The Journal of Neuroscience November 1!386, 6(11): 3258-3264 Somatostatin mRNA: Regional Variation in Hybridization Densities in Individual Neurons George R. Uhl and Cathrine A. Sasek Howard Hughes Medical Institute at Harvard Medical School and Massachusetts General Hospital, and Department of Neurology, Massachusetts General Hospital, Boston, Massachusetts 02114 Somatostatin mRNA is detected by in situ hybridization of %- a meansof measuringgene expression and may reflect the func- labeled single-strandedcDNA probesto coronal sectionsof the tional transmitter-synthesizing and releasingactivity of pepti- rat brain that include the periventricular nucleus of the hypo- dergic neurons under certain circumstances(Mocchetti et al., thalamus. Features supporting hybridization specificity include 1984; Tang et al., 1983; Uhl and Reppert, 1986; Uhl et al., its anatomic distribution, the results of studies using multiple 1985). In situ hybridization techniquescan allow identification cDNA probes, RNAase experiments, competition studies, and of neurons containing peptide-specific mRNAs and relative correlations with patterns of somatostatinpeptide immunostain- quantitation of hybridization densities in individual neurons ing in adjacent sections.The hybridization densitiesvary strik- located in different brain regions (Gee and Roberts, 1983; Gee ingly from region to region, with highest densities in the peri- et al., 1983; McAllister et al., 1984; Uhl et al., 1985; Wolfson ventricular nucleusand more modest levels in areassuch as the et al., 1985). cerebral cortex and the striatum. On the basisof the results of We now report development and validation of an in situ in situ and immunohistochemicalapproaches, we suggestthat technique to detect somatostatinmRNAs in sectionsof rat brain. this variation is due to regional differences in the density of Using this technique, we describeregion-to-region variations in hybridization per positive cell, as well as to regional variation brain hybridization densities. We have focused on analysis of in the densitiesof somatostatinergicperikarya. 3 somatostatin-containing regions implicated in different brain functions: (1) the periventricular nucleus of the hypothalamus, Somatostatin is a neuropeptide family whoseprincipal member involved in neuroendocrine function; (2) the striatum, impli- is a 14 amino acid peptide that is found in neurons lying in cated in motor regulation; and (3) the entorhinal cerebral cortex, many nuclei distributed widely through the neuraxis (Bennett- implicated in “higher cortical” functions (Reichlin, 1983). Using Clark et al., 1980; Elde and Parsons, 1975; Finley et al., 1981; film autoradiography, emulsion autoradiography, and correla- Johanssonet al., 1984). Somatostatin-containing neurons lying tions with immunohistochemical staining in adjacent sections, in different zones have been differentially implicated in such we have adduced evidence for 2 sourcesfor this regional vari- brain activities as neuroendocrine, motor, autonomic, sensory, ation in hybridization densities.First, regional differencesin the and higher cortical functions (Reichlin, 1983). It is frequently densities of neuronal perikarya containing somatostatin and its assumedthat somatostatin-containing neuronslying within one mRNA contribute to some of this variability. Second, we de- brain nucleus will display greater functional similarity to one scriberegion-to-region differencesin the density of somatostatin another than to somatostatin-containing neurons that lie in a hybridization per positive cell. We discussthe implications of different brain nucleus (Bjorklund and HGkfelt, 1984a, b; Em- these findings for ascribing differential functional activities to son, 1983; Uhl, 1986a). However, meansfor direct demonstra- somatostatin neurons located in different brain regions. tion of differential functions for cells using somatostatin but lying in different brain regions have not been readily available. Materials and Methods Recent identification of the nucleotide sequencecoding for preprosomatostatinhas facilitated study of somatostatinmRNA Tissue using hybridization techniques (Goodman et al., 1980, 1983; Male Sprague-Dawley rats, 200 gm, were maintained on a 12 hr: 12 hr Montminy et al., 1984; Shen and Rutter, 1984). In several dif- day-night cycle with ad lib access to food and water. Forty-eight hours ferent neuroendocrine systems,known variations in the activ- before sacrifice, some animals were injected in the lateral ventricle with ities of peptidergic cells are closely paralleled by alterations in 50 rg of colchicine in 2 ~1 of saline solution over 5 min. Analysis of their expression of the genesfor their secretedproducts (Bar- tissue from 6 animals is reported here: 2 individuals injected with col- inaga et al., 1983; Burbach et al., 1984; Chin et al., 1985; Dia- chicine, 2 “sham-operated” controls in whom the cannulae was inserted mond and Goodman, 1985; Eberwine and Roberts, 1984; Kil- but only vehicle administered, and 2 unoperated controls. Similar results patrick et al., 1984; Murdoch et al., 1983). Assessmentof the were obtained in 10 other animals. Rats were anesthetized with chloral hydrate, and perfused with 2% depolymerized paraformaldehyde/0.5% cellular content of transmitter-specific mRNAs can thus provide glutaraldehydeI75 mM polylysine/37.5 mM sodium phosphate, pH 7.41 10 mM sodium periodate to approximately 1 ml/gm of body weight (Uhl et al., 1985). Brains were removed, postfixed in the same fixative Received Jan. 24, 1986; revised May 13, 1986; accepted May 14, 1986. for 90 min at PC, soaked for an additional 2 hr in 15% sucrose in We gratefully acknowledge discussions with Drs. J. Habener and G. Heimich; phosphate buffer, cut into slabs, and rapidly frozen on cryostat chucks excellent technical assistance from K. Hill and J. Evans, and careful assistance with the manuscript by S. Cronin. This work, from the Howard Hughes Medical using powdered dry ice or liquid nitrogen. Five (colchicine-pretreated) Institute Neuroscience Laboratories, Boston, was also supported by the M&night or 10 (non-colchicinized) micron sections of appropriate areas were cut Foundation, Sloan Foundation, American Parkinson Disease Associatidn, NIMH, on a Bright/Hacker cryostat and thaw-mounted onto slides. Thinner and NINCDS. sections of colchicine-pretreated brains were cut to allow identification Correspondence should be addressed to Dr. George R. Uhl, Wellman 405, of some single neurons in each of 2 serial sections, allowing correlations Massachusetts General Hospital, 50 Blossom Street, Boston, MA 02114 between immunohistochemistry and in situ hybridization. Slides pre- Copyright 0 1986 Society for Neuroscience 0270-6474/86/l 13258-07502.00/O treated by acetylation and coated with Denhardt’s solution (0.02% BSA/ The Journal of Neuroscience Somatostatin mRNA In Situ Hybridization 3259 0.02% Ficol/O.O2% polyvinylpirolidine) were used for sections destined confirmed by analysis of toluidine blue-stained adjacent sections. A for in situ hybridization. Sections destined for immunohistochemistry linear relationship between film optical density and radioactivity was were thaw-mounted onto chrome alum/gelatin-coated slides. confirmed using ‘S-brain paste standards of known activity, cut at 5 Sections for in situ hybridization were pretreated for 20 min at 22°C or 10 pm as appropriate (Uhl et al., 1985). with 0.2 M HCl, and 50 min at 37°C with proteinase K (1 &ml in 10 Two modes of microscopic analysis were applied to the immuno- mM Tris HCl, pH 7.4/2 mM calcium chloride) as described (Brahic and histochemical and emulsion autoradiographic images generated in the Haase, 1978; Uhl et al., 1985). In some experiments, slides were in- study. In both cases, selection of sections through anterior portions of cubated for 30 min in 25 &ml ribonuclease A to establish a control the periventricular hypothalamus allowed study of spatially separated (Gee and Roberts, 1983). Slides were then dehydrated in ethanol and neurons that were positive for somatostatin markers. In serial sections air-dried at room temperature. of tissue taken from colchicine-pretreated animals, correlative immu- nohistochemicallin situ hybridization analyses were undertaken (meth- Probe preparation od A). mRNA-sense template oligonucleotides corresponding to 2 regions of In method A, somatostatin-immunoreactive neurons were localized the rat preprosomatostatin mRNA extending from codon 26 to the first in immunohistochemically stained sections and their relationships to base codon of 41 (SMS 20) and from codon 46 to the first base of codon adjacent landmarks (blood vessels, aspects of the third ventricle, rhinal 6 1 (SMS 21) (Goodman et al., 1983) were synthesized chemically using sulcus, etc.) noted. Attempts were then made to identify the same neuron an Applied Biosystems oligonucleotide synthesizer. Antisense 17 base in the adjacent toluidine blue-stained section underlying the in situ cDNA “primers” complementary to regions beginning 6 bases from the hybridization emulsion autoradiogram. Only neurons whose represen- 5’ end of the “template” strand mRNA were synthesized. These com- tation in the toluidine blue-stained sections included their nuclei were plementary DNA sequences were allowed to hybridize to each other, included. After determination of an appropriate cell, the plane of focus and the antisense strand extended using Klenow fragment of DNA was moved from the level of the tissue to the level of the emulsion, and polymerase I, ?S-labeled