved in the temporary storage of declarative memories (1, 2), the neocortex is believed to store remote memories (3–6). However, remarkably little knowledge regarding the sites and dynamics of remote memory storage has been revealed at the cellular level owing to the complexity of the connections and the large number of neurons within the cortical circuit.In vivo electrophysiological recording of neuronal firing revolutionized neurobiology by linking neuronal activity with animal behavior. The small number of neurons recorded by the electrodes, however, was a limitation, as information coding and decoding may use an army of neurons forming neuronal assemblies (7, 8). Efforts to record the activity of larger populations of neurons in cortical volumes have been actively pursued by either increasing the number of electrode probes (7, 9–11) or using calcium indicator–based imaging (12–15) and immediate early gene (IEG)-based reporters (16–18). The expression of IEGs is correlated with the averaged neuronal activation on external stimuli (19, 20), implying that the marked neurons are involved in behavior (1, 21–25). Studies using in vivo imaging of IEGs have revealed cortical coding in the visual cortex and in other cortical areas, reflecting electrical activation in individual neurons (16, 17). Among IEGs, the expression of early growth response protein 1 (EGR1, also known as zif268) is associated with high-frequency stimulation and the induction of long-term plasticity during learning (26, 27). To measure neuronal activation in cortical circuits during a behavioral task, we used an EGR1 expression reporter mouse line in which the expression of the EGFP protein is under the control of the Egr1 gene promoter. We designed offline recording strategies to monitor task-associated neuronal activity by quantifying changes in cellular EGFP signals in the mouse cortex. Patterns of activated neuronal assemblies during different tasks were visualized in the entire cortical volume. Furthermore, through computer recognition-based reconstruction, we were able to track the activity-related cellular EGFP signals from multiple cortical areas for 2 mo to reveal memory-related changes in the cortical circuit.     

Loss of Miro1-directed mitochondrial movement results in a novel murine model for neuron disease

Defective mitochondrial distribution in neurons is proposed to cause ATP depletion and calcium-buffering deficiencies that compromise cell function. However, it is unclear whether aberrant mitochondrial motility and distribution alone are sufficient to cause neurological disease. Calcium-binding mitochondrial Rho (Miro) GTPases attach mitochondria to motor proteins for anterograde and retrograde transport in neurons. Using two new KO mouse models, we demonstrate that Miro1 is essential for development of cranial motor nuclei required for respiratory control and maintenance of upper motor neurons required for ambulation. Neuron-specific loss of Miro1 causes depletion of mitochondria from corticospinal tract axons and progressive neurological deficits mirroring human upper motor neuron disease. Although Miro1-deficient neurons exhibit defects in retrograde axonal mitochondrial transport, mitochondrial respiratory function continues. Moreover, Miro1 is not essential for calcium-mediated inhibition of mitochondrial movement or mitochondrial calcium buffering. Our findings indicate that defects in mitochondrial motility and distribution are sufficient to cause neurological disease.Motor neuron diseases (MNDs), including ALS and spastic paraplegia (SP), are characterized by the progressive, length-dependent degeneration of motor neurons, leading to muscle atrophy, paralysis, and, in some cases, premature death. There are both inherited and sporadic forms of MNDs, which can affect upper motor neurons, lower motor neurons, or both. Although the molecular and cellular causes of most MNDs are unknown, many are associated with defects in axonal transport of cellular components required for neuron function and maintenance (1–6).A subset of MNDs is associated with impaired mitochondrial respiration and mitochondrial distribution. This observation has led to the hypothesis that neurodegeneration results from defects in mitochondrial motility and distribution, which, in turn, cause subcellular ATP depletion and interfere with mitochondrial calcium ([Ca²⁺]_m) buffering at sites of high synaptic activity (reviewed in ref. 7). It is not known, however, whether mitochondrial motility defects are a primary cause or a secondary consequence of MND progression. In addition, it has been difficult to isolate the primary effect of mitochondrial motility defects in MNDs because most mutations that impair mitochondrial motility in neurons also affect transport of other organelles and vesicles (1, 8–11).In mammals, the movement of neuronal mitochondria between the cell body and the synapse is controlled by adaptors called trafficking kinesin proteins (Trak1 and Trak2) and molecular motors (kinesin heavy chain and dynein), which transport the organelle in the anterograde or retrograde direction along axonal microtubule tracks (7, 12–24). Mitochondrial Rho (Miro) GTPase proteins are critical for transport because they are the only known surface receptors that attach mitochondria to these adaptors and motors (12–15, 18, 25, 26). Miro proteins are tail-anchored in the outer mitochondrial membrane with two GTPase domains and two predicted calcium-binding embryonic fibroblast (EF) hand motifs facing the cytoplasm (12, 13, 25, 27, 28). A recent Miro structure revealed two additional EF hands that were not predicted from the primary sequence (29). Studies in cultured cells suggest that Miro proteins also function as calcium sensors (via their EF hands) to regulate kinesin-mediated mitochondrial “stopping” in axons (15, 16, 26). Miro-mediated movement appears to be inhibited when cytoplasmic calcium is elevated in active synapses, effectively recruiting mitochondria to regions where calcium buffering and energy are needed. Despite this progress, the physiological relevance of these findings has not yet been tested in a mammalian animal model. In addition, mammals ubiquitously express two Miro orthologs, Miro1 and Miro2, which are 60% identical (12, 13). However, the individual roles of Miro1 and Miro2 in neuronal development, maintenance, and survival have no been evaluated.We describe two new mouse models that establish the importance of Miro1-mediated mitochondrial motility and distribution in mammalian neuronal function and maintenance. We show that Miro1 is essential for development/maintenance of specific cranial neurons, function of postmitotic motor neurons, and retrograde mitochondrial motility in axons. Loss of Miro1-directed retrograde mitochondrial transport is sufficient to cause MND phenotypes in mice without abrogating mitochondrial respiratory function. Furthermore, Miro1 is not essential for calcium-mediated inhibition of mitochondrial movement or [Ca²⁺]_m buffering. These findings have an impact on current models for Miro1 function and introduce a specific and rapidly progressing mouse model for MND.     

Receptive field focus of visual area V4 neurons determines responses to illusory surfaces

Illusory figures demonstrate the visual system’s ability to infer surfaces under conditions of fragmented sensory input. To investigate the role of midlevel visual area V4 in visual surface completion, we used multielectrode arrays to measure spiking responses to two types of visual stimuli: Kanizsa patterns that induce the perception of an illusory surface and physically similar control stimuli that do not. Neurons in V4 exhibited stronger and sometimes rhythmic spiking responses for the illusion-promoting configurations compared with controls. Moreover, this elevated response depended on the precise alignment of the neuron’s peak visual field sensitivity (receptive field focus) with the illusory surface itself. Neurons whose receptive field focus was over adjacent inducing elements, less than 1.5° away, did not show response enhancement to the illusion. Neither receptive field sizes nor fixational eye movements could account for this effect, which was present in both single-unit signals and multiunit activity. These results suggest that the active perceptual completion of surfaces and shapes, which is a fundamental problem in natural visual experience, draws upon the selective enhancement of activity within a distinct subpopulation of neurons in cortical area V4.Visual illusions are valuable stimuli for studying the neural basis of visual processing because they reveal the brain’s internal mechanisms for interpreting sensory input. Illusory figures, for example, exploit the visual system’s capacity to construct contours, shapes, and surfaces despite the lack of a continuous physical border (1, 2). Illusory figures are perceived by a range of phylogenetically diverse species, including monkeys, cats, owls, and bees, pointing to perceptual completion as a fundamental aspect of natural vision (3).Neural correlates of illusory figures have been found in a wide range of brain areas. Recordings in monkeys revealed that illusory figures evoke spiking responses from neurons in visual areas as early as V1 and V2 and as late as the inferotemporal cortex (4–9). Neuroimaging studies in humans similarly found responses to illusory figures throughout visual cortex (10–13).Several theoretical models postulate mechanisms of illusory figure perception (14–19). A common feature of these models is spatial integration of the inducing elements combined with an active interpolation to complete the surface. These processes are frequently assigned to neurons in midlevel areas, whose receptive fields are large enough to cover separate elements yet sensitive enough to distinguish between local features such as orientation, curvature, and colinearity (20, 21). A range of evidence suggests that visual area V4 in particular may play an active role in surface completion. First, the receptive fields of V4 neurons are large by comparison with V1 and V2 receptive fields and are therefore able to integrate information across spatially separated stimulus components (22). Second, psychophysical studies demonstrate that the perception of certain similar illusory figures varies over visual space in a manner consistent with the retinotopy of V4 (23, 24). Third, both human (10–13) and nonhuman primate (25) functional imaging studies reveal responses to illusory contours and surfaces in area V4. Fourth, ablation of area V4 in the macaque selectively impairs performance on discrimination tasks that involve illusory contours (26).Here we investigate the neural representation of illusory surfaces in macaque area V4 using Kanizsa patterns known to give rise to the perception of illusory surfaces. Illusion-promoting patterns elicited electrophysiological responses that were often rhythmic and were significantly enhanced in their firing rate compared with physically similar control patterns that did not promote the illusion. This enhancement depended critically on the spatial alignment of the illusory surface with the point of peak V4 receptive field sensitivity, or “RF focus.” Only neurons with receptive fields focused on the illusory surface showed elevated responses to the illusory surface, whereas those with receptive fields focused on the inducing elements did not. This effect was observed for neurons whose receptive fields, as defined by conventional mapping techniques, were several degrees in size and overlapped with both the illusory surface and the inducer elements. These observations suggest that V4 neurons play an active role in the representation of illusory surfaces and are sensitive to stimulus details much finer than would be predicted based on receptive field size alone.