Thomas G. Sherman

In situ hybridization histochemistry with synthetic oligonucleotides: Strategies and methods

In situ hybridization histochemistry is a useful method for localizing specific mRNA and studying the regulation of gene expression in an anatomical context. Previously, classical recombinant DNA and microbiological techniques have been required to identify and nick-translate the cloned DNAs necessary for in situ hybridization experiments. These requirements can be circumvented by the use of synthetic oligonucleotides complementary to the mRNA of interest. Compared to cloned cDNA probes, oligonucleotides are easy to manufacture, penetrate tissue much more easily, can be made to correspond to a sequence at any point in a known cDNA structure, and allow for the design of more precise controls for in situ studies. We describe a number of considerations in oligonucleotide probe design, including unique probe design from cDNA sequences and mixed probe design from protein primary structure data. The issues of species specificity, G-C content, probe length, tissue-specific mRNA expression, repeated sequences, non-coding region specific probes, and gene family homologies are discussed in an in situ hybridization context. Alternative strategies for mixed probe design are also considered. Information on the synthesis, purification, and sequence confirmation of oligonucleotides is then presented, followed by methods for labeling and using these probes for in situ hybridization histochemistry. The special considerations of specificity controls are addressed, including combined in situ hybridization histochemistry and immunocytochemistry, competition studies, the use of multiple hybridization probes, Tm studies, and Northern analysis of extracted RNA. The current and future directions of research with this technique are considered, with emphasis on the need to improve quantitation in order to facilitate the study of gene expression and regulation at the single cell level.

Coordinate Expression of Hypothalamic Pro-Dynorphin and Pro-Vasopressin mRNAs with Osmotic Stimulation

Peptides derived from pro-dynorphin and pro-vasopressin precursors coexist within neurosecretory vesicles of magnocellular neurons in the rat hypothalamus projecting to the posterior pituitary. The secretory activity of these neurons can be stimulated using physiological manipulations known to increase plasma vasopressin levels, such as dehydration and salt-loading. With chronic osmotic challenge, the mRNAs for both pro-dynorphin and pro-vasopressin increase in parallel in the supraoptic and paraventricular nuclei of the hypothalamus, but not within the nonmagnocellular suprachiasmatic nucleus cell groups projecting elsewhere than the neural lobe. The results indicate an example of coordinate regulation of mRNA expression for coexisting peptides within the brain.

Rapid expression of novel proteins in goldfish retina following optic nerve crush

Polyadenylated messenger RNA was isolated from goldfish retinas at various times following unilateral crush of the optic nerve. RNA was translated in a cell-free system and product proteins analyzed by two-dimensional gel electrophoresis and autofluorography. Poly(A)+ mRNA-directed protein synthesis revealed an 8-fold increase in the labeling of polypeptides of about 30 kd Mr and a pI of 5.5 in retinas 2 d following optic nerve crush, compared with control retina mRNA translation products. In vitro labeling of retinal proteins revealed the enhanced synthesis of comparable 30 kd proteins in 2 d post-crush retinas. Evidence presented suggests that this 30 kd protein cluster may correspond to fish 30 kd stress or heat-shock proteins (hsp-30).

Regulation of mRNA in Peptidergic Systems: Quantitative and In Situ Studies

Understanding the physiology of the brain is the ultimate goal of the neurosciences. A wide variety of tools are used In the attempt to delve into the biology of the CNS, included among them are such disparate tools as those used in anatomy, physiology, protein chemistry, and molecular genetics. On the face of it several of these methods give such different types of information that they would appear to be almost unrelated. Yet in the last few years it has become abundantly clear that the integration of these methods, at differing levels of discourse, has aided powerfully in our Increasing understanding of brain biochemistry, anatomy, and to some degree its functioning.

Opioid Peptides and Vasopressin

During the past several years, several of the neuroscience disciplines have increasingly assumed both the character and methodologies of classical molecular biology. In large part, this inundation reflects our need to investigate and understand the “pretranslational” events governing the regulation of peptide hormone and receptor expression throughout the nervous system. As a result, it has become evident that many components of the regulation of neuronal function and neurotransmission take place at sites removed from synapses. One such site is the regulation of specific messenger RNA (mRNA) expression. This has received increasing recent attention in light of the pivotal roles that these molecules play in neurotransmission and neuroendocrine functions.