Wilbrand Knee Revisited.

BACKGROUND: Over a century ago, German ophthalmologist Hermann Wilbrand reported inferonasal crossing fibers within the chiasm curve anteriorly into the contralateral optic nerve. This anatomic bend, "Wilbrand knee," is classically cited as the explanation for the "junctional scotoma," a contralateral superotemporal visual field defect associated with lesions affecting the optic nerve at its junction with the chiasm. More recent reports have called into question the existence of Wilbrand knee or suggested that it may simply be an artifact. METHODS: Four human optic chiasms (obtained from cadaver donors with no reported premortem visual pathology) and 2 monkey chiasms were fixed and thin sectioned (40 µm), then examined using anisotropic scattering imaging, a novel technique that takes advantage of the fact that light reflects off well-defined linear structures (i.e., axonal tracts) in a predictable manner based on their orientation. Using this technique, tissue structures oriented in different directions can be distinguished at high resolution without the need for tissue staining. RESULTS: In all 4 human optic chiasms, thin fiber tracts consistent with, but less prominent than, those Wilbrand had described were observed. No such tracts were found in the monkey chiasms. CONCLUSIONS: Wilbrand knee exists in humans but is modest in its anterior projection. Wilbrand knee does not seem to be present in monkeys, however, which may explain conflicting reports in the literature regarding its existence.

GABA releases from parvalbumin-expressing and unspecific GABAergic neurons onto CA1 pyramidal cells are differentially modulated by presynaptic GABAB receptors in mouse hippocampus.

Hippocampus CA1 pyramidal cells receive γ-aminobutyric acid (GABA) release from multiple GABAergic interneurons. Combining optogenetic strategy and whole-cell recordings, we demonstrate that baclofen, a specific GABAB receptor agonist, depresses monosynaptic GABAA receptor-mediated transmission from parvalbumin (PV)-expressing interneuron terminals onto pyramidal cells with less efficacy than that from the unspecific GABAergic terminals. The depression from PV neuron terminals is mainly mediated by presynaptic P/N type calcium channels. The results suggest that GABAB receptors are widely expressed on GABAergic interneurons, where they exert inhibition onto pyramidal cells by GABA release with different efficacy. The data strengthen the proposal that diverse GABA neurons play different roles in modulating CA1 pyramidal cell excitability.

Ncm, a Photolabile Group for Preparation of Caged Molecules: Synthesis and Biological Application.

Ncm, 6-nitrocoumarin-7-ylmethyl, is a photolabile protective group useful for making "caged" molecules. Ncm marries the reliable photochemistry of 2-nitrobenzyl systems with the excellent stability and spectroscopic properties of the coumarin chromophore. From simple, commercially available starting materials, preparation of Ncm and its caged derivatives is both quick and easy. Photorelease of Ncm-caged molecules occurs on the microsecond time scale, with quantum efficiencies of 0.05-0.08. We report the synthesis and physical properties of Ncm and its caged derivatives. The utility of Ncm-caged glutamate for neuronal photostimulation is demonstrated in cultured hippocampal neurons and in brain slice preparations.

A Postsynaptic Role for Short-Term Neuronal Facilitation in Dendritic Spines.

Synaptic plasticity is a fundamental component of information processing in the brain. Presynaptic facilitation in response to repetitive stimuli, often referred to as paired-pulse facilitation (PPF), is a dominant form of short-term synaptic plasticity. Recently, an additional cellular mechanism for short-term facilitation, short-term postsynaptic plasticity (STPP), has been proposed. While a dendritic mechanism was described in hippocampus, its expression has not yet been demonstrated at the levels of the spine. Furthermore, it is unknown whether the mechanism can be expressed in other brain regions, such as sensory cortex. Here, we demonstrated that a postsynaptic response can be facilitated by prior spine excitation in both hippocampal and cortical neurons, using 3D digital holography and two-photon calcium imaging. The coordinated action of pre- and post-synaptic plasticity may provide a more thorough account of information processing in the brain.

The Shaping of Two Distinct Dendritic Spikes by A-Type Voltage-Gated K(+) Channels.

Dendritic ion channels have been a subject of intense research in neuroscience because active ion channels in dendrites shape input signals. Ca(2+)-permeable channels including NMDA receptors (NMDARs) have been implicated in supralinear dendritic integration, and the IA conductance in sublinear integration. Despite their essential roles in dendritic integration, it has remained uncertain whether these conductance coordinate with, or counteract, each other in the process of dendritic integration. To address this question, experiments were designed in hippocampal CA1 neurons with a recent 3D digital holography system that has shown excellent performance for spatial photoactivation. The results demonstrated a role of IA as a key modulator for two distinct dendritic spikes, low- and high-threshold Ca(2+) spikes, through a preferential action of IA on Ca(2+)-permeable channel-mediated currents, over fast AMPAR-mediated currents. It is likely that the rapid kinetics of IA provides feed-forward inhibition to counteract the regenerative Ca(2+) channel-mediated dendritic excitability. This research reveals one dynamic ionic mechanism of dendritic integration, and may contribute to a new understanding of neuronal hyperexcitability embedded in several neural diseases such as epilepsy, fragile X syndrome and Alzheimer's disease.

Interlamellar CA1 network in the hippocampus.

To understand the cellular basis of learning and memory, the neurophysiology of the hippocampus has been largely examined in thin transverse slice preparations. However, the synaptic architecture along the longitudinal septo-temporal axis perpendicular to the transverse projections in CA1 is largely unknown, despite its potential significance for understanding the information processing carried out by the hippocampus. Here, using a battery of powerful techniques, including 3D digital holography and focal glutamate uncaging, voltage-sensitive dye, two-photon imaging, electrophysiology, and immunohistochemistry, we show that CA1 pyramidal neurons are connected to one another in an associational and well-organized fashion along the longitudinal axis of the hippocampus. Such CA1 longitudinal connections mediate reliable signal transfer among the pyramidal cells and express significant synaptic plasticity. These results illustrate a need to reconceptualize hippocampal CA1 network function to include not only processing in the transverse plane, but also operations made possible by the longitudinal network. Our data will thus provide an essential basis for future computational modeling studies on information processing operations carried out in the full 3D hippocampal network that underlies its complex cognitive functions.

The kinetics of multibranch integration on the dendritic arbor of CA1 pyramidal neurons.

The process by which synaptic inputs separated in time and space are integrated by the dendritic arbor to produce a sequence of action potentials is among the most fundamental signal transformations that takes place within the central nervous system. Some aspects of this complex process, such as integration at the level of individual dendritic branches, have been extensively studied. But other aspects, such as how inputs from multiple branches are combined, and the kinetics of that integration have not been systematically examined. Using a 3D digital holographic photolysis technique to overcome the challenges posed by the complexities of the 3D anatomy of the dendritic arbor of CA1 pyramidal neurons for conventional photolysis, we show that integration on a single dendrite is fundamentally different from that on multiple dendrites. Multibranch integration occurring at oblique and basal dendrites allows somatic action potential firing of the cell to faithfully follow the driving stimuli over a significantly wider frequency range than what is possible with single branch integration. However, multibranch integration requires greater input strength to drive the somatic action potentials. This tradeoff between sensitivity and temporal precision may explain the puzzling report of the predominance of multibranch, rather than single branch, integration from in vivo recordings during presentation of visual stimuli.

Wilbrand knee.

Wilbrand and Saenger(1) studied optic chiasms after unilateral enucleation, noting inferonasal crossing fibers curved anteriorly into the contralateral optic nerve (Wilbrand knee; figure, A). This explains contralateral superotemporal visual field defects (junctional scotomas) with optic nerve lesions at the chiasmal junction. However, Wilbrand knee may be an enucleation artifact.(2) The anisotropic light-reflecting properties of myelinated axons permitted imaging of normal human chiasms. Thin sections (25 µm) were illuminated and digitally imaged from 3 incident angles. Each of the images was pseudocolored (red, green, or blue) and merged, revealing an anomalously oriented fiber tract (appearing white) that reversed direction at the optic nerve-chiasm junction, found in inferior (figure, C) but not in superior sections (figure, B), consistent with Wilbrand and Saenger's original description.

Coherence-gated Doppler: a fiber sensor for precise localization of blood flow.

Miniature optical sensors that can detect blood vessels in front of advancing instruments will significantly benefit many interventional procedures. Towards this end, we developed a thin and flexible coherence-gated Doppler (CGD) fiber probe (O.D. = 0.125 mm) that can be integrated with minimally-invasive tools to provide real-time audio feedback of blood flow at precise locations in front of the probe. Coherence-gated Doppler (CGD) is a hybrid technology with features of laser Doppler flowmetry (LDF) and Doppler optical coherence tomography (DOCT). Because of its confocal optical design and coherence-gating capabilities, CGD provides higher spatial resolution than LDF. And compared to DOCT imaging systems, CGD is simpler and less costly to produce. In vivo studies of rat femoral vessels using CGD demonstrate its ability to distinguish between artery, vein and bulk movement of the surrounding soft tissue. Finally, by placing the CGD probe inside a 30-gauge needle and advancing it into the brain of an anesthetized sheep, we demonstrate that it is capable of detecting vessels in front of advancing probes during simulated stereotactic neurosurgical procedures. Using simultaneous ultrasound (US) monitoring from the surface of the brain we show that CGD can detect at-risk blood vessels up to 3 mm in front of the advancing probe. The improved spatial resolution afforded by coherence gating combined with the simplicity, minute size and robustness of the CGD probe suggest it may benefit many minimally invasive procedures and enable it to be embedded into a variety of surgical instruments.

Dendritic hold and read: a gated mechanism for short term information storage and retrieval.

Two contrasting theories have been proposed to explain the mechanistic basis of short term memory. One theory posits that short term memory is represented by persistent neural activity supported by reverberating feedback networks. An alternate, more recent theory posits that short term memory can be supported by feedforward networks. While feedback driven memory can be implemented by well described mechanisms of synaptic plasticity, little is known of possible molecular and cellular mechanisms that can implement feedforward driven memory. Here we report such a mechanism in which the memory trace exists in the form of glutamate-bound but Mg(2+)-blocked NMDA receptors on the thin terminal dendrites of CA1 pyramidal neurons. Because glutamate dissociates from subsets of NMDA receptors very slowly, excitatory synaptic transmission can leave a silent residual trace that outlasts the electrical activity by hundreds of milliseconds. Read-out of the memory trace is possible if a critical level of these bound-but-blocked receptors accumulates on a dendritic branch that will allow these quasi-stable receptors to sustain a regenerative depolarization when triggered by an independent gating signal. This process is referred to here as dendritic hold and read (DHR). Because the read-out of the input is not dependent on repetition of the input and information flows in a single-pass manner, DHR can potentially support a feedforward memory architecture.

Three-dimensional holographic photostimulation of the dendritic arbor.

Digital holography is an emerging technology that can generate complex light patterns for controlling the excitability of neurons and neural circuits. The strengths of this technique include a high efficiency with which available light can be effectively utilized and the ability to deliver highly focused light to multiple locations simultaneously. Here we demonstrate another strength of digital holography: the ability to generate instantaneous three-dimensional light patterns. This capability is demonstrated with the photolysis of caged glutamate on the dendritic arbor of hippocampal neurons, to study the nature of the integration of inputs arriving on multiple dendritic branches.

Patterned photostimulation with digital micromirror devices to investigate dendritic integration across branch points.

Light is a versatile and precise means to control neuronal excitability. The recent introduction of light sensitive effectors such as channel-rhodopsin and caged neurotransmitters have led to interests in developing better means to control patterns of light in space and time that are useful for experimental neuroscience. One conventional strategy, employed in confocal and 2-photon microscopy, is to focus light to a diffraction limited spot and then scan that single spot sequentially over the region of interest. This approach becomes problematic if large areas have to be stimulated within a brief time window, a problem more applicable to photostimulation than for imaging. An alternate strategy is to project the complete spatial pattern on the target with the aid of a digital micromirror device (DMD). The DMD approach is appealing because the hardware components are relatively inexpensive and is supported by commercial interests. Because such a system is not available for upright microscopes, we will discuss the critical issues in the construction and operations of such a DMD system. Even though we will be primarily describing the construction of the system for UV photolysis, the modifications for building the much simpler visible light system for optogenetic experiments will also be provided. The UV photolysis system was used to carryout experiments to study a fundamental question in neuroscience, how are spatially distributed inputs integrated across distal dendritic branch points. The results suggest that integration can be non-linear across branch points and the supralinearity is largely mediated by NMDA receptors.

A forward-imaging needle-type OCT probe for image guided stereotactic procedures.

A forward-imaging needle-type optical coherence tomography (OCT) probe with Doppler OCT (DOCT) capability has the potential to solve critical challenges in interventional procedures. A case in point is stereotactic neurosurgery where probes are advanced into the brain based on predetermined coordinates. Laceration of blood vessels in front of the advancing probe is an unavoidable complication with current methods. Moreover, cerebrospinal fluid (CSF) leakage during surgery can shift the brain rendering the predetermined coordinates unreliable. In order to address these challenges, we developed a forward-imaging OCT probe (740 µm O.D.) using a gradient-index (GRIN) rod lens that can provide real-time imaging feedback for avoiding at-risk vessels (8 frames/s with 1024 A-scans per frame for OCT/DOCT dual imaging) and guiding the instrument to specific targets with 12 µm axial resolution (100 frames/s with 160 A-scans per frame for OCT imaging only). The high signal-to-background characteristic of DOCT provides exceptional sensitivity in detecting and quantifying the blood flow within the sheep brain parenchyma in real time. The OCT/DOCT dual imaging also demonstrated its capability to differentiate the vessel type (artery/vein) on rat's femoral vessels. We also demonstrated in ex vivo human brain that the location of the tip of the OCT probe can be inferred from micro-anatomical landmarks in OCT images. These findings demonstrate the suitability of OCT guidance during stereotactic procedures in the brain and its potential for reducing the risk of cerebral hemorrhage.

Optical coherence tomography guided neurosurgical procedures in small rodents.

The delivery of therapeutic agents directly to targets deep within the brain is becoming an important tool in the treatment of a variety of neurological disorders. Currently, the standard method to accomplish this is by using stereotactic procedures. While this existing method is adequate for many experimental situations, it is essentially a blind procedure that cannot provide real-time feedback on whether the actual location deviated from the intended location or whether the therapeutic agent was actually delivered. Here we describe an optical guidance technique that is designed to work in conjunction with existing stereotactic procedures to provide the needed real-time feedback for therapeutic delivery in live animals. This real-time feedback is enabled by a technology called catheter-based optical coherence tomography (OCT). In this study we show that OCT can provide real-time position feedback based on microanatomic landmarks from the live rodent brain. We show that OCT can provide the necessary guidance to perform microsurgery such as the selective transection of the Schaffer collateral inputs to the CA1 region of the hippocampus with minimal perturbation of overlying structures. We also show that OCT allows visual monitoring of the successful delivery of viral vectors to specific subregions of the hippocampus.

Thinking inside the graft: applications of optical coherence tomography in coronary artery bypass grafting.

Recent advances in catheter-based optical coherence tomography (OCT) have provided the necessary resolution and acquisition speed for high-quality intravascular imaging. Complications associated with clearing blood from the vessel of a living patient have prevented its wider acceptance. We identify a surgical application that takes advantage of the vascular imaging powers of OCT but that circumvents the difficulties. Coronary artery bypass grafting (CABG) is the most commonly performed major surgery in America. A critical determinant of its outcome has been postulated to be injury to the conduit vessel incurred during the harvesting procedure or pathology preexistent in the harvested vessel. As a test of feasibility, intravascular OCT imaging is obtained from the radial arteries (RAs) and/or saphenous veins (SVs) of 35 patients scheduled for CABG. Pathologies detected by OCT are compared to registered histological sections obtained from discarded segments of each graft. OCT reliably detects atherosclerotic lesions in the RAs and discerns plaque morphology as fibrous, fibrocalcific, or fibroatheromatous. OCT is also used to assess intimal trauma and residual thrombi related to endoscopic harvest and the quality of the distal anastomosis. We demonstrate the feasibility of OCT imaging as an intraoperative tool to select conduit vessels for CABG.

Hyperexcitability of distal dendrites in hippocampal pyramidal cells after chronic partial deafferentation.

Traumatic injury to the CNS results in chronic partial deafferentation of subsets of surviving neurons. Such injuries are often followed by a delayed but long-lasting period of aberrant hyperexcitability. The cellular mechanisms underlying this delayed hyperexcitability are poorly understood. We developed an in vitro model of deafferentation and reactive hyperexcitability using organotypic hippocampal slice cultures to study the underlying cellular mechanisms. One week after transection of the Schaffer collateral and temporoammonic afferents to CA1 neurons, brief tetanic stimulation of the residual excitatory synapses produced abnormally prolonged depolarizations, compared with responses in normally innervated neurons. Responses to weak stimulation, in contrast, were unaffected after deafferentation. Direct stimulation of distal apical dendrites using focal photolysis of caged glutamate triggered abnormally prolonged plateau potentials in the deafferented neurons when strong stimulation was given, but responses to weak stimulation were not different from controls. An identical phenotype was produced by chronic "chemical deafferentation" with glutamate receptor antagonists. Responses to strong synaptic and photolytic stimulation were selectively prolonged by small-conductance (SK-type) calcium-activated potassium channel blockers in normally innervated cells but not after deafferentation. No significant changes in SK2 mRNA or protein levels, GABAergic inhibition, glutamate receptor function, input resistance, or action potential parameters were observed after chronic deafferentation. We suggest that a posttranslational downregulation of SK channel function in thin distal dendrites is a significant contributor to deafferentation-induced reactive hyperexcitability.

Catheter-based infrared light scanner as a tool to assess conduit quality in coronary artery bypass surgery.

OBJECTIVES: Endothelial disruption within saphenous vein and radial artery grafts increases thrombosis risk. However, no clinically applicable method for imaging the intima currently exists. We used a novel infrared imaging technology, optical coherence tomography (OCT; LightLab Imaging, Inc, Westford, Mass), to visualize the intima within harvested conduits. METHODS: Conduits were procured endoscopically (37 saphenous vein grafts and 8 radial artery grafts) or with the open technique (9 radial artery grafts) from 50 patients. Surplus segments were analyzed by means of OCT for evidence of preexisting pathology or traumatic injury. Focal plaques in radial artery grafts and the intimal hyperplasia area in saphenous vein grafts were quantified as having an intimal/medial thickness ratio of greater than 0.5. Biopsy specimens were obtained for histologic confirmation and to analyze matrix metalloproteinase 2 levels (saphenous vein grafts) and prostacyclin/nitric oxide metabolites (radial artery grafts). Interobserver kappa coefficients and a Bland-Altman analysis were used to determine the reproducibility and accuracy of OCT interpretations. RESULTS: Radial artery imaging revealed plaque in 76%. Endoscopically harvested vessels showed intraluminal clot (38%) and intimal tears ranging from severe (6%) to mild (88%). In saphenous vein grafts intimal thickening was detected in 86% and intraluminal clotting in 68%. The intimal/medial thickness ratio determined by means of OCT correlated directly with matrix metalloproteinase 2 levels (R = 0.6804) in saphenous vein grafts and inversely with metabolites of prostacyclin (R = -0.55) and nitric oxide (R = -0.58) in radial artery grafts. OCT imaging was reproducible (interobserver kappa coefficients of >0.81 for the characterization of plaque types) and showed a strong correlation with histology (R = 0.8, P < .001). CONCLUSIONS: OCT imaging provides an accurate, real-time, and reproducible means for assessing saphenous vein graft and radial artery graft bypass conduits. As a quality assurance tool, this technology might afford a more objective basis for conduit selection.

Photolysis of caged neurotransmitters: theory and procedures for light delivery.

Photolysis of "caged" compounds is a technique for releasing biologically active compounds in which the timing, rate, and spatial profile of release are controlled by light. Issues relating to the delivery of light for single-photon photolysis are presented. Specific discussions include the theories relating to how light interacts with biological tissue to produce scattering and phototoxicity, as well as the issues involved in choosing the appropriate light source. Several approaches and optical designs are presented for delivering the output of a laser to a microscopic specimen. The criteria for choosing an approach are presented. The commercial sources for the parts needed to build a photolysis system are also provided. This unit will be particularly useful for investigators interested in single-photon photolysis of caged neurotransmitters in brain slices.

Optical coherence tomography in the diagnosis and treatment of neurological disorders.

Optical contrast is often the limiting factor in the imaging of live biological tissue. Studies were conducted in postmortem human brain to identify clinical applications where the structures of interest possess high intrinsic optical contrast and where the real-time, high-resolution imaging capabilities of optical coherence tomography (OCT) may be critical. Myelinated fiber tracts and blood vessels are two structures with high optical contrast. The ability to image these two structures in real time may improve the efficacy and safety of a neurosurgical procedure to treat Parkinson's disease called deep brain stimulation (DBS). OCT was evaluated as a potential optical guidance system for DBS in 25 human brains. The results suggest that catheter-based OCT has the resolution and contrast necessary for DBS targeting. The results also demonstrate the ability of OCT to detect blood vessels with high sensitivity, suggesting a possible means to avoid their laceration during DBS. Other microscopic structures in the human brain with high optical contrast are pathological vacuoles associated with transmissible spongiform encephalopathy (TSE). TSE include diseases such as Mad Cow disease and Creutzfeldt-Jakob disease (CJD) in humans. OCT performed on the brain from a woman who died of CJD was able to detect clearly the pathological vacuoles.

Long-term potentiation of exogenous glutamate responses at single dendritic spines.

Long-term increases in the strength of excitatory transmission at Schaffer collateral-CA1 cell synapses of the hippocampus require the insertion of new alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPARs) into the synapse, but the kinetics of this process are not well established. Using microphotolysis of caged glutamate to activate receptors at single dendritic spines in hippocampal CA1 cells, we report the long-lasting potentiation of AMPAR-mediated currents with only a single pairing of photoreleased glutamate and brief postsynaptic depolarization. This potentiation was N-methyl-d-aspartate receptor (NMDAR)-dependent and was reversed with low-frequency photostimulation in an NMDAR-dependent manner, suggesting that it is mediated by the same mechanism(s) as conventional synaptic long-term potentiation. Potentiation of photolytic responses developed rapidly in a stepwise manner after a brief and variable delay (<60 s) at spines, but could not be induced at extrasynaptic sites on the dendritic shaft. Potentiation was accompanied by a concomitant decrease in postsynaptic, polyamine-dependent paired-pulse facilitation of the photolytic currents, indicating that a change in the subunit composition of the AMPARs underlying the response contributed to the potentiation. These changes are consistent with an increase in the proportion of GluR2-containing AMPARs in the spine head. These results demonstrate that activation of postsynaptic glutamate receptors by glutamate is not only necessary, but sufficient, for the induction of NMDAR-dependent long-term potentiation and reveal additional aspects of its expression.