Qualitative and quantitative analysis of monocyte transendothelial migration by confocal microscopy and three-dimensional image reconstruction.

A novel method for qualitative and quantitative analysis of monocyte transendothelial migration is described. By labeling monocytes and endothelial cells with different fluorophores, and utilizing confocal microscopy and three-dimensional image reconstruction, transmigrating monocytes were resolved and quantified within a subendothelial collagen gel. Comparison of monocyte migration across endothelial monolayers derived from human brain microvessels versus umbilical veins revealed diapedesis across brain endothelium to be significantly delayed. Inclusion of astrocytes within the subendothelial collagen gel resulted in the formation of an array of astrocytic processes that simulated the glia limitans surrounding brain microvessels in situ, thus yielding a more physiologic paradigm of the blood-brain barrier. By virtue of its unique capacity to provide information on the total number of migrating cells, this analytic approach overcomes significant caveats associated with sampling only aspects of the migration process. The potential adaptability of this method to computer-assisted analysis further enhances its prospective use in high-throughput screening.

Odors elicit three different oscillations in the turtle olfactory bulb.

We measured the spatiotemporal aspects of the odor-induced population response in the turtle olfactory bulb using a voltage-sensitive dye, RH414, and a 464-element photodiode array. In contrast with previous studies of population activity using local field potential recordings, we distinguished four signals in the response. The one called DC covered almost the entire area of the olfactory bulb; in addition, three oscillations, named rostral, middle, and caudal according to their locations, occurred over broad regions of the bulb. In a typical odor-induced response, the DC signal appeared almost immediately after the start of the stimulus, followed by the middle oscillation, the rostral oscillation, and last, the caudal oscillation. The initial frequencies of the three oscillations were 14.1, 13.0, and 6.6 Hz, respectively. When the rostral and caudal oscillations occurred together, their frequencies differed by a factor of 1.99 +/- 0.01. The following evidence suggests that the four signals are functionally independent: (1) in different animals some signals could be easily detected whereas others were undetectable; (2) the four signals had different latencies and frequencies; (3) the signals occurred in different locations and propagated in different directions; (4) the signals responded differently to changes in odor concentration; (5) the signals had different shapes; and (6) the rostral and caudal signals added in a simple, linear manner in regions where the location of the two signals overlapped. However, the finding that the frequency of the rostral oscillation is precisely two times that of the caudal oscillation suggests a significant relationship between the two. The location of the caudal oscillation in the bulb changed from cycle to cycle, implying that different groups of neurons are active in different cycles. This result is consistent with the earlier findings in the olfactory system of the locust (). Our results suggest an additional complexity of parallel processing of olfactory input by multiple functional population domains.