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Visuospatial information in the retinotectal system of xenopus before correct image formation by the developing eye.
The retinotectal pathway of Xenopus laevis is a well-established experimental model for studying activity-dependent processes during visual system development. Such processes can be guided by stimulus-evoked activity patterns, which depend on the refractive characteristics of the eye. Previous work has shown that many animals are hyperopic at early developmental stages due to immature refractive properties. Whether this is also the case for Xenopus laevis is unknown. Here, we measure the focal length of the lens and the size of the eye of embryos at different stages and find that Xenopus laevis exhibits a similar shift from hyperopia to emmetropia. At early stages, immediately after innervation of the tectum by the optic nerve, Xenopus embryos are hyperopic. Soon afterwards the focal length of the lens decreases and the eye converges to a state of emmetropia. Despite being hyperopic we find that some visuospatial information is available to the young circuit. Calculations based on the optical properties of the eye show that even when the animals are hyperopic the blurred retinal image provides a crude spatial resolution. Furthermore, using whole-cell recordings in the optic tectum combined with visual stimulation through the intact eye, we show that tectal neurons in hyperopic embryos have spatially restricted glutamatergic receptive fields. Our data demonstrate that Xenopus laevis eyes undergo a process of developmental emmetropization, and suggest that despite an initial stage of suboptimal image formation there is potentially enough information to guide activity-dependent refinements of the retinotectal pathway from the onset of vision.
The X-linked mental retardation protein oligophrenin-1 is required for dendritic spine morphogenesis.
Of 11 genes involved in nonspecific X-linked mental retardation (MRX), three encode regulators or effectors of the Rho GTPases, suggesting an important role for Rho signaling in cognitive function. It remains unknown, however, how mutations in Rho-linked genes lead to MRX. Here we report that oligophrenin-1, a Rho-GTPase activating protein that is absent in a family affected with MRX, is required for dendritic spine morphogenesis. Using RNA interference and antisense RNA approaches, we show that knock-down of oligophrenin-1 levels in CA1 neurons in rat hippocampal slices significantly decreases spine length. This phenotype can be recapitulated using an activated form of RhoA and rescued by inhibiting Rho-kinase, indicating that reduced oligophrenin-1 levels affect spine length by increasing RhoA and Rho-kinase activities. We further demonstrate an interaction between oligophrenin-1 and the postsynaptic adaptor protein Homer. Our findings provide the first insight into how mutations in a Rho-linked MRX gene may compromise neuronal function.
Responses of neurons in neonatal cortex and thalamus to patterned visual stimulation through the naturally closed lids.
In studies of the developing mammalian visual system, it has been axiomatic that visual experience begins with eye-opening. Any role for neuronal activity earlier in development has been attributed to the patterned spontaneous activity found in retina and lateral geniculate nucleus (LGN). Here we show that, as early as 2 wk before eye-opening, visual stimuli presented through the closed eyelids can drive neuronal activity in LGN and striate cortex of the ferret. At this age, spontaneous activity in cortex is much lower than in LGN, and the visual responses of many cortical, but not geniculate, neurons depend on the orientation of a moving grating. Furthermore the selectivity of cortical neurons to the orientation of gratings presented through the closed eyelids improves with age. Thus neuronal activity patterned by visual experience, rather than by spontaneous retinal activity, is present in visual cortex much earlier than previously thought. This could have important implications for the self-organization of visual cortex.
Visually driven regulation of intrinsic neuronal excitability improves stimulus detection in vivo.
Neurons adapt their electrophysiological properties to maintain stable levels of electrical excitability when faced with a constantly changing environment. We find that exposing freely swimming Xenopus tadpoles to 4-5 hr of persistent visual stimulation increases the intrinsic excitability of optic tectal neurons. This increase is correlated with enhanced voltage-gated Na+ currents. The same visual stimulation protocol also induces a polyamine synthesis-dependent reduction in Ca2+-permeable AMPAR-mediated synaptic drive, suggesting that the increased excitability may compensate for this reduction. Accordingly, the change in excitability was prevented by blocking polyamine synthesis during visual stimulation and was rescued when Ca2+-permeable AMPAR-mediated transmission was selectively reduced. The changes in excitability also rendered tectal cells more responsive to synaptic burst stimuli, improving visual stimulus detection. The synaptic and intrinsic adaptations function together to keep tectal neurons within a constant operating range, while making the intact visual system less responsive to background activity yet more sensitive to burst stimuli.
Mapping neurogenesis onset in the optic tectum of Xenopus laevis.
Neural progenitor cells have a central role in the development and evolution of the vertebrate brain. During early brain development, neural progenitors first expand their numbers through repeated proliferative divisions and then begin to exhibit neurogenic divisions. The transparent and experimentally accessible optic tectum of Xenopus laevis is an excellent model system for the study of the cell biology of neurogenesis, but the precise spatial and temporal relationship between proliferative and neurogenic progenitors has not been explored in this system. Here we construct a spatial map of proliferative and neurogenic divisions through lineage tracing of individual progenitors and their progeny. We find a clear spatial separation of proliferative and neurogenic progenitors along the anterior-posterior axis of the optic tectum, with proliferative progenitors located more posteriorly and neurogenic progenitors located more anteriorly. Since individual progenitors are repositioned toward more anterior locations as they mature, this spatial separation likely reflects an increasing restriction in the proliferative potential of individual progenitors. We then examined whether the transition from proliferative to neurogenic behavior correlates with cellular properties that have previously been implicated in regulating neurogenesis onset. Our data reveal that the transition from proliferation to neurogenesis is associated with a small change in cleavage plane orientation and a more pronounced change in cell cycle kinetics in a manner reminiscent of observations from mammalian systems. Our findings highlight the potential to use the optic tectum of Xenopus laevis as an accessible system for the study of the cell biology of neurogenesis. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1328-1341, 2016.
Relay of visual information to the lateral geniculate nucleus and the visual cortex in albino ferrets.
The abnormal organization of the central visual pathways in the albino ferret has been characterized anatomically and physiologically. Recordings in dorsal lateral geniculate nucleus of the albino ferret show that lamina A1, which receives an aberrant projection from the contralateral eye, contains an extensive representation of the ipsilateral visual hemifield with receptive fields located up to 35 degrees from the vertical meridian. This is not the case in pigmented ferrets, for which the vast majority of units, activated through either the contralateral or ipsilateral eye, have receptive fields confined to the contralateral hemifield. The few fields found in the ipsilateral hemifield are driven through the contralateral eye and none is more than 10 degrees from the midline. Cortical topography was studied by making closely spaced electrode penetrations across the area 17/18 border. In pigmented animals, the reversal of topography at the border is characterized by units with receptive fields centered a few degrees into the ipsilateral hemifield. In 22 of 25 albinos, the "Boston" aberrant topography was found: the representation of the vertical meridian is within area 17, rather than at the area 17/18 border. Instead, at the area 17/18 border, there is a reversal in the topographic progression at up to 30 degrees into the ipsilateral hemifield. This pattern was most pronounced in the upper visual field. In agreement with the "Boston" physiology, injections of retrograde tracer made in area 17 usually label neurons in either lamina A or the part of lamina A1 that is aberrantly innervated by the contralateral eye. A column of labeled cells extending through all geniculate layers is rarely seen in albinos, although this is commonly the pattern in pigmented ferrets.
State-dependent mechanisms of LTP expression revealed by optical quantal analysis.
The expression mechanism of long-term potentiation (LTP) remains controversial. Here we combine electrophysiology and Ca(2+) imaging to examine the role of silent synapses in LTP expression. Induction of LTP fails to change p(r) at these synapses but instead mediates an unmasking process that is sensitive to the inhibition of postsynaptic membrane fusion. Once unmasked, however, further potentiation of formerly silent synapses leads to an increase in p(r). The state of the synapse thus determines how LTP is expressed.
Clonal relationships impact neuronal tuning within a phylogenetically ancient vertebrate brain structure.
Understanding how neurons acquire specific response properties is a major goal in neuroscience. Recent studies in mouse neocortex have shown that "sister neurons" derived from the same cortical progenitor cell have a greater probability of forming synaptic connections with one another and are biased to respond to similar sensory stimuli. However, it is unknown whether such lineage-based rules contribute to functional circuit organization across different species and brain regions. To address this question, we examined the influence of lineage on the response properties of neurons within the optic tectum, a visual brain area found in all vertebrates. Tectal neurons possess well-defined spatial receptive fields (RFs) whose center positions are retinotopically organized. If lineage relationships do not influence the functional properties of tectal neurons, one prediction is that the RF positions of sister neurons should be no more (or less) similar to one another than those of neighboring control neurons. To test this prediction, we developed a protocol to unambiguously identify the daughter neurons derived from single tectal progenitor cells in Xenopus laevis tadpoles. We combined this approach with in vivo two-photon calcium imaging in order to characterize the RF properties of tectal neurons. Our data reveal that the RF centers of sister neurons are significantly more similar than would be expected by chance. Ontogenetic relationships therefore influence the fine-scale topography of the retinotectal map, indicating that lineage relationships may represent a general and evolutionarily conserved principle that contributes to the organization of neural circuits.
Adenosine release during seizures attenuates GABAA receptor-mediated depolarization.
Seizure-induced release of the neuromodulator adenosine is a potent endogenous anticonvulsant mechanism, which limits the extension of seizures and mediates seizure arrest. For this reason several adenosine-based therapies for epilepsy are currently under development. However, it is not known how adenosine modulates GABAergic transmission in the context of seizure activity. This may be particularly relevant as strong activation of GABAergic inputs during epileptiform activity can switch GABA(A) receptor (GABA(A)R) signaling from inhibitory to excitatory, which is a process that plays a significant role in intractable epilepsies. We used gramicidin-perforated patch-clamp recordings to investigate the role of seizure-induced adenosine release in the modulation of postsynaptic GABA(A)R signaling in pyramidal neurons of rat hippocampus. Consistent with previous reports, GABA(A)R responses during seizure activity transiently switched from hyperpolarizing to depolarizing and excitatory. We found that adenosine released during the seizure significantly attenuated the depolarizing GABA(A)R responses and also reduced the extent of the after-discharge phase of the seizure. These effects were mimicked by exogenous adenosine administration and could not be explained by a change in chloride homeostasis mechanisms that set the reversal potential for GABA(A)Rs, or by a change in the conductance of GABA(A)Rs. Rather, A(1)R-dependent activation of potassium channels increased the cell's membrane conductance and thus had a shunting effect on GABA(A)R currents. As depolarizing GABA(A)R signaling has been implicated in seizure initiation and progression, the adenosine-induced attenuation of depolarizing GABA(A)R signaling may represent an important mechanism by which adenosine can limit seizure activity.
Depolarizing GABAergic conductances regulate the balance of excitation to inhibition in the developing retinotectal circuit in vivo.
Neurotransmission during development regulates synaptic maturation in neural circuits, but the contribution of different neurotransmitter systems is unclear. We investigated the role of GABAA receptor-mediated Cl- conductances in the development of synaptic responses in the Xenopus visual system. Intracellular Cl- concentration ([Cl-]i) was found to be high in immature tectal neurons and then falls over a period of several weeks. GABAergic synapses are present at early stages of tectal development and, when activated by optic nerve stimulation or visual stimuli, induce sustained depolarizing Cl- conductances that facilitate retinotectal transmission by NMDA receptors. To test whether depolarizing GABAergic inputs cooperate with NMDA receptors during activity-dependent maturation of glutamatergic synapses, we prematurely reduced [Cl-]i in tectal neurons in vivo by expressing the Cl- transporter KCC2. This blocked the normal developmental increase in AMPA receptor-mediated retinotectal transmission and increased GABAergic synaptic input to tectal neurons. Therefore, depolarizing GABAergic transmission plays a pivotal role in the maturation of excitatory transmission and controls the balance of excitation and inhibition in the developing retinotectal circuit.
Calcium-dependent neuroepithelial contractions expel damaged cells from the developing brain.
Both developing and adult organisms need efficient strategies for wound repair. In adult mammals, wounding triggers an inflammatory response that can exacerbate tissue injury and lead to scarring. In contrast, embryonic wounds heal quickly and with minimal inflammation, but how this is achieved remains incompletely understood. Using in vivo imaging in the developing brain of Xenopus laevis, we show that ATP release from damaged cells and subsequent activation of purinergic receptors induce long-range calcium waves in neural progenitor cells. Cytoskeletal reorganization and activation of the actomyosin contractile machinery in a Rho kinase-dependent manner then lead to rapid and pronounced apical-basal contractions of the neuroepithelium. These contractions drive the expulsion of damaged cells into the brain ventricle within seconds. Successful cell expulsion prevents the death of nearby cells and an exacerbation of the injury. Cell expulsion through neuroepithelial contraction represents a mechanism for rapid wound healing in the developing brain.
Spatial and temporal properties of visual responses in the thalamus of the developing ferret.
Spatiotemporal patterning of neural activity is thought to influence the development of connections in the visual pathway. This patterning can arise spontaneously or through sensory experience. Here, we use a combination of natural and simple stimuli to investigate which elements of the visual environment modulate the earliest responses in the primary visual pathway of developing ferrets. Recordings were made during the first 2 weeks of visual responsiveness, which, in the ferret, overlaps with the period that the eyelids have not yet opened. Even when the eyelids are closed, both thalamic and cortical activity was found to be temporally modulated under conditions of natural visual stimulation. The modulations correlated with temporal changes in stimulus contrast but also reflected spatial structure in the visual scene. Simple stimuli were used to show that early responses to naturalistic stimuli are influenced by the localization and structure of through-the-eyelid receptive fields. The early visual responses were also characterized by substantial variability in the ability of the cells to detect stimuli of different duration and different intensity, in a temporally precise manner. These temporal and spatial properties should constrain how plasticity mechanisms interpret naturally patterned activity.
Visual experience before eye-opening and the development of the retinogeniculate pathway.
Visual experience before eye-opening is not usually thought to have any developmental significance. Here we show that naturalistic visual stimuli presented through unopened eyelids robustly activate neurons in the ferret dorsal lateral geniculate nucleus. Further, dark-rearing prior to natural eye-opening has striking effects upon geniculate physiology. Receptive field maps after dark-rearing show increased convergence of On- and Off-center responses, and neurons frequently respond to both bright and dark phases of drifting gratings. There is also increased selectivity for the orientation of the gratings. These abnormalities of On-Off segregation can be explained by the finding that the responses of immature On and Off cells to naturalistic stimuli are strongly anticorrelated.
Control of axon branch dynamics by correlated activity in vivo.
To determine how patterned visual activity regulates the development of axonal projections, we collected in vivo time-lapse images of retinal axons from albino Xenopus tadpoles in which binocular innervation of the optic tectum was induced. Axons added branch tips with nearly equal probability in all territories, but eliminated them preferentially from territory dominated by the opposite eye. This selective branch elimination was abolished by blockade of N-methyl-D-aspartate receptors. These results describe a correlation-based mechanism by which visual experience directly governs axon branch dynamics that contribute to the development of topographic maps.
Sensory-Evoked Spiking Behavior Emerges via an Experience-Dependent Plasticity Mechanism.
The ability to generate action potentials (spikes) in response to synaptic input determines whether a neuron participates in information processing. How a developing neuron becomes an active participant in a circuit or whether this process is activity dependent is not known, especially as spike-dependent plasticity mechanisms would not be available to non-spiking neurons. Here we use the optic tectum of awake Xenopus laevis tadpoles to determine how a neuron becomes able to generate sensory-driven spikes in vivo. At the onset of vision, many tectal neurons do not exhibit visual spiking behavior, despite being intrinsically excitable and receiving visuotopically organized synaptic inputs. However, a brief period of visual stimulation can drive these neurons to start generating stimulus-driven spikes. This conversion relies upon a selective increase in glutamatergic input and requires depolarizing GABAergic transmission and NMDA receptor activation. This permissive form of experience-dependent plasticity enables a neuron to start contributing to circuit function.
In vivo spike-timing-dependent plasticity in the optic tectum of Xenopus laevis.
Spike-timing-dependent plasticity (STDP) is found in vivo in a variety of systems and species, but the first demonstrations of in vivo STDP were carried out in the optic tectum of Xenopus laevis embryos. Since then, the optic tectum has served as an excellent experimental model for studying STDP in sensory systems, allowing researchers to probe the developmental consequences of this form of synaptic plasticity during early development. In this review, we will describe what is known about the role of STDP in shaping feed-forward and recurrent circuits in the optic tectum with a focus on the functional implications for vision. We will discuss both the similarities and differences between the optic tectum and mammalian sensory systems that are relevant to STDP. Finally, we will highlight the unique properties of the embryonic tectum that make it an important system for researchers who are interested in how STDP contributes to activity-dependent development of sensory computations.
The potassium-chloride cotransporter 2 promotes cervical cancer cell migration and invasion by an ion transport-independent mechanism.
K(+)-Cl(-) cotransporters (KCCs) play a fundamental role in epithelial cell function, both in the context of ionic homeostasis and also in cell morphology, cell division and locomotion. Unlike other ubiquitously expressed KCC isoforms, expression of KCC2 is widely considered to be restricted to neurons, where it is responsible for maintaining a low intracellular chloride concentration to drive hyperpolarising postsynaptic responses to the inhibitory neurotransmitters GABA and glycine. Here we report a novel finding that KCC2 is widely expressed in several human cancer cell lines including the cervical cancer cell line (SiHa). Membrane biotinylation assays and immunostaining showed that endogenous KCC2 is located on the cell membrane of SiHa cells. To elucidate the role of KCC2 in cervical tumuorigenesis, SiHa cells with stable overexpression or knockdown of KCC2 were employed. Overexpression of KCC2 had no significant effect on cell proliferation but dramatically suppressed cell spreading and stress fibre organization, while knockdown of KCC2 showed opposite effects. In addition, insulin-like growth factor 1 (IGF-1)-induced cell migration and invasiveness were significantly increased by overexpression of KCC2. KCC2-induced cell migration and invasion were not dependent on KCC2 transport function since overexpression of an activity-deficient mutant KCC2 still increased IGF-1-induced cell migration and invasion. Moreover, overexpression of KCC2 significantly diminished the number of focal adhesions, while knockdown of KCC2 increased their number. Taken together, our data establish that KCC2 expression and function are not restricted to neurons and that KCC2 serves to increase cervical tumourigenesis via an ion transport-independent mechanism.
Assessing similarity to primary tissue and cortical layer identity in induced pluripotent stem cell-derived cortical neurons through single-cell transcriptomics.
Induced pluripotent stem cell (iPSC)-derived cortical neurons potentially present a powerful new model to understand corticogenesis and neurological disease. Previous work has established that differentiation protocols can produce cortical neurons, but little has been done to characterize these at cellular resolution. In particular, it is unclear to what extent in vitro two-dimensional, relatively disordered culture conditions recapitulate the development of in vivo cortical layer identity. Single-cell multiplex reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) was used to interrogate the expression of genes previously implicated in cortical layer or phenotypic identity in individual cells. Totally, 93.6% of single cells derived from iPSCs expressed genes indicative of neuronal identity. High proportions of single neurons derived from iPSCs expressed glutamatergic receptors and synaptic genes. And, 68.4% of iPSC-derived neurons expressing at least one layer marker could be assigned to a laminar identity using canonical cortical layer marker genes. We compared single-cell RNA-seq of our iPSC-derived neurons to available single-cell RNA-seq data from human fetal and adult brain and found that iPSC-derived cortical neurons closely resembled primary fetal brain cells. Unexpectedly, a subpopulation of iPSC-derived neurons co-expressed canonical fetal deep and upper cortical layer markers. However, this appeared to be concordant with data from primary cells. Our results therefore provide reassurance that iPSC-derived cortical neurons are highly similar to primary cortical neurons at the level of single cells but suggest that current layer markers, although effective, may not be able to disambiguate cortical layer identity in all cells.
Neuronal Chloride Regulation via KCC2 Is Modulated through a GABAB Receptor Protein Complex.
GABAB receptors are G-protein-coupled receptors that mediate inhibitory synaptic actions through a series of downstream target proteins. It is increasingly appreciated that the GABAB receptor forms part of larger signaling complexes, which enable the receptor to mediate multiple different effects within neurons. Here we report that GABAB receptors can physically associate with the potassium-chloride cotransporter protein, KCC2, which sets the driving force for the chloride-permeable ionotropic GABAA receptor in mature neurons. Using biochemical, molecular, and functional studies in rodent hippocampus, we show that activation of GABAB receptors results in a decrease in KCC2 function, which is associated with a reduction in the protein at the cell surface. These findings reveal a novel "crosstalk" between the GABA receptor systems, which can be recruited under conditions of high GABA release and which could be important for the regulation of inhibitory synaptic transmission.SIGNIFICANCE STATEMENT Synaptic inhibition in the brain is mediated by ionotropic GABAA receptors (GABAARs) and metabotropic GABAB receptors (GABABRs). To fully appreciate the function and regulation of these neurotransmitter receptors, we must understand their interactions with other proteins. We describe a novel association between the GABABR and the potassium-chloride cotransporter protein, KCC2. This association is significant because KCC2 sets the intracellular chloride concentration found in mature neurons and thereby establishes the driving force for the chloride-permeable GABAAR. We demonstrate that GABABR activation can regulate KCC2 at the cell surface in a manner that alters intracellular chloride and the reversal potential for the GABAAR. Our data therefore support an additional mechanism by which GABABRs are able to modulate fast synaptic inhibition.
Random synaptic feedback weights support error backpropagation for deep learning.
The brain processes information through multiple layers of neurons. This deep architecture is representationally powerful, but complicates learning because it is difficult to identify the responsible neurons when a mistake is made. In machine learning, the backpropagation algorithm assigns blame by multiplying error signals with all the synaptic weights on each neuron's axon and further downstream. However, this involves a precise, symmetric backward connectivity pattern, which is thought to be impossible in the brain. Here we demonstrate that this strong architectural constraint is not required for effective error propagation. We present a surprisingly simple mechanism that assigns blame by multiplying errors by even random synaptic weights. This mechanism can transmit teaching signals across multiple layers of neurons and performs as effectively as backpropagation on a variety of tasks. Our results help reopen questions about how the brain could use error signals and dispel long-held assumptions about algorithmic constraints on learning.