Attenuation of visual activity in the superficial layers (SLs), stratum griseum superficiale and stratum opticum, of the superior colliculus during saccades may contribute to reducing perceptual blur during saccades and also may help prevent subsequent unwanted saccades. GABAergic neurons in the intermediate, premotor, layer (SGI), stratum griseum intermedium, send an inhibitory input to SL. This pathway provided the basis for a model proposing that the SGI premotor cells that project to brainstem gaze centers and discharge before saccades also activate neighboring GABAergic neurons that suppress saccade-induced visual activity in SL. The in vitro method allowed us to test this model. We made whole-cell patch-clamp recordings in collicular slices from either rats or GAD67–GFP knock-in mice, in which GABAergic neurons could be identified by their expression of green fluorescence protein (GFP). Antidromic electrical stimulation of SGI premotor cells was produced by applying pulse currents in which their axons congregate after exiting the superior colliculus. The stimulation evoked monosynaptic EPSCs in SGI GABAergic neurons that project to SL, as would be predicted if these neurons receive excitatory input from the premotor cells. Second, IPSCs were evoked in SL neurons, some of which project to the visual thalamus. These IPSCs were polysynaptically mediated by the GABAergic neurons that were excited by the antidromically activated SGI neurons. These results support the hypothesis that collaterals of premotor neuron axons excite GABAergic neurons that inhibit SL visuosensory cells.

Southern Subanen, spoken on the Philippine island of Mindanao, is the only Philippine language known to have contrastive aspiration, which is a rarity in the Austronesian family. While aspirated consonants are common in the world’s languages, Southern Subanen provides us with an uncommon glimpse at how aspirated consonants can develop. Their unique historical derivation in Southern Subanen is such that, in certain environments, aspiration marks semantic contrasts in verbal prefixes and even functions as a marker of nominalization. In this paper, we will analyze the historical sources of this aspiration and its realization in the modern language.

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.

Here we survey a molecular genetic approach for imaging synaptic inhibition. This approach is based on measuring intracellular chloride concentration ([Cl − ] i ) with the fluorescent chloride indicator protein, Clomeleon. We first describe several different ways to express Clomeleon in selected populations of neurons in the mouse brain. These methods include targeted viral gene transfer, conditional expression controlled by Cre recombination, and transgenesis based on the neuron-specific promoter, thy1. Next, we evaluate the feasibility of using different lines of thy1::Clomeleon transgenic mice to image synaptic inhibition in several different brain regions: the hippocampus, the deep cerebellar nuclei (DCN), the basolateral nucleus of the amygdala, and the superior colliculus (SC). Activation of hippocampal interneurons caused [Cl − ] i to rise transiently in individual postsynaptic CA1 pyramidal neurons. [Cl − ] i increased linearly with the number of electrical stimuli in a train, with peak changes as large as 4 mM. These responses were largely mediated by GABA receptors because they were blocked by antagonists of GABA receptors, such as GABAzine and bicuculline. Similar responses to synaptic activity were observed in DCN neurons, amygdalar principal cells, and collicular premotor neurons. However, in contrast to the hippocampus, the responses in these three regions were largely insensitive to antagonists of inhibitory neurotransmitter receptors. This indicates that synaptic activity can also cause Cl − influx through alternate pathways that remain to be identified. We conclude that Clomeleon imaging permits non-invasive, spatiotemporally precise recordings of [Cl − ] i in a large variety of neurons, and provides new opportunities for imaging synaptic inhibition and other forms of neuronal chloride signaling.

AbstractTissue cross‐reactivity studies provide a rational in vitro assay to determine the range and intensity of distribution of potential epitopes reactive with a monoclonal antibody test article prior to its administration to humans. The predictive value of the assay lies in incorporating the characteristics of the monoclonal antibody (isotype, subtype, and other molecular modifications) with the biological activity of the molecule itself, and the potential in vivo distribution of it. This article outlines important considerations in the conduct of the tissue cross‐reactivity study and provides best practices guidelines for interpreting the information gained from this assay relative to human safety.

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.

Neurons in the superficial gray layer (SGS) of the superior colliculus receive visual input and excite intermediate layer (SGI) neurons that play a critical role in initiating rapid orienting movements of the eyes, called saccades. In the present study, two types of experiments demonstrate that a population of SGI neurons gives rise to a reciprocal pathway that inhibits neurons in SGS. First, in GAD67-GFP knockin mice, GABAergic SGI neurons that expressed GFP fluorescence were injected with the tracer biocytin to reveal their axonal projections. Axons arising from GFP-positive neurons in SGI terminated densely in SGS. Next, SGI neurons in rats and mice were stimulated by using the photolysis of caged glutamate, and in vitro whole-cell patch-clamp recordings were used to measure the responses evoked in SGS cells. Large, synaptically mediated outward currents were evoked in SGS neurons. These currents were blocked by gabazine, confirming that they were GABA(A) receptor-mediated inhibitory postsynaptic currents. This inhibitory pathway from SGI transiently suppresses visual activity in SGS, which in turn could have multiple effects. These effects could include reduction of perceptual blurring during saccades as well as prevention of eye movements that might be spuriously triggered by the sweep of the visual field across the retina.

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 microm of the soma in the intermediate layer; smaller responses could occasionally be evoked from sites as distant as 500 microm. 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.

A pathway from the superficial visual layers to the intermediate premotor layers of the superior colliculus has been proposed to mediate visually guided orienting movements. In these experiments, we combined photostimulation using "caged" glutamate with in vitro whole cell patch-clamp recording to demonstrate this pathway in the rat. Photostimulation in the superficial gray and optic layers (SGS and SO, respectively) evoked synaptic responses in intermediate gray layer (SGI) cells. The responses comprised individual excitatory postsynaptic currents (EPSCs) or EPSC clusters. Blockade of these EPSCs by TTX confirmed that they were synaptically mediated. Stimulation within a column (approximately 500 microm diam) extending superficially from the recorded cell evoked the largest and most reliable responses, but off-axis stimuli were effective as well. The EPSCs could be evoked by stimuli 1,000 microm off-axis from the postsynaptic neuron. The dimensions of this wider region (approximately 2 mm diam) corresponded to those of the dendrites of superficial layer wide-field neurons. SGI neurons differed in their input from SGS and SO; neurons in the middle of the intermediate layer (SGIb) were less likely to respond to visual layer photostimulation than were those in sublayers just above and below them. However, focal stimulation within SGIa did evoke responses within SGIb, indicating that SGIb neurons may receive input from the visual layers indirectly. These results demonstrate a columnar pathway that may mediate visually guided orienting movements, but the results also reveal spatial attributes of the pathway which imply that it also plays a more complex role in visuomotor integration.

Surgical biopsies of the liver were obtained from woodchuck hepatitis virus (WHV)-infected neonatal woodchucks at 2 time points before the self-limited or chronic outcomes became obvious by serologic criteria. Following segregation of outcomes, livers were analyzed for intrahepatic type 1 cytokine messenger RNAs (mRNAs) (interleukin 2 [IL-2], interferon gamma [IFN-gamma], tumor necrosis factor-alpha [TNF-alpha]) and leukocyte inflammatory phenotype (IgG+ plasma cells, lysozyme+ macrophages, CD3+ T cells). Baselines were assessed using age-matched uninfected control livers. At week 8 (early acute phase), intrahepatic type 1 cytokine mRNAs were similarly low in both outcome settings and no different from age-matched uninfected controls. This was consistent with the minimal initial viral loads and lack of histologic inflammation at this time. At week 14 (mid-acute phase), changes in viral load between outcome groups related inversely to the intrahepatic inflammatory responses. Animals that eventually became resolved had increased intrahepatic expression of IFN-gamma and TNF-alpha mRNAs and robust inflammation by CD3+ T cells, plasma cells, and macrophages. At the same time point of infection, animals that eventually became chronic carriers had an acute hepatitis involving the same cell types, but at diminished levels, and markedly deficient intrahepatic expression of IFN-gamma and TNF-alpha mRNAs. IL-2 mRNA remained at baseline control levels in both outcome groups. These cotemporal comparisons map a critical deviation in host response to the acute stage of an evolving chronic infection. They strongly suggest that increasing viral load and chronicity as an outcome of neonatal WHV infection result from a temporal deficiency in the acute intrahepatic effector mechanisms mediated by IFN-gamma and TNF-alpha.

Stratum griseum superficiale (SGS) of the superior colliculus receives a dense cholinergic input from the parabigeminal nucleus. In this study, we examined in vitro the modulatory influence of acetylcholine (ACh) on the responses of SGS neurons that project to the visual thalamus in the rat. We used whole-cell patch-clamp recording to measure the responses of these projection neurons to electrical stimulation of their afferents in the stratum opticum (SO) before and during local pressure injections of ACh. These colliculothalamic projection neurons (CTNs) were identified during the in vitro experiments by prelabeling them from the thalamus with the retrograde axonal tracer wheat germ agglutinin-apo-HRP-gold. In a group of cells that included the prelabeled neurons, EPSCs evoked by SO stimulation were significantly reduced by the application of ACh, whereas IPSC amplitudes were significantly enhanced. Similar effects were observed when the nicotinic ACh receptor agonist lobeline was used. Application of the selective GABA(B) receptor antagonist 3-[[(3,4-dichlorophenyl)-methyl]amino]propyl]( diethoxymethyl)phosphinic acid blocked ACh-induced reduction in the evoked response. In contrast, the ACh-induced reduction was insensitive to application of the GABA(A) receptor antagonist bicuculline. The ACh-induced reduction was also diminished by bath application of muscimol at the low concentrations that selectively activate GABA(C) receptors. Because GABA(C) receptors may be specifically expressed by GABAergic SGS interneurons (Schmidt et al., 2001), our results support the hypothesis that ACh reduces CTN activity by nicotinic receptor-mediated excitation of local GABAergic interneurons. These interneurons in turn use GABA(B) receptors to inhibit the CTNs.