Edmund M. Glaser

Analysis of thick brain sections by obverse—Reverse computer microscopy: Application of a new, high clarity Golgi—Nissl stain

Exceptionally clear Golgi-Nissl sections of 300 micron thickness have been morphometrically studied by light microscopy using oil immersion objectives. The clarity results from a new variation of a staining procedure that combines Golgi and Nissl images in one section. A viewing technique has been developed that permits a histologic preparation to be examined from its obverse (or normally viewed) side and its reverse (or under) side. The technique was designed for use with a computer microscope but can be employed with any light microscope whose stage position can be measured within 100 micron. Sections thicker than 300 micron can be studied dependent on the working distance of the objective lens, provided that the clarity of the material permits it.

A Semi-Automatic Computer-Microscope for the Analysis of Neuronal Morphology

Quantitative microscopic studies of individual neurons of the central nervous system, especially of their elaborate dendritic and axonal patterns, can be done only with great difficulty using conventional biological microscope instrumentation. In order to simplify and speed the gathering of such data, a computing light microscope has been developed. This instrument functions as a unit under the control of the investigator examining the histologic preparations. It is capable of measuring accurately distances in all three coordinate axes. Measurement of the length of dendrite branches is performed by means of a chord approximation. Computation is performed by means of conventional electronic analog techniques. Chord distances are computed according to the Pythagorean theorem by means of squaring, summing and square rooting. The initial coordinates of the chord are held in capacitor hold-circuits. The input to the computer section of the instrument is, by means of linear-motion transducers, fixed to the stage of the microscope along the three coordinate axes. There are two output devices, 1) a digital printer which prints on tape the distance measurements in micra (?), and 2) a plotting board on which is drawn a two-dimensional projection (in the plane of section) of the neuron. The distances measured range roughly from 3 to 100?, the accuracy of the measurement is ±l? or ±9 per cent, whichever is greater. Analysis times are reduced from the approximately 24 hours required by camera lucida techniques and hand calculation to 30 min with this new instrument.

Tonotopic organization of rabbit auditory cortex

Abstract The representation of sound frequency within the auditory cortex of New Zealand and Dutch Belted rabbits was investigated using tangential microelectrode penetrations. Both multiunit and evoked potential responses to tone bursts were examined. The auditory cortex of the rabbit was found to lie caudal to the genu of the rhinal sulcus on the lateral surface of the temporal cortex. Two contiguous and tonotopically organized fields were found: a large primary field and a smaller secondary field dorsal and anterior to it. Within the primary field there is a complete and orderly representation of sound frequency. The highest best frequencies (ca. 30.5 kHz) are represented in the dorsal part of the field; the lowest best frequencies (ca 0.4 kHz) are represented in the ventral part of the field. The frequency range of 1 to 18 kHz was represented in greatest detail. The primary field was coextensive with cortex exhibiting cytoarchitectonic features characteristic of sensory cortex: a wide, cell-dense lamina III/IV, a broad, sparsely cellular lamina V, and a well-developed lamina VI. The secondary field also contains an orderly tonotopic organization but it is reverse to that of the primary field. Its highest best frequencies were located ventrally and its lowest best frequencies dorsally. The auditory region in the rabbit is compared with that of two previously mapped, smooth-brain mammals—the squirrel and the guinea pig.

Tangential orientation and spatial order in dendrites of cat auditory cortex: A computer microscope study of Golgi-impregnated material

SummaryIn the tangential plane (parallel to the pial surface) dendrites in the primary auditory cortex (A1) of cat were found to exhibit preferentially oriented growth. This was shown by means of a computer microscope study of Golgi-Cox stained neurons as seen in 100 μm and 300 μm thick tangential sections. Two techniques were used to represent the 3-dimensional structure of dendrites: the “dendritic stick” and the “dendritic trumpet”. The former dismembers a dendrite into its individual segments; the latter considers a dendrite as an entity and represents it by its centroid, its moments and the spatial dispersion of its branches. Both statistical and Fourier analyses of the data show that within the tangential plane there is a significant and consistent orientation of the dendritic sticks in a dorso-ventral direction which seems correlated with the cortical isofrequency contours observed in electrophysiological maps of the A1 region. The dendritic trumpet analyses also show a distinctly non-random vertical distribution of pyramidal cell basal dendrites but not of stellate cell dendrites.

Cortical EEG power spectra associated with sleep-awake behavior in the rat

Power spectral analyses were used to study cortical EEG activities during sleep-awake behavior in the rat. EEG spectra, both long-time and sequential short-time, derived from EEG during the states of wakefulness, sleep, and REM sleep were qualitatively and quantitatively different. The degree of inter- and intrasubject variability between these spectra was minimal. This experimental model with the rat should allow quantitative delineation of cortical EEG changes produced by psychotropic drugs.

Auditory cortical responses to neonatal deafening: pyramidal neuron spine loss without changes in growth or orientation

SummaryNeonatal rabbits were unilaterally deafened at birth by surgical removal of the stapes, aspiration of the cochlear lymph, and kanamycin injection into the oval window. At 60 days of age, all rabbits were screened with brain stem evoked response tests in order to establish the efficacy of the deafening procedure. The auditory cortex contralateral to the destroyed cochlea was processed according to Golgi-Cox/Nissl procedures. Temporal bone histology revealed nearly complete outer hair cell loss in the damaged cochlea. The dendritic system of lamina III/IV pyramidal neurons contralateral to the deafened ear was digitized from frontal sections using a computer microscope system. Spine counts were also made along the basal dendrites. Spine counts revealed that neonatally deafened rabbits had 38.7% fewer spines along their basal dendrites. No differences between experimental and control rabbits were found in terms of soma cross-sectional area, total number of basal dendrites, total number of dendritic branches and total basal dendritic length. A fan-in projection of the dendritic system revealed no changes in the radial growth of basal dendrites resulting from the early acoustic trauma. In a prior study, spine-free nonpyramidal neurons in the same sections revealed altered dendritic growth and abnormally recurved dendrites. The separate response of pyramidal and nonpyramidal cell types to early cochlear damage is evidence for the different role of epigenetic determinants of dendritic form and orientation in sensory neocortical neurons.

The fan-in projection method for analyzing dendrite and axon systems

The fan-in projection is a computer graphical method of projecting onto a half plane the branching patterns of dendrites and axons that have already been 3-dimensionally digitized. It is suited for neurons possessing an axis of orientation (or elongation), such as cortical neurons. This axis is taken as the polar axis of a spherical coordinate system whose center is the soma. For cortical neurons, the equatorial plane corresponds to the tangential plane. Co-latitude is measured with respect to the positive polar axis. Longitude is discarded. What results is a projection in which dendrites and axons appear to be growing in a half plane whose boundary is the polar axis. The projection eliminates many of the distorting effects of depth foreshortening seen in conventional projections. In so doing it helps one to visualize branching properties that would otherwise be obscure.

Morphine self-administration and EEG power spectra in the rat.

Power spectral analyses were used to study changes in cortical EEG during morphine self-administration in freely-moving dependent rats prepared with chronic cortical and muscle electrodes and with permanent indwelling IV cannulae. As time progressed from a morphine self-injection toward another injection, a significant spectral shift of the EEG to lower frequencies occurred during successive REM sleep episodes. Each morphine self-injection reinstated the predominance of higher frequencies in the EEG spectra. These EEG changes which preceded lever pressing may reflect changes in morphine plasma levels and in the state of the CNS that precede drug-seeking behavior.

A simple ventilator suitable for neurophysiologic studies of small animals.

Abstract An inexpensive ventilator which produces intermittent positive pressure ventilation of the lungs is described. The device is usable with any respiratory gas mixture and permits continual adjustments of rate, stroke volume and inspiratory-expiratory duty ratio during operation. As judged by electrical, acoustical and blood gas measurements, the ventilator has proved to be quite satisfactory for the long term maintenance (up to 6 hr) of proper rodent respiration during microelectrode studies of the central auditory system.

A binary identification system for use in tracing and analyzing dichotomously branching dendrite and axon systems

Abstract A binary labeling scheme is described for identifying the nodes and branches of dichotomously branching structures, those of neurons in particular. Other local structural features of the branches can also be labeled by the procedure which is intended for use in the neuronal tracing operations that are performed in computer microscopy. The labeling scheme facilitates not only data acquisition but also a variety of morphometric analyses of branching patterns.