William Guido

ClearT: a detergent- and solvent-free clearing method for neuronal and non-neuronal tissue

We describe a clearing method for enhanced visualization of cell morphology and connections in neuronal and non-neuronal tissue. Using ClearT or ClearT2, which are composed of formamide or formamide/polyethylene glycol, respectively, embryos, whole mounts and thick brain sections can be rapidly cleared with minimal volume changes. Unlike other available clearing techniques, these methods do not use detergents or solvents, and thus preserve lipophilic dyes, fluorescent tracers and immunohistochemical labeling, as well as fluorescent-protein labeling.

Attention to pattern orientation: Negative cortical potentials, reaction time, and the selection process ☆

Abstract Six subjects were instructed to give an RT response to one of 3 stimuli randomly presented at a rate of 1/720–1180 msec. The stimuli were diffuse light flashes and grating light flashes with 9′ black and white bars oriented either horizontally or vertically. Attention was controlled by having the subject give an RT response within 375 msec following the relevant stimulus. Evoked potentials to the relevant and irrelevant stimuli were recorded monopolarly over the occipital (O z ), central (C z ), and frontal (F z ) areas of the cortex. Occipital potentials following relevant, as compared to irrelevant, flashes were relatively more negative between 150 and 250 msec poststimulus (O z :N235 potential). The early portion of the negative potential (150–200 msec) reflected selection between diffuse and patterned flashes but not between the orientations of the pattern in the flashes. The later portion of the negative potential (200–250 msec) reflected selection between the orientations of the pattern in the flashes. These results support the hypothesis that occipital potentials following relevant and irrelevant stimuli reflect not only differential processing of relevant and irrelevant information but also reflect a progressive change in the unit or specificity of the differential processing — early and late portions of the negative potential reflecting differential processing on the basis of first the general and then the specific characteristics of the relevant stimulus respectively. In contrast, frontal cortical potentials following relevant stimuli were slightly positive between 150 and 250 msec poststimulus (F z :P235 potential). The unique scalp distribution of these early attention effects suggests that O z :N235 reflects an intramodality underlying process localized in extrastriate visual cortex. A late positive potential peaking at about 370 msec following relevant stimuli (P370) was evident at all electrodes, although it had a central-occipital maximum. Since this potential followed the RT responses, it appeared to reflect processes other than those directly involved in the selective response to relevant stimuli.

Structural and functional composition of the developing retinogeniculate pathway in the mouse.

The advent of transgenic mice has made the developing retinogeniculate pathway a model system for targeting potential mechanisms that underlie the refinement of sensory connections. However, a detailed characterization of the form and function of this pathway is lacking. Here we use a variety of anatomical and electrophysiological techniques to delineate the structural and functional changes occurring in the lateral geniculate nucleus (LGN) of dorsal thalamus of the C57/BL6 mouse. During the first two postnatal weeks there is an age-related recession in the amount of terminal space occupied by retinal axons arising from the two eyes. During the first postnatal week, crossed and uncrossed axons show substantial overlap throughout most of the LGN. Between the first and second week retinal arbors show significant pruning, so that by the time of natural eye opening (P12-14) segregation is complete and retinal projections are organized into distinct eye-specific domains. During this time of rapid anatomical rearrangement, LGN cells could be readily distinguished using immunocytochemical markers that stain for NMDA receptors, GABA receptors, L-type Ca2+ channels, and the neurofilament protein SMI-32. Moreover, the membrane properties and synaptic responses of developing LGN cells are remarkably stable and resemble those of mature neurons. However, there are some notable developmental changes in synaptic connectivity. At early ages, LGN cells are binocularly responsive and receive input from as many as 11 different retinal ganglion cells. Optic tract stimulation also evokes plateau-like depolarizations that are mediated by the activation of L-type Ca2+ channels. As retinal inputs from the two eyes segregate into nonoverlapping territories, there is a loss of binocular responsiveness, a decrease in retinal convergence, and a reduction in the incidence of plateau potentials. These data serve as a working framework for the assessment of phenotypes of genetically altered strains as well as provide some insight as to the molecular mechanisms underlying the refinement of retinogeniculate connections.

Receiver operating characteristic (ROC) analysis of neurons in the cat's lateral geniculate nucleus during tonic and burst response mode.

Relay cells of the lateral geniculate nucleus respond to visual stimuli in one of two modes: burst and tonic. The burst mode depends on the activation of a voltage-dependent, Ca2+ conductance underlying the low threshold spike. This conductance is inactivated at depolarized membrane potentials, but when activated from hyperpolarized levels, it leads to a large, triangular, nearly all-or-none depolarization. Typically, riding its crest is a high-frequency barrage of action potentials. Low threshold spikes thus provide a nonlinear amplification allowing hyperpolarized relay neurons to respond to depolarizing inputs, including retinal EPSPs. In contrast, the tonic mode is characterized by a steady stream of unitary action potentials that more linearly reflects the visual stimulus. In this study, we tested possible differences in detection between response modes of 103 geniculate neurons by constructing receiver operating characteristic (ROC) curves for responses to visual stimuli (drifting sine-wave gratings and flashing spots). Detectability was determined from the ROC curves by computing the area under each curve, known as the ROC area. Most cells switched between modes during recording, evidently due to small shifts in membrane potential that affected the activation state of the low threshold spike. We found that the more often a cell responded in burst mode, the larger its ROC area. This was true for responses to optimal and nonoptimal visual stimuli, the latter including nonoptimal spatial frequencies and low stimulus contrasts. The larger ROC areas associated with burst mode were due to a reduced spontaneous activity and roughly equivalent level of visually evoked response when compared to tonic mode. We performed a within-cell analysis on a subset of 22 cells that switched modes during recording. Every cell, whether tested with a low contrast or high contrast visual stimulus exhibited a larger ROC area during its burst response mode than during its tonic mode. We conclude that burst responses better support signal detection than do tonic responses. Thus, burst responses, while less linear and perhaps less useful in providing a detailed analysis of visual stimuli, improve target detection. The tonic mode, with its more linear response, seems better suited for signal analysis rather than signal detection.

Failure to Maintain Eye-Specific Segregation in nob, a Mutant with Abnormally Patterned Retinal Activity

Axon terminals from the two eyes initially overlap in the dorsal-lateral geniculate nucleus (dLGN) but subsequently refine to occupy nonoverlapping territories. Retinal activity is required to establish and maintain this segregation. We show that despite the presence of retinal activity, segregated projections desegregate when the structure of activity is altered. Early in development, spontaneous retinal activity in the no b-wave (nob) mouse is indistinguishable from that of wild-type mice, and eye-specific segregation proceeds normally. But, around eye-opening, spontaneous and visually evoked activity in nob retinas become abnormal, coincident with a failure to preserve precise eye-specific territories. Dark-rearing studies suggest that altered visual experience is not responsible. Transgenic rescue of the mutated protein (nyctalopin) within nob retinal interneurons, without rescuing expression in either retinal projection neurons or their postsynaptic targets in the dLGN, restores spontaneous retinal activity patterns and prevents desegregation. Thus, normally structured spontaneous retinal activity stabilizes newly refined retinogeniculate circuitry.

Refinement of the retinogeniculate pathway

Much of our present understanding about the mechanisms contributing to the activity‐dependent refinement of sensory connections comes from experiments done in the retinogeniculate pathway. In recent years the mouse has emerged as a model system of study. This review outlines the major changes in connectivity that occur in this species and a potential mechanism that can account for such remodelling. During early postnatal life when spontaneous activity of retinal ganglion cells sweeps across the retina in waves, retinal projections from the two eyes to the dorsal lateral geniculate nucleus (LGN) segregate to form non‐overlapping eye‐specific domains. There is a loss of binocular innervation, a pruning of excitatory inputs from a dozen or more to one or two, and the emergence of inhibitory circuitry. Many of these changes underlie the development of precise eye‐specific visual maps and receptive field structure of LGN neurons. Retinal activity plays a major role both in the induction and maintenance of this refinement. The activity‐dependent influx of Ca2+ through L‐type channels and associated activation of CREB signalling may underlie the pruning and stabilization of developing retinogeniculate connections.

Synaptic dysfunction in the hippocampus accompanies learning and memory deficits in human immunodeficiency virus type-1 Tat transgenic mice.

BACKGROUND Human immunodeficiency virus (HIV) associated neurocognitive disorders (HAND), including memory dysfunction, continue to be a major clinical manifestation of HIV type-1 infection. Viral proteins released by infected glia are thought to be the principal triggers of inflammation and bystander neuronal injury and death, thereby driving key symptomatology of HAND. METHODS We used a glial fibrillary acidic protein-driven, doxycycline-inducible HIV type-1 transactivator of transcription (Tat) transgenic mouse model and examined structure-function relationships in hippocampal pyramidal cornu ammonis 1 (CA1) neurons using morphologic, electrophysiological (long-term potentiation [LTP]), and behavioral (Morris water maze, fear-conditioning) approaches. RESULTS Tat induction caused a variety of different inclusions in astrocytes characteristic of lysosomes, autophagic vacuoles, and lamellar bodies, which were typically present within distal cytoplasmic processes. In pyramidal CA1 neurons, Tat induction reduced the number of apical dendritic spines, while disrupting the distribution of synaptic proteins (synaptotagmin 2 and gephyrin) associated with inhibitory transmission but with minimal dendritic pathology and no evidence of pyramidal neuron death. Electrophysiological assessment of excitatory postsynaptic field potential at Schaffer collateral/commissural fiber-CA1 synapses showed near total suppression of LTP in mice expressing Tat. The loss in LTP coincided with disruptions in learning and memory. CONCLUSIONS Tat expression in the brain results in profound functional changes in synaptic physiology and in behavior that are accompanied by only modest structural changes and minimal pathology. Tat likely contributes to HAND by causing molecular changes that disrupt synaptic organization, with inhibitory presynaptic terminals containing synaptotagmin 2 appearing especially vulnerable.

The brain-stem parabrachial region controls mode of response to visual stimulation of neurons in the cat’s lateral geniculate nucleus

We recorded the responses of neurons from the cats lateral geniculate nucleus to drifting sine-wave grating stimuli both before and during electrical stimulation of the parabrachial region of the midbrain. The parabrachial region provides a mostly cholinergic input to the lateral geniculate nucleus, and our goal was to study its effect on responses of geniculate cells to visual stimulation. Geniculate neurons respond to visual stimuli in one of two modes. At relatively hyperpolarized membrane potentials, low threshold (LT) Ca2+ spikes are activated, leading to high-frequency burst discharges (burst mode). At more depolarized levels, the low threshold Ca2+ spike is inactivated, permitting a more tonic response (relay or tonic mode). During our intracellular recordings of geniculate cells, we found that, at initially hyperpolarized membrane potentials, LT spiking in response to visual stimulation was pronounced, but that parabrachial activation abolished this LT spiking and associated burst discharges. Coupled with the elimination of LT spiking, parabrachial activation also led to a progressive increase in tonic responsiveness. Parabrachial activation thus effectively switched the responses to visual stimulation of geniculate neurons from the burst to relay mode. Accompanying this switch was a gradual depolarization of resting membrane potential by about 5-10 mV and a reduction in the hyperpolarization that normally occurs in response to the inhibitory phase of the visual stimulus. Presumably, the membrane depolarization was sufficient to inactivate the LT spikes. We were able to extend and confirm our intracellular observations on the effects of parabrachial activation to a sample of cells recorded extracellularly. This was made possible by adopting empirically determined criteria to distinguish LT bursts from tonic responses solely on the basis of the temporal pattern of action potentials. During parabrachial activation, every cell responded only in the relay mode, an effect that corresponds to our intracellular observations. We quantified the effects of parabrachial activation on various response measures. The fundamental Fourier response amplitude (F1) was calculated separately for the total response, the tonic response component, and the LT burst component. Parabrachial activation resulted in an increased F1 amplitude for the total response. This increase was due to an increase in the tonic response component. For a subset of cells showing epochs of LT bursting, parabrachial activation concurrently reduced LT bursting and increased the amplitude of the tonic response. Parabrachial activation, by eliminating LT bursting, also caused cells to respond with more linearity.(ABSTRACT TRUNCATED AT 400 WORDS)

Morphologically Distinct Classes of Relay Cells Exhibit Regional Preferences in the Dorsal Lateral Geniculate Nucleus of the Mouse

A fundamental feature of the mammalian visual system is the presence of separate channels that work in parallel to efficiently extract and analyze specific elements of a visual scene. Despite the extensive use of the mouse as a model system, it is not clear whether such parallel organization extends beyond the retina to subcortical structures, such as the dorsal lateral geniculate (dLGN) of thalamus. To begin to address this, we examined the morphology of biocytin-filled relay cells recorded in dLGN of mice. Based on a quantitative assessment of their dendritic architecture, we found that even at early postnatal ages relay cells could be readily classified as X-like (biconical), Y-like (symmetrical), or W-like (hemispheric) and that each cell type was regionally specified in dLGN. X-like cells were confined primarily to the monocular ventral region of dLGN. Y-like cells occupied a central core that also contained ipsilateral eye projections, whereas W-like cells were found along the perimeter of dLGN. Similar to cat, Y-like cells were more prevalent than X- and W-like cells, and X-like cells tended to be smaller than other cell types. However, the dendritic fields of X- and W-like cells did not exhibit an orientation bias with respect to optic tract or boundaries of dLGN. Although we found clear morphological differences among relay cells, an analysis of their electrophysiological properties did not reveal any additional distinguishing characteristics. Overall, these data coupled with recent observations in the retina suggest that the mouse has many of the hallmark features of a system-wide parallel organization.

Synaptic development of the mouse dorsal lateral geniculate nucleus

The dorsal lateral geniculate nucleus (dLGN) of the mouse has emerged as a model system in the study of thalamic circuit development. However, there is still a lack of information regarding how and when various types of retinal and nonretinal synapses develop. We examined the synaptic organization of the developing mouse dLGN in the common pigmented C57/BL6 strain, by recording the synaptic responses evoked by electrical stimulation of optic tract axons, and by investigating the ultrastructure of identified synapses. At early postnatal ages (P14), when optic tract stimulation routinely evoked an excitatory postsynaptic potential/inhibitory postsynaptic potential (EPSP/IPSP) sequence, with the latter having both a GABAA and GABAB component. Electrophysiological and ultrastructural observations were consistent. At P7, many synapses were present, but synaptic profiles lacked the ultrastructural features characteristic of the adult dLGN, and little γ‐aminobutyric acid (GABA) could be detected by using immunocytochemical techniques. In contrast, by P14, GABA staining was robust, mature synaptic profiles of retinal and nonretinal origin were easily distinguished, and the size and proportion of synaptic contacts were similar to those of the adult. The emergence of nonretinal synapses coincides with pruning of retinogeniculate connections, and the transition of retinal activity from spontaneous to visually driven. These results indicate that the synaptic architecture of the mouse dLGN is similar to that of other higher mammals, and thus provides further support for its use as a model system for visual system development. J. Comp. Neurol. 518:622–635, 2010.