Beating heart cells: Using cultured cardiomyocytes to study cellular structure and contractility in laboratory exercises.

Heart muscle cells, or cardiomyocytes, exhibit intrinsic contractility in vitro. We found that commercially-available mammalian cardiomyocytes serve as an excellent model system for studying the cytoskeleton and cellular contractility, fundamental topics in undergraduate cell and molecular biology courses. Embryonic rat cardiomyocytes were plated on cell culture dishes or glass coverslips and visualized using an inverted phase-contrast microscope. The cardiomyocytes began contracting within 1-2 days after plating and continued to contract for many weeks, allowing their use in multiple laboratory sessions. Following background reading and instruction, students fixed and triple-stained the cardiomyocytes to examine the relative distributions of actin filaments and microtubules and the position of nuclei. Analysis and image capture with fluorescence microscopy provided striking examples of highly organized cytoskeletal elements. Students then designed experiments in which cardiomyocyte intrinsic contractility was explored. Changes in contraction rates were examined after treatment with signaling molecules, such as epinephrine. The addition of epinephrine to the culture medium, within a usable concentration window, increased the rate of contraction. These adaptable exercises provide undergraduate cell and molecular biology students with the exciting opportunity to study cardiomyocytes using standard cell culture and microscopy techniques.

Tyrosine hydroxylase-producing neurons in the human cerebral cortex do not colocalize with calcium-binding proteins or the serotonin 3A receptor.

Interneurons of the cerebral cortex play a significant role in cortical information processing and are of clinical interest due to their involvement in neurological disorders. In the human neocortex, three subsets of interneurons can be identified based on the production of the calcium-binding proteins parvalbumin, calretinin or calbindin. A subset of interneurons in the mouse cortex expresses the serotonin 3A receptor (5-HT3AR). Previous work in humans has also demonstrated the presence of a subgroup of cortical neurons that produces the catecholaminergic enzyme tyrosine hydroxylase (TH). Many TH-producing cells in the rat cortex coexpress calretinin and are adjacent to blood vessels. However, little is known about the phenotype of these TH interneurons in humans. Here we immunohistochemically examined the coexpression of TH with parvalbumin, calretinin, calbindin or 5-HT3AR in human Brodmann's areas 10 and 24, cortical regions with high densities of TH-containing neurons. Colocalization of TH with these calcium-binding proteins and with 5-HT3AR was not detected in either area. Cortical TH cells were rarely apposed to blood vessels, denoted by immunolabeling for the gliovascular marker aquaporin-4. Our results suggest that the TH-immunoreactive cells in the human cortex do not overlap with any known neurochemically-defined subsets of interneurons and provide further evidence of differences in the phenotype of these cells across species.

Increasing proportions of tyrosine hydroxylase-immunoreactive interneurons colocalize with choline acetyltransferase or vasoactive intestinal peptide in the developing rat cerebral cortex.

Cortical interneurons are critical for information processing, and their dysfunction has been implicated in neurological disorders. One subset of this diverse cell population expresses tyrosine hydroxylase (TH) during postnatal rat development. Cortical TH-immunoreactive neurons appear at postnatal day (P) 16. The number of TH cells sharply increases between P16 and P20 and subsequently decreases to adult values. The absence of apoptotic markers in these cells suggests that the reduction in cell number is not due to cell death but is due to a decline in TH production. Cortical TH cells lack all additional catecholaminergic enzymes, and many coexpress GABA and calretinin, but little else is known about their phenotype or function. Because interneurons containing choline acetyltransferase (ChAT) or vasoactive intestinal peptide (VIP) share characteristics with cortical TH neurons, the coexpression of TH with ChAT or VIP was examined throughout the neocortex at P16, P20, and P30. The proportions of TH cell profiles double-labeled for ChAT or VIP significantly increased between P16 and P30. Based on their proximity to blood vessels, intrinsic cholinergic and VIPergic cells have been hypothesized to regulate cortical microcirculation. Labeling with the gliovascular marker aquaporin-4 revealed that at least half of the TH cells were apposed to microvessels at these ages, and many of these cells contained ChAT or VIP. Cortical TH neurons did not coproduce nitric oxide synthase. These results suggest that increasing proportions of cortical TH neurons express ChAT or VIP developmentally and that a subset of these TH neurons may regulate local blood flow.

Neurochemical characterization of tyrosine hydroxylase-immunoreactive interneurons in the developing rat cerebral cortex.

Understanding the development of cortical interneuron phenotypic diversity is critical because interneuron dysfunction has been implicated in several neurodevelopmental disorders. Here, tyrosine hydroxylase (TH)-immunoreactive neurons in the developing and adult rat cortex were characterized in light of findings regarding interneuron neurochemistry and development. Cortical TH-immunoreactive neurons were first observed 2 weeks postnatally and peaked in number 3 weeks after birth. At subsequent ages, the number of these cell profiles was gradually reduced, and they were seen less frequently in adults. No DNA fragmentation or active caspase 3 was observed in cortical TH cells at any age examined, eliminating cell death as an explanation for the decrease in cell number. Although cortical TH cells reportedly fail to produce subsequent catecholaminergic enzymes, we found that the majority of these cells at all ages contained phosphorylated TH, suggesting that the enzyme may be active and producing L-DOPA as an end-product. Morphological criteria and colocalization of some TH cells with glutamic acid decarboxylase suggest that these cells are interneurons. Previously, parvalbumin, somatostatin, and calretinin were demonstrated in non-overlapping subsets of interneurons. Cortical TH neurons colocalized with calretinin but not with parvalbumin or somatostatin. These findings suggest that the transitory increase in TH cell number is not due to cell death but possibly due to alterations in the amount of detectable TH present in these cells, and that at least some cortical TH-producing interneurons belong to the calretinin-containing subset of interneurons that originate developmentally in the caudal ganglionic eminence.