Tectal Circuits Research Articles

Activation patterns of dopaminergic cell populations reflect different learning scenarios in a cichlid fish, Pseudotropheus zebra

Dopamine is present in all vertebrates and the functional roles of the subsystems are assumed to be similar. Whereas the effect of dopaminergic modulation is well investigated in different target systems, less is known about the factors that are causing the modulation of dopaminergic cells. Using the zebra mbuna, Pseudotropheus zebra, a cichlid fish from Lake Malawi as a model system, we investigated the activation of specific dopaminergic cell populations detected by double-labeling with TH and pS6 antibodies while the animals were solving different learning tasks. Specifically, we compared an intense avoidance learning situation, an instrumental learning task, and a non-learning isolated group and found strong activation of different dopaminergic cell populations. Preoptic-hypothalamic cell populations respond to the stress component in the avoidance task, and the forced movement/locomotion may be responsible for activation in the posterior tubercle. The instrumental learning task had little stress component, but the activation of the raphe superior in this group may be correlated with attention or arousal during the training sessions. At the same time, the weaker activation of the nucleus of the posterior commissure may be related to positive reward acting onto tectal circuits. Finally, we examined the co-activation patterns across all dopaminergic cell populations and recovered robust differences across experimental groups, largely driven by hypothalamic, posterior tubercle, and brain stem regions possibly encoding the valence and salience associated with stressful stimuli. Taken together, our results offer some insights into the different functions of the dopaminergic cell populations in the brain of a non-mammalian vertebrate in correlation with different behavioral conditions, extending our knowledge for a more comprehensive view of the mechanisms of dopaminergic modulation in vertebrates.

Input from torus longitudinalis drives binocularity and spatial summation in zebrafish optic tectum

BackgroundA continued effort in neuroscience aims to understand the way brain circuits consisting of diverse neuronal types generate complex behavior following sensory input. A common feature of vertebrate visual systems is that lower-order and higher-order visual areas are reciprocally connected. Feedforward projections confer visual responsiveness to higher-order visual neurons while feedback projections likely serve to modulate responses of lower-order visual neurons in a context-dependent manner. Optic tectum is the largest first-order visual brain area in zebrafish and is reciprocally connected with the torus longitudinalis (TL), a second-order visual brain area that does not receive retinal input. A functional role for feedback projections from TL to tectum has not been identified. Here we aim to understand how this feedback contributes to visual processing.ResultsIn this study, we demonstrate that TL feedback projections to tectum drive binocular integration and spatial summation in a defined tectal circuit. We performed genetically targeted, cell type-specific functional imaging in tectal pyramidal neurons (PyrNs) and their two input neuron populations: retinal ganglion cells (RGCs) and neurons in TL. We find that PyrNs encode gradual changes in scene luminance using a complement of three distinct response classes that encode different light intensity ranges. Functional imaging of RGC inputs to tectum suggest that these response classes originate in the retina and RGC input specifies PyrN functional classes. In contrast, TL input serves to endow PyrNs with large, compound receptive fields that span both retinal hemifields.ConclusionsThese findings reveal a novel role for the zebrafish TL in driving binocular integration and spatial summation in tectal PyrNs. The neural circuit we describe generates a population of tectal neurons with large receptive fields tailored for detecting changes in the visual scene.

Environmental enrichment increases the number of telencephalic but not tectal cells of angelfish (Pterophyllum scalare): an exploratory investigation using optical fractionator

Based on previous evidence that environmental enrichment is associated with telencephalic cellular proliferation and that stable visuotopic tectal circuits are essential for discrimination of placement and identity of stationary or moving objects in the visual field, differential plasticity is expected in these areas. Here we tested this hypothesis in the Angelfish (Pterophyllum scalare), a species of ornamental fish with great value in the aquarist trade. We hypothesized that total telencephalic cell number would increase under the influence of an enriched environment whereas the tectal cell number would not change. To test this hypothesis, 12 aquaria of 80 l each were used, with six fish in each. The aquaria had either an enriched environment (EE) including stones, plants, sand and the presence of another fish from the Loricariidae family for interspecific social interaction, or an impoverished environment (IE), in which stimuli were limited to intraspecific interactions in a barren aquarium. After 62 days, six fish from each treatment were euthanized, and their brains were fixed and sectioned for Nissl staining. Then, stereological estimates of the total number of cells were performed. The fish showed no differences in weight gain, feed conversion ratio, condition factor, specific growth rate or survival. Animals kept in the enriched environment had a higher number of total telencephalic cells than animals kept in the impoverished environment (1,038,555 ± 65,357 vs. 758,331 ± 51,587, bilateral t-test, p = 0.008), but a similar number of tectal optical cells (EE 424,097 ± 29,914 vs. IE 471,409 ± 50,850, bilateral t-test, p = 0.445752). We concluded that cell proliferation in response to stimulation by the enriched environment is differentially expressed in the telencephalon and tectal areas of Pterophyllum scalare.

Enhanced visual experience rehabilitates the injured brain in Xenopus tadpoles in an NMDAR-dependent manner.

Traumatic brain injuries introduce functional and structural circuit deficits that must be repaired for an organism to regain function. We developed an injury model in which Xenopus laevis tadpoles are given a penetrating stab wound that damages the optic tectal circuit and impairs visuomotor behavior. In tadpoles, as in other systems, injury induces neurogenesis. The newly generated neurons are thought to integrate into the existing circuit; however, whether they integrate via the same mechanisms that govern normal neuronal maturation during development is not understood. Development of the functional visuomotor circuit in Xenopus is driven by sensory activity. We hypothesized that enhanced visual experience would improve recovery from injury by facilitating integration of newly generated neurons into the tectal circuit. We labeled newly generated neurons in the injured tectum by green fluorescent protein expression and examined their circuit integration using electrophysiology and in vivo imaging. Providing animals with brief bouts of enhanced visual experience starting 24 h after injury increased synaptogenesis and circuit integration of new neurons and facilitated behavioral recovery. To investigate mechanisms of neuronal integration and behavioral recovery after injury, we interfered with N-methyl-d-aspartate (NMDA) receptor function. Ifenprodil, which blocks GluN2B-containing NMDA receptors, impaired dendritic arbor elaboration. GluN2B blockade inhibited functional integration of neurons generated in response to injury and prevented behavioral recovery. Furthermore, tectal GluN2B knockdown blocked the beneficial effects of enhanced visual experience on functional plasticity and behavioral recovery. We conclude that visual experience-mediated rehabilitation of the injured tectal circuit occurs by GluN2B-containing NMDA receptor-dependent integration of newly generated neurons. NEW & NOTEWORTHY Recovery from brain injury is difficult in most systems. The study of regenerative animal models that are capable of injury repair can provide insight into cellular and circuit mechanisms underlying repair. Using Xenopus tadpoles, we show enhanced sensory experience rehabilitates the injured visual circuit and that this experience-dependent recovery depends on N-methyl-d-aspartate receptor function. Understanding the mechanisms of rehabilitation in this system may facilitate recovery in brain regions and systems where repair is currently impossible.

Principles of Functional Circuit Connectivity: Insights From Spontaneous Activity in the Zebrafish Optic Tectum

The brain is continuously active, even in the absence of external stimulation. In the optic tectum of the zebrafish larva, this spontaneous activity is spatially organized and reflects the circuit’s functional connectivity. The structure of the spontaneous activity displayed patterns associated with aspects of the larva’s preferences when engaging in complex visuo-motor behaviors, suggesting that the tectal circuit is adapted for the circuit’s functional role in detecting visual cues and generating adequate motor behaviors. Further studies in sensory deprived larvae suggest that the basic structure of the functional connectivity patterns emerges even in the absence of retinal inputs, but that its fine structure is affected by visual experience.

Direct intertectal inputs are an integral component of the bilateral sensorimotor circuit for behavior in Xenopus tadpoles.

The circuit controlling visually guided behavior in nonmammalian vertebrates, such as Xenopus tadpoles, includes retinal projections to the contralateral optic tectum, where visual information is processed, and tectal motor outputs projecting ipsilaterally to hindbrain and spinal cord. Tadpoles have an intertectal commissure whose function is unknown, but it might transfer information between the tectal lobes. Differences in visual experience between the two eyes have profound effects on the development and function of visual circuits in animals with binocular vision, but the effects on animals with fully crossed retinal projections are not clear. We tested the effect of monocular visual experience on the visuomotor circuit in Xenopus tadpoles. We show that cutting the intertectal commissure or providing visual experience to one eye (monocular visual experience) is sufficient to disrupt tectally mediated visual avoidance behavior. Monocular visual experience induces asymmetry in tectal circuit activity across the midline. Repeated exposure to monocular visual experience drives maturation of the stimulated retinotectal synapses, seen as increased AMPA-to-NMDA ratios, induces synaptic plasticity in intertectal synaptic connections, and induces bilaterally asymmetric changes in the tectal excitation-to-inhibition ratio (E/I). We show that unilateral expression of peptides that interfere with AMPA or GABAA receptor trafficking alters E/I in the transfected tectum and is sufficient to degrade visuomotor behavior. Our study demonstrates that monocular visual experience in animals with fully crossed visual systems produces asymmetric circuit function across the midline and degrades visuomotor behavior. The data further suggest that intertectal inputs are an integral component of a bilateral visuomotor circuit critical for behavior. NEW & NOTEWORTHY The developing optic tectum of Xenopus tadpoles represents a unique circuit in which laterally positioned eyes provide sensory input to a circuit that is transiently monocular, but which will be binocular in the animal's adulthood. We challenge the idea that the two lobes of tadpole optic tectum function independently by testing the requirement of interhemispheric communication and demonstrate that unbalanced sensory input can induce structural and functional plasticity in the tectum sufficient to disrupt function.

Hypothalamic Projections to the Optic Tectum in Larval Zebrafish.

The optic tectum of larval zebrafish is an important model for understanding visual processing in vertebrates. The tectum has been traditionally viewed as dominantly visual, with a majority of studies focusing on the processes by which tectal circuits receive and process retinally-derived visual information. Recently, a handful of studies have shown a much more complex role for the optic tectum in larval zebrafish, and anatomical and functional data from these studies suggest that this role extends beyond the visual system, and beyond the processing of exclusively retinal inputs. Consistent with this evolving view of the tectum, we have used a Gal4 enhancer trap line to identify direct projections from rostral hypothalamus (RH) to the tectal neuropil of larval zebrafish. These projections ramify within the deepest laminae of the tectal neuropil, the stratum album centrale (SAC)/stratum griseum periventriculare (SPV), and also innervate strata distinct from those innervated by retinal projections. Using optogenetic stimulation of the hypothalamic projection neurons paired with calcium imaging in the tectum, we find rebound firing in tectal neurons consistent with hypothalamic inhibitory input. Our results suggest that tectal processing in larval zebrafish is modulated by hypothalamic inhibitory inputs to the deep tectal neuropil.

Spatiotemporal interplay between multisensory excitation and recruited inhibition in the lamprey optic tectum.

Animals integrate the different senses to facilitate event-detection for navigation in their environment. In vertebrates, the optic tectum (superior colliculus) commands gaze shifts by synaptic integration of different sensory modalities. Recent works suggest that tectum can elaborate gaze reorientation commands on its own, rather than merely acting as a relay from upstream/forebrain circuits to downstream premotor centers. We show that tectal circuits can perform multisensory computations independently and, hence, configure final motor commands. Single tectal neurons receive converging visual and electrosensory inputs, as investigated in the lamprey - a phylogenetically conserved vertebrate. When these two sensory inputs overlap in space and time, response enhancement of output neurons occurs locally in the tectum, whereas surrounding areas and temporally misaligned inputs are inhibited. Retinal and electrosensory afferents elicit local monosynaptic excitation, quickly followed by inhibition via recruitment of GABAergic interneurons. Multisensory inputs can thus regulate event-detection within tectum through local inhibition without forebrain control.

Experience-dependent plasticity of excitatory and inhibitory intertectal inputs in Xenopus tadpoles

Communication between optic tecta/superior colliculi is thought to be required for sensorimotor behaviors by comparing inputs across the midline, however the development of and the role of visual experience in the function and plasticity of intertectal connections are unclear. We combined neuronal tracing, in vivo time-lapse imaging, and electrophysiology to characterize the structural and functional development of intertectal axons and synapses in Xenopus tadpole optic tectum. We find that intertectal connections are established early during optic tectal circuit development. We determined the neurotransmitter identity of intertectal neurons using both rabies virus-mediated tracing combined with post-hoc immunohistochemistry, and electrophysiology. Excitatory and inhibitory intertectal neuronal somata are similarly distributed throughout the tectum. Excitatory and inhibitory intertectal axons are structurally similar and elaborate broadly in the contralateral tectum. We demonstrate that intertectal and retinotectal axons converge onto tectal neurons by recording postsynaptic currents after stimulating intertectal and retinotectal inputs. Cutting the intertectal commissure removes synaptic responses to contralateral tectal stimulation. In vivo time-lapse imaging demonstrated that visual experience drives plasticity in intertectal bouton size and dynamics. Finally, visual experience coordinately drives the maturation of excitatory and inhibitory intertectal inputs by increasing AMPA- and GABA-receptor mediated currents, comparable to experience-dependent maturation of retinotectal inputs. These data indicate that visual experience regulates plasticity of excitatory and inhibitory intertectal inputs, maintaining the excitatory: inhibitory ratio of intertectal input. These studies place intertectal inputs as key players in tectal circuit development and suggest that they may play a role in sensory information processing critical to sensorimotor behaviors.

Lamination Speeds the Functional Development of Visual Circuits

SummaryA common feature of the brain is the arrangement of synapses in layers. To examine the significance of this organizational feature, we studied the functional development of direction-selective (DS) circuits in the tectum of astray mutant zebrafish in which lamination of retinal ganglion cell (RGC) axons is lost. We show that although never laminar, the tuning of DS-RGC axons targeting the mutant tectum is normal. Analysis of mutant tectal neurons at late developmental stages reveals that directional tuning is indistinguishable from wild-type larvae. Furthermore, we show that structural plasticity of tectal dendrites and RGC axons compensates for the loss of lamination, establishing connectivity between DS-RGCs and their normal tectal targets. However, tectal direction selectivity is severely perturbed at earlier developmental stages. Thus, the formation of synaptic laminae is ultimately dispensable for the correct wiring of direction-selective tectal circuits, but it is crucial for the rapid assembly of these networks.Video

Acute Synthesis of CPEB Is Required for Plasticity of Visual Avoidance Behavior in Xenopus

Neural plasticity requires protein synthesis, but theidentity of newly synthesized proteins generated in response to plasticity-inducing stimuli remains unclear. We used invivo bio-orthogonal noncanonical amino acid tagging (BONCAT) with the methionine analog azidohomoalanine (AHA) combined with themultidimensional protein identification technique (MudPIT) to identify proteins that are synthesized inthe tadpole brain over 24hr. We induced conditioning-dependent plasticity of visual avoidance behavior, which required N-methyl-D-aspartate (NMDA) and Ca(2+)-permeable α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, αCaMKII, and rapid protein synthesis. Combining BONCAT with western blots revealed that proteins including αCaMKII, MEK1, CPEB, and GAD65 are synthesized during conditioning. Acute synthesis of CPEB during conditioning is required for behavioral plasticity as well asconditioning-induced synaptic and structural plasticity in the tectal circuit. We outline a signaling pathway that regulates protein-synthesis-dependent behavioral plasticity in intact animals, identify newly synthesized proteins induced by visual experience, and demonstrate a requirement for acute synthesis of CPEB in plasticity.

Emergent properties of the optic tectum revealed by population analysis of direction and orientation selectivity.

How local circuits within the brain process visual information has classically been addressed at the single neuron level. Such reductionist approaches, however, struggle to capture the full scope of functional properties associated with even "simple" brain nuclei. Using population functional calcium imaging, we aim to describe how local circuits within the zebrafish optic tectum process visual information. Specifically, how are previously identified direction-selective (DS) and orientation-selective (OS) retinal ganglion cell (RGC) inputs (Nikolaou et al., 2012) represented in tectal cells? First, we identify an emergent population of DS tectal cell with a direction preference not explicitly present in any one of the RGC inputs. Second, this is associated with a striking shift from a tiled and triangular representation of directional space (RGC inputs) into an overlapping cardinal representation by tectal cell populations. Third, and in contrast, we find that orientation space is represented similarly in both the RGC input and tectal cell populations illustrating feature-dependent differences in how tectal circuits process their inputs. Finally, we identify OS and two populations of DS cells at the superficial border of the tectal neuropil, one of which is an emergent population. This study, together with our previous one (Nikolaou et al., 2012), demonstrate that direction-selectivity is established in both the retina and tectum.

Parametric Functional Maps of Visual Inputs to the Tectum

How features of the visual scene are encoded in the population activity of retinal ganglion cells (RGCs) targeting specific regions of the brain is not well understood. To address this, we have used a genetically encoded reporter of presynaptic function (SyGCaMP3) to record visually evoked activity in the population of RGC axons innervating the zebrafish tectum. Using unbiased voxel-wise analysis of SyGCaMP3 signals, we identify three subtypes of direction-selective and two subtypes of orientation-selective retinal input. Composite parametric functional maps generated across many larvae show laminar segregation of direction- and orientation-selective responses and unexpected retinotopic biases in the distribution of functional subtypes. These findings provide a systematic description of the form, organization, and dimensionality of visual inputs to the brain and will serve as a platform for understanding emergent properties in tectal circuits associated with visually driven behavior.

Inhibition to excitation ratio regulates visual system responses and behavior in vivo

The balance of inhibitory to excitatory (I/E) synaptic inputs is thought to control information processing and behavioral output of the central nervous system. We sought to test the effects of the decreased or increased I/E ratio on visual circuit function and visually guided behavior in Xenopus tadpoles. We selectively decreased inhibitory synaptic transmission in optic tectal neurons by knocking down the γ2 subunit of the GABA(A) receptors (GABA(A)R) using antisense morpholino oligonucleotides or by expressing a peptide corresponding to an intracellular loop of the γ2 subunit, called ICL, which interferes with anchoring GABA(A)R at synapses. Recordings of miniature inhibitory postsynaptic currents (mIPSCs) and miniature excitatory PSCs (mEPSCs) showed that these treatments decreased the frequency of mIPSCs compared with control tectal neurons without affecting mEPSC frequency, resulting in an ∼50% decrease in the ratio of I/E synaptic input. ICL expression and γ2-subunit knockdown also decreased the ratio of optic nerve-evoked synaptic I/E responses. We recorded visually evoked responses from optic tectal neurons, in which the synaptic I/E ratio was decreased. Decreasing the synaptic I/E ratio in tectal neurons increased the variance of first spike latency in response to full-field visual stimulation, increased recurrent activity in the tectal circuit, enlarged spatial receptive fields, and lengthened the temporal integration window. We used the benzodiazepine, diazepam (DZ), to increase inhibitory synaptic activity. DZ increased optic nerve-evoked inhibitory transmission but did not affect evoked excitatory currents, resulting in an increase in the I/E ratio of ∼30%. Increasing the I/E ratio with DZ decreased the variance of first spike latency, decreased spatial receptive field size, and lengthened temporal receptive fields. Sequential recordings of spikes and excitatory and inhibitory synaptic inputs to the same visual stimuli demonstrated that decreasing or increasing the I/E ratio disrupted input/output relations. We assessed the effect of an altered I/E ratio on a visually guided behavior that requires the optic tectum. Increasing and decreasing I/E in tectal neurons blocked the tectally mediated visual avoidance behavior. Because ICL expression, γ2-subunit knockdown, and DZ did not directly affect excitatory synaptic transmission, we interpret the results of our study as evidence that partially decreasing or increasing the ratio of I/E disrupts several measures of visual system information processing and visually guided behavior in an intact vertebrate.

Focusing on optic tectum circuitry through the lens of genetics

The visual pathway is tasked with processing incoming signals from the retina and converting this information into adaptive behavior. Recent studies of the larval zebrafish tectum have begun to clarify how the 'micro-circuitry' of this highly organized midbrain structure filters visual input, which arrives in the superficial layers and directs motor output through efferent projections from its deep layers. The new emphasis has been on the specific function of neuronal cell types, which can now be reproducibly labeled, imaged and manipulated using genetic and optical techniques. Here, we discuss recent advances and emerging experimental approaches for studying tectal circuits as models for visual processing and sensorimotor transformation by the vertebrate brain.

Neurodevelopmental effects of chronic exposure to elevated levels of pro-inflammatory cytokines in a developing visual system

BackgroundImbalances in the regulation of pro-inflammatory cytokines have been increasingly correlated with a number of severe and prevalent neurodevelopmental disorders, including autism spectrum disorder, schizophrenia and Down syndrome. Although several studies have shown that cytokines have potent effects on neural function, their role in neural development is still poorly understood. In this study, we investigated the link between abnormal cytokine levels and neural development using the Xenopus laevis tadpole visual system, a model frequently used to examine the anatomical and functional development of neural circuits.ResultsUsing a test for a visually guided behavior that requires normal visual system development, we examined the long-term effects of prolonged developmental exposure to three pro-inflammatory cytokines with known neural functions: interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α. We found that all cytokines affected the development of normal visually guided behavior. Neuroanatomical imaging of the visual projection showed that none of the cytokines caused any gross abnormalities in the anatomical organization of this projection, suggesting that they may be acting at the level of neuronal microcircuits. We further tested the effects of TNF-α on the electrophysiological properties of the retinotectal circuit and found that long-term developmental exposure to TNF-α resulted in enhanced spontaneous excitatory synaptic transmission in tectal neurons, increased AMPA/NMDA ratios of retinotectal synapses, and a decrease in the number of immature synapses containing only NMDA receptors, consistent with premature maturation and stabilization of these synapses. Local interconnectivity within the tectum also appeared to remain widespread, as shown by increased recurrent polysynaptic activity, and was similar to what is seen in more immature, less refined tectal circuits. TNF-α treatment also enhanced the overall growth of tectal cell dendrites. Finally, we found that TNF-α-reared tadpoles had increased susceptibility to pentylenetetrazol-induced seizures.ConclusionsTaken together our data are consistent with a model in which TNF-α causes premature stabilization of developing synapses within the tectum, therefore preventing normal refinement and synapse elimination that occurs during development, leading to increased local connectivity and epilepsy. This experimental model also provides an integrative approach to understanding the effects of cytokines on the development of neural circuits and may provide novel insights into the etiology underlying some neurodevelopmental disorders.

Metaplasticity Governs Natural Experience-Driven Plasticity of Nascent Embryonic Brain Circuits

During embryogenesis, brain neurons receiving the same sensory input may undergo potentiation or depression. While the origin of variable plasticity in vivo is unknown, it plays a key role in shaping dynamic neural circuit refinement. Here, we investigate effects of natural visual stimuli on neuronal firing within the intact, awake, developing brain using calcium imaging of 100 s of central neurons in the Xenopus retinotectal system. We find that specific patterns of visual stimuli shift population responses toward either potentiation or depression in an N-methyl-D-aspartate receptor (NMDA-R)-dependent manner. In agreement with Bienenstock-Cooper-Munro metaplasticity, our results show that functional potentiation or depression can be predicted by individual neurons' specific receptive field properties and historic firing rates. Interestingly, this activity-dependent metaplasticity is itself NMDA-R dependent. Furthermore, network analysis reveals increased correlated firing of neurons that undergo potentiation. These findings implicate metaplasticity as a natural property regulating experience-dependent refinement of nascent embryonic brain circuits.

The cellular architecture of the larval zebrafish tectum, as revealed by Gal4 enhancer trap lines

We have carried out a Gal4 enhancer trap screen in zebrafish, and have generated 184 stable transgenic lines with interesting expression patterns throughout the nervous system. Of these, three display clear expression in the tectum, each with a distinguishable and stereotyped distribution of Gal4 expressing cells. Detailed morphological analysis of single cells, using a genetic “Golgi-like” labelling method, revealed four common cell types (superficial, periventricular, shallow periventricular, and radial glial), along with a range of other less common neurons. The shallow periventricular (PV) and a subset of the PV neurons are tectal efferent neurons that target various parts of the reticular formation. We find that it is specifically PV neurons with dendrites in the deep tectal neuropil that target the reticular formation. This indicates that these neurons receive the tectum's highly processed visual information (which is fed from the superficial retinorecipient layers), and relay it to premotor regions. Our results show that the larval tectum, both broadly and at the single cell level, strongly resembles a miniature version of its adult counterpart, and that it has all of the necessary anatomical characteristics to inform motor responses based on sensory input. We also demonstrate that mosaic expression of GFP in Gal4 enhancer trap lines can be used to describe the types and abundance of cells in an expression pattern, including the architectures of individual neurons. Such detailed anatomical descriptions will be an important part of future efforts to describe the functions of discrete tectal circuits in the generation of behavior.

Time-lapse in vivo imaging of the morphological development of Xenopus optic tectal interneurons.

Excitatory and inhibitory interneurons play a key role in the establishment of neuronal responses and circuit properties in the brain. Despite their importance in brain function, the structural development of interneurons has not been well studied. We used in vivo time-lapse imaging in intact anesthetized Xenopus tadpoles to determine the morphological events underlying the development of interneuron dendritic arbor structure. Single optic tectal neurons were labeled with DiI and imaged at daily intervals over 4 days in intact albino Xenopus tadpoles. The same neurons were also imaged at shorter intervals to determine the dynamic rearrangements in arbor branches that accompany large-scale arbor growth. Tectal interneurons, like projection neurons, develop from neuroepithelial cells located near the ventricular layer. They elaborate complex dendritic arbors over a period of 2 days. Short-interval time-lapse images reveal that tectal interneuron arbors have rapid rates of branch additions and retractions. We identified four patterns of interneuron arbor development, based on the cell morphology and types of structural rearrangements that occur over the development of the neuronal arbor. A surprising feature of interneuronal development is the large extent of structural rearrangements: many interneurons extend transient processes so that the neuronal structure is dramatically different from one day to the next. Because the majority of synaptic contacts are formed on dendrites, the structural changes we observed in interneuronal dendritic arbors suggest that optic tectal circuits are extremely plastic during early stages of development.

Distribution of GABA immunoreactivity in the optic tectum of the frog: A light and electron microscopic study

GABA immunoreactivity was studied in the optic tectum of the frog, Rana esculenta, by postembedding immunohistochemical methods at the light and electron microscopic levels. Nearly one-third of the total population of tectal cells appeared to be GABA-immunoreactive. The proportion of stained neurons was highest in layer 9 (61%), and they occurred less frequently in layers 7 (21%) and 6 (27%). Stained perikarya represented a population of small neurons with a diameter of 8–10 μm. Large cell bodies in layer 7 or at the top of layer 6, and cells of origin of the mesencephalic trigeminal tract in layer 2, were devoid of labelling. Axon terminals and dendrites displaying immunoreactivity for GABA were observed in all of the plexiform layers. On the basis of ultrastructural characteristics two types of GABA-positive axon terminals and two variations of GABA-immunoreactive dendrites were distinguished. Synaptic relations of GABA-immunoreactive and GABA-negative axons as well as dendrites were also studied. Besides a wide variety of axodendritic synapses, dendrodendritic synaptic appositions were also revealed. The results suggest that various inhibitory mechanisms are involved in tectal circuits, which have to be incorporated into future neuronal models concerning visual information processing in the optic tectum of the frog.