William C. Hall

High-speed mapping of synaptic connectivity using photostimulation in Channelrhodopsin-2 transgenic mice

To permit rapid optical control of brain activity, we have engineered multiple lines of transgenic mice that express the light-activated cation channel Channelrhodopsin-2 (ChR2) in subsets of neurons. Illumination of ChR2-positive neurons in brain slices produced photocurrents that generated action potentials within milliseconds and with precisely timed latencies. The number of light-evoked action potentials could be controlled by varying either the amplitude or duration of illumination. Furthermore, the frequency of light-evoked action potentials could be precisely controlled up to 30 Hz. Photostimulation also could evoke synaptic transmission between neurons, and, by scanning with a small laser light spot, we were able to map the spatial distribution of synaptic circuits connecting neurons within living cerebral cortex. We conclude that ChR2 is a genetically based photostimulation technology that permits analysis of neural circuits with high spatial and temporal resolution in transgenic mammals.

Superior Colliculus of the Tree Shrew: A Structural and Functional Subdivision into Superficial and Deep Layers

Superficial lesions of the superior colliculus produced deficits in form discrimination, while deeper lesions produced, in addition, an inability to track objects. These two syndromes were related to an anatomical subdivision: Superficial lesions resulted in anterograde degeneration in the visual thalamus, whereas lesions confined to the deeper layers produced degeneration in the nonvisual thalamus and in brainstem motor areas.

Visual cortex in a reptile, the turtle (Pseudem ys scripta and Chrysem ys picta)

The afferent and efferent connections of general cortex were studied in two species of turtle,Pseudemys scripta and Chrysemys picta. The thalamic distribution of labeled cells following cortical applications of horseradish peroxidase (HRP) indicated that at least 3 dorsal thalamic nuclei, the dorsal lateral geniculate nucleus, nucleus ventralis and the nucleus dorsolateralis anterior, project to general cortex. When cortical applications of HRP were combined with intraocular injections of tritiated proline in the same animal, autoradiographically labeled retinal terminations were found among the dendrites of lateral geniculate neurons containing the HRP reaction product. These experiments demonstrated that the dorsal lateral geniculate nucleus not only projects to general cortex, but also receives retinal input. Thus, gereral cortex in the turtle is a target of a visual pathway which relays in the dorsal thalamus. Cortical lesions produced anterograde degeneration in the same thalamic nuclei which the HRP experiments demonstrated project to cortex, thereby indicating that the dorsal thalamus and general cortex have reciprocal connections in the turtle. These same experiments with cortical lesions demonstrated that general cortex also sends projections to the optic tectum and tegmentum of the midbrain. These afferent and efferent connections of general cortex in the turtle are compared with the connections of general cortex in other reptilian groups and with those of neocortex in mammals.

Evolution of the Pulvinar (Part 1 of 2)

Our evidence from both cytoarchitecture and studies of connections suggests that LE GROS CLARK was correct in postulating that the lateral posterior nucleus of primitive mammals is the homologue to the primate pulvinar. It seems evident that during mammalian evolution both this region of the thalamus and its cortical target have grown larger and more complex. Truly intrinsic subdivisions may have appeared not only in the primates but also independently in other mammalian lines of descent, such as the carnivores. One main goal of our laboratory is to ask how functional subdivisions are related to the increasing subdivisions of structure. Our hope is that this line of inquiry might contribute to an understanding of those functions most recently evolved in mammalian evolution.

Exploring the superior colliculus in vitro.

The superior colliculus plays an important role in the translation of sensory signals that encode the location of objects in space into motor signals that encode vectors of the shifts in gaze direction called saccades. Since the late 1990s, our two laboratories have been applying whole cell patch-clamp techniques to in vitro slice preparations of rodent superior colliculus to analyze the structure and function of its circuitry at the cellular level. This review describes the results of these experiments and discusses their contributions to our understanding of the mechanisms responsible for sensorimotor integration in the superior colliculus. The experiments analyze vertical interactions between its superficial visuosensory and intermediate premotor layers and propose how they might contribute to express saccades and to saccadic suppression. They also compare and contrast the circuitry within each of these layers and propose how this circuitry might contribute to the selection of the targets for saccades and to the build-up of the premotor commands that precede saccades. Experiments also explore in vitro the roles of extrinsic inputs to the superior colliculus, including cholinergic inputs from the parabigeminal and parabrachial nuclei and GABAergic inputs from the substantia nigra pars reticulata, in modulating the activity of the collicular circuitry. The results extend and clarify our understanding of the multiple roles the superior colliculus plays in sensorimotor integration.

Laminar origin of projections from the superficial layers of the superior colliculus in the tree shrew, Tupaia glis

The laminar origin of the efferent projections from the superior colliculus to the pulvinar and to the dorsal and ventral lateral geniculate nuclei has been studied using the retrograde axonal transport of horseradish peroxidase. Following injections in either the dorsal or the ventral lateral geniculate nucleus, cells heavily labeled with the horseradish peroxidase reaction product are restricted primarily to the upper stratum griseum superficiale. These cells have small, fusiform somas with dendrites which extend dorsally and ventrally, perpendicular to the pial surface. In contrast, following injections in the pulvinar, cells labeled with reaction product are restricted primarily to the lower stratum griseum superficiale and to the most superficial part of stratum opticum. These cells typically have larger somas than cells in the upper stratum griseum superficiale, and often have dendrites which emerge horizontally from the cell body. When taken together with previous electrophysiological and anatomical studies, the present findings suggest that there is a laminar subdivision of the tree shrew stratum griseum superficiale, and that these subdivisions project selectively to different thalamic targets.

Projections from the superior colliculus to the dorsal lateral geniculate nucleus of the grey squirrel (Sciurus carolinensis)

The present report provides evidence that there is a lamina in the dorsal lateral geniculate nucleus of the grey squirrel which receives projections both from the retina and from stratum griseum superficiale of the superior colliculus. To demonstrate these projections, it was necessary to rely on several different methods for tracing pathways including anterograde degeneration, and anterograde and retrograde axonal

Reciprocal connections between the zona incerta and the pretectum and superior colliculus of the cat

The goal of the present experiments was to examine the relationships of the zona incerta with two structures associated with visuomotor behavior, the superior colliculus and pretectum. The experiments were carried out in the cat, a species commonly used in studies of visuomotor integration, and utilized wheat germ agglutinin horseradish peroxidase and biocytin as retrograde and anterograde neuronal tracers. Retrograde axonal transport demonstrated that most cells in the ventral subdivision of the zona incerta project to the superior colliculus. Anterograde tracers demonstrated that the incertotectal terminal field is most dense in the intermediate gray layer, which is the primary source of the descending pathway from the superior colliculus to brainstem gaze centers. Further experiments showed that scattered cells within the intermediate gray layer give rise to a reciprocal pathway that terminates in both the dorsal and ventral subdivisions of the zona incerta. The distribution of both labeled incertotectal cells and tectoincertal terminals extends dorsolateral to the zona incerta proper, between the reticular thalamic nucleus and the external medullary lamina. Electron microscopic examination of labeled tectoincertal terminals demonstrated that they contain mainly spherical vesicles and have slightly asymmetric to symmetric synaptic densities. Labeled terminals were observed contacting labeled cells in the zona incerta, suggesting that the reciprocal pathway may be monosynaptic. The zona incerta is also reciprocally interconnected with the pretectum. The anterior pretectal nucleus provides a dense projection to the ventral part of the zona incerta and receives a sparse reciprocal projection. The posterior pretectal nucleus and nucleus of the optic tract may also project to the zona incerta. The pretectoincertal fibers form terminals that contain primarily spherical vesicles and make distinctly asymmetric synaptic contacts. In summary, these results indicate that the deep layers of the superior colliculus, which are important for controlling saccades, are the target of a projection from the ventral subdivision of the zona incerta. Like the substantia nigra, the zona incerta may play a permissive role in the tectal initiation of saccadic eye movements. The incertotectal terminal field in the cat is less dense than that observed previously in the rat, suggesting species differences in the development of this pathway. An additional finding of this study is that one of the main sources of input to these incertotectal cells is the anterior pretectal nucleus. This pretectal incertal tectal pathway is likely to play a role in the guidance of tectally initiated saccades by somatosensory stimuli.

The sources of the nigrotectal pathway

The nigrotectal pathway plays a role in the generation of saccade related responses by cells in the deep layers of the superior colliculus. By using a retrograde horseradish peroxidase technique that homogeneously fills neurons, the present experiments demonstrate that the source of the nigrotectal projection to the intermediate gray layer of the grey squirrel (Sciurus carolinensis) is a heterogeneous population of neurons whose somas and dendrites are concentrated in the rostral pole of pars reticulata. This region of pars reticulata receives projections from the posterior caudate, which in turn is a target of both the pulvinar and visual cortex. In addition, these experiments reveal the presence of a second, distinct set of neurons projecting to the midbrain tectum that are located in pars lateralis of the substantia nigra. These neurons can be distinguished from those in pars reticulata by their homogeneity and by their prominent basal dendrites. Furthermore, pars lateralis of the squirrel substantia nigra is, on cytoarchitectonic and immunocytochemical grounds, a distinct subdivision that does not receive projections from the posterior caudate. We conclude that both pars reticulata and lateralis are sources of the nigrotectal pathway. In addition, our results suggest, on connectional grounds, that the rostral pole of pars reticulata may be specialized to subserve the visual guidance of orienting movements.

An In Vitro Study of Horizontal Connections in the Intermediate Layer of the Superior Colliculus

Some models propose that the spatial and temporal distributions of premotor activity in the intermediate layer of the superior colliculus are shaped by neuronal ensembles that give rise to local excitatory and distant inhibitory connections. One function proposed for these connections is to mediate a “winner-take-all” network; the short-range excitatory connections build up the activity of neighboring cells that command orienting movements in one direction, whereas the wide-ranging inhibitory projections attenuate the activity of remote cells that command incompatible movements. We used in vitro photostimulation and whole-cell patch-clamp recording to test these models by measuring the spatial extent of synaptic interactions within the rat intermediate layer. Uncaging glutamate over whole-cell patch-clamped cells in the intermediate layer elicited long-lasting inward currents, resulting from direct activation of glutamate receptors expressed by the cells, and brief synaptic currents evoked by activation of presynaptic neurons. The synaptic responses comprised clusters of excitatory and inhibitory currents. The size of these responses depended on the location of the stimulus with respect to the clamped cell. Large responses were commonly evoked by stimuli within 200 μm of the soma in the intermediate layer; smaller responses could occasionally be evoked from sites as distant as 500 μm. Responses evoked by stimulation beyond this distance were rare. Although the results demonstrated powerful local excitatory and inhibitory connections, they did not support the pattern of short-range excitation and widespread inhibition predicted by the winner-take-all hypothesis.