David M. Senseman

Odor-elicited activity monitored simultaneously from 124 regions of the salamander olfactory bulb using a voltage-sensitive dye.

In response to controlled, odor pulse stimulation of the olfactory receptor mucosa, large fluorescence signals were recorded simultaneously from 124 contiguous anatomical regions of the salamander olfactory bulb using the potentiometric probe RH 414. The amplitudes and waveforms of the signals varied systematically across the bulbar surface in apparent correspondence with the laminae of the bulbar neurons. Qualitatively similar results were obtained using both intact and decorporate preparations, although fluorescence signals obtained from intact animals were distorted by optical noise generated by mechanical disturbances related to the functioning cardiovascular system. These results indicate that multiple site optical recording can be used to obtain information about spatio-temporal patterning of bulbar electrical activity evoked by physiological odor stimulation of the receptor mucosa. This is the first demonstration that activity elicited by a single, one second odor stimulus at physiological concentration and duration can be measured across many elements in the olfactory bulb. Information provided by this approach, in combination with complementary data derived from 2-deoxyglucose and single unit studies, may yield a better understanding of how the vertebrate central nervous system extracts quality and concentration information from olfactory afferent input.

Correspondence between visually evoked voltage-sensitive dye signals and synaptic activity recorded in cortical pyramidal cells with intracellular microelectrodes

Fast, multiple-site optical recording of voltage-sensitive dye (VSD) signals and intracellular microelectrode recordings were combined to characterize visually evoked neuronal responses in the visual cortex of the pond turtle, Pseudemys scripta. By using an in vitro, eye-brain preparation stained with the merocyanine oxazolone voltage-sensitive dye, NK-2495 or a close analog, NK-2761, large VSD signals relatively free of vibrational noise could be recorded in single trials following a stroboscopic light flash to the contralateral eye. VSD signals recorded from the same cortical location in repeated trials exhibited considerable variability in the onset, duration, and amplitude of secondary depolarizations. Because of this variability, secondary depolarizations were largely absent in signal-averaged responses. Superposition of VSD signals with intracellular recordings obtained from cortical pyramidal cells revealed a close correspondence between their signal waveforms. The two signals were virtually identical in their onset, initial rate of rise, and time-to-peak. At later periods (> 500 ms), the correspondence was less close, especially for large cortical depolarizations. Some of this disparity could be attributed to contamination of the VSD signal by a large intrinsic optical response. A second contribution was a failure of the VSD signal to register asynchronous regenerative effects occurring in single pyramidal cells. It is suggested that the close correspondence between the microelectrode and optical recordings in the early phase of the response may reflect the organization of pyramidal cells into clusters that receive virtually identical synaptic inputs.

Spatiotemporal structure of depolarization spread in cortical pyramidal cell populations evoked by diffuse retinal light flashes

The spatiotemporal structure of cortical activity evoked by diffuse light flashes was investigated in an isolated eyecup-brain preparation of the pond turtle, Pseudemys scripta. By combining a photomicroscopic image of the preparation with voltage-sensitive dye signals recorded by a 464-element photodiode array, the spread of depolarization within different cortical areas could be directly visualized with millisecond temporal resolution. Diffuse stimulation of the contralateral eyecup initially depolarized the visual cortex at the junction between its lateral and medial divisions in a small area rostral of the ventricular eminence. From this point, the depolarization spread at different velocities (10-100 microm/ms) depending upon the direction of travel. Since the initial depolarization was always in the rostral pole, the largest spread invariably occurred in a rostral --> caudal direction. Within the confines of the medial visual cortex, depolarization spread at a constant velocity but slowed after entering the adjoining medial cortex. Increasing the stimulus illuminance increased the velocity of spread. Rostrocaudal spread of depolarization was also observed in response to electrical stimulation of the geniculocortical pathway and by direct focal stimulation of the cortical sheet. These data suggest that excitatory connections between pyramidal cell clusters play a prominent role in the initial activation of the cortex by diffuse retinal stimulation.

Physiologically evoked neuronal current MRI in a bloodless turtle brain: detectable or not?

Contradictory reports regarding the detection of neuronal currents have left the feasibility of neuronal current MRI (ncMRI) an open question. Most previous ncMRI studies in human subjects are suspect due to their inability to separate or eliminate hemodynamic effects. In this study, we used a bloodless turtle brain preparation that eliminates hemodynamic effects, to explore the feasibility of detecting visually-evoked ncMRI signals at 9.4 T. Intact turtle brains, with eyes attached, were dissected from the cranium and placed in artificial cerebral spinal fluid. Light flashes were delivered to the eyes to evoke neuronal activity. Local field potential (LFP) and MRI signals were measured in an interleaved fashion. Robust visually-evoked LFP signals were observed in turtle brains, but no significant signal changes synchronized with neuronal currents were found in the ncMRI images. In this study, detection thresholds of 0.1% and 0.1 degrees were set for MRI magnitude and phase signal changes, respectively. The absence of significant signal changes in the MRI images suggests that visually-evoked ncMRI signals in the turtle brain are below these detectable levels.

Visualizing differences in movies of cortical activity

This paper discusses techniques for visualizing structure in video data and other data sets that represent time snapshots of physical phenomena. Individual frames of a movie are treated as vectors and projected onto a low-dimensional subspace spanned by principal components. Movies can be compared and their differences visualized by analyzing the nature of the subspace and the projections of multiple movies onto the same subspace. The approach is demonstrated on an application in neurobiology in which the electrical response of a visual cortex to optical stimulation is imaged onto a high-speed photodiode array to produce a cortical movie. Techniques for sampling movies over a single trial and multiple trials are discussed. The approach provides the traditional benefits of principal component analysis (compression, noise reduction and classification) and also allows the visual separation of spatial and temporal behavior.

Extracting Wave Structure from Biological Data with Application to Responses in Turtle Visual Cortex

Waves have long been thought to be a fundamental mechanism for communicating information within a medium and are widely observed in biological systems. However, a quantitative analysis of biological waves is confounded by the variability and complexity of the response. This paper proposes a robust technique for extracting wave structure from experimental data by calculating “wave subspaces” from the KL decomposition of the data set. If a wave subspace contains a substantial portion of the data set energy during a particular time interval, one can deduce the structure of the wave and potentially isolate its information content. This paper uses the wave subspace technique to extract and compare wave structure in data from three different preparations of the turtle visual cortex. The paper demonstrates that wave subspace caricatures from the three cortical preparations have qualitative similarities. In the numerical model, where information about the underlying dynamics is available, wave subspace landmarks are related to activation and changes in behavior of other dynamic variables besides membrane potential.

Recordings from human myenteric neurons using voltage-sensitive dyes.

Voltage-sensitive dye (VSD) imaging became a powerful tool to detect neural activity in the enteric nervous system, including its routine use in submucous neurons in freshly dissected human tissue. However, VSD imaging of human myenteric neurons remained a challenge because of limited visibility of the ganglia and dye accessibility. We describe a protocol to apply VSD for recordings of human myenteric neurons in freshly dissected tissue and myenteric neurons in primary cultures. VSD imaging of guinea-pig myenteric neurons was used for reference. Electrical stimulation of interganglionic fiber tracts and exogenous application of nicotine or elevated KCl solution was used to evoke action potentials. Bath application of the VSDs Annine-6Plus, Di-4-ANEPPS, Di-8-ANEPPQ, Di-4-ANEPPDHQ or Di-8-ANEPPS revealed no neural signals in human tissue although most of these VSD worked in guinea-pig tissue. Unlike methylene blue and FM1-43, 4-Di-2-ASP did not influence spike discharge and was used in human tissue to visualize myenteric ganglia as a prerequisite for targeted intraganglionic VSD application. Of all VSDs, only intraganglionic injection of Di-8-ANEPPS by a volume controlled injector revealed neuronal signals in human tissue. Signal-to-noise ratio increased by addition of dipicrylamine to Di-8-ANEPPS (0.98±0.16 vs. 2.4±0.62). Establishing VSD imaging in primary cultures of human myenteric neurons led to a further improvement of signal-to-noise ratio. This allowed us to routinely record spike discharge after nicotine application. The described protocol enabled reliable VSD recordings from human myenteric neurons but may also be relevant for the use of other fluorescent dyes in human tissues.

Fast Multisite Optical Recording of Mono- and Polysynaptic Activity in the Hamster Suprachiasmatic Nucleus Evoked by Retinohypothalamic Tract Stimulation

Responses of the hamster suprachiasmatic nucleus (SCN) to retinohypothalamic tract (RHT) stimulation were studied in horizontal hypothalamic slices using fast multisite optical recording techniques. A 124-element photodiode detector array provided high-speed monitoring (0.5 ms/frame) of evoked neural activity in the SCN, while a larger 464-element photodiode array yielded improved spatial imaging with some loss in temporal resolution (1.6 ms/frame). Brief electrical stimulation of the optic nerves evoked a propagated compound action potential that was recorded optically as a single transient depolarization in many slice regions, including the SCN. Only within the SCN, however, was this optic tract signal followed by additional voltage-dependent optical responses which exhibited a fast and a slow depolarizing component. The initial upstroke of the fast component was Ca(2+)-insensitive and is presumed to reflect activity in presynaptic RHT afferents. The remainder of the fast depolarization and the slow depolarization were Ca(2+)-sensitive. These responses were labeled the early population excitatory postsynaptic potential (Early P.E.P.S.P.) and the Late P.E.P.S.P. respectively. The Late P.E.P.S.P. was not enhanced by K+ channel blockade, suggesting that glial depolarization is not the primary source of this component. Drugs known to suppress RHT-evoked SCN field potentials also suppressed the Early and Late P.E.P.S.P.s recorded optically in the SCN. Unexpectedly, the Early P.E.P.S.P. was also reduced by the GABAA antagonist, bicuculline. Surface plots of normalized peak amplitudes showed that both SCN components had similar spatial distributions within the SCN, although the Early P.E.P.S.P. tended to be slightly more prominent within the medial SCN in some preparations. It is suggested that the Early P.E.P.S.P. represents firing of monosynaptically activated SCN neurons, while the Late P.E.P.S.P. reflects polysynaptic activity within the intrinsic SCN neuronal network that may be involved in the light entrainment of the circadian oscillator.

Animated Pseudocolor Activity Maps (Pam’s): Scientific Visualization of Brain Electrical Activity

Major advances in neuroscience have often followed directly from the application of new and more powerful methodological approaches to the study of brain structure and function (Clarke & Jacyna, 1987). Within the last decade a new technique has been developed that allows both brain structure and function to be studied in a closely integrated and highly complimentary fashion. This technique is multiple-site optical recording of membrane potential, or more simply, optical recording. Optical recording is based upon the ability of certain vital dyes (potentiometric probes) to optically signal changes in intracellular membrane potential. By viewing brain tissue stained with a voltage-sensitive dye with a suitable light detector system, changes in neuronal activity can be monitored simultaneously from a 100 or more contiguous anatomical regions (cf. Grinvald et al., 1988).

Modeling oxygen effects in tissue-preparation neuronal-current MRI

Tissue‐preparation neuronal‐current MRI (ncMRI) was recently developed to directly detect neuronal activity without hemodynamic contamination. However, as a paramagnetic substance, the oxygen molecules present in the tissue may also alter the ncMRI signal through relaxivity and susceptibility effects. To study the effects of oxygen on the ncMRI signal and estimate their impact on tissue‐preparation experiments, oxygen‐induced MRI signal changes were formulated as a function of oxygen concentration (OC) of gas, oxygen consumption rate, and imaging parameters. Under favorable conditions of these parameters, the maximum oxygen‐induced signal magnitude and phase change were estimated to be 0.32% and 3.85°, respectively. Considering that the ncMRI signal changes obtained in previous tissue‐preparation experiments (3–5% in magnitude, 0.8–1.7° in phase) were tens or hundreds of times larger than the corresponding oxygen‐induced signal changes (0.03% in magnitude, 0.03–0.07° in phase), it is concluded that the oxygen had negligible effects in the previous experiments. Magn Reson Med 58:407–412, 2007.