Receptors: Model Systems and Specific Receptors

volume.

Methods in Neurosciences

Volume 1. Gene Probes

Edited by P. Michael Conn

Volume 2. Cell Culture

Edited by P. Michael Conn

Volume 3. Quantitative and Qualitative Microscopy

Edited by P. Michael Conn

Volume 4. Electrophysiology and Microinjection

Edited by P. Michael Conn

Volume 5. Neuropeptide Technology: Gene Expression and Neuropeptide Receptors

Edited by P. Michael Conn

Volume 6. Neuropeptide Technology: Synthesis, Assay, Purification, and Processing

Edited by P. Michael Conn

Volume 7. Lesions and Transplantation

Edited by P. Michael Conn

Volume 8. Neurotoxins

Edited by P. Michael Conn

Volume 9. Gene Expression in Neural Tissues

Edited by P. Michael Conn

Volume 10. Computers and Computations in the Neurosciences

Edited by P. Michael Conn

Volume 11. Receptors: Model Systems and Specific Receptors

Edited by P. Michael Conn

Volume 12. Receptors: Molecular Biology, Receptor Subclasses, Localization, and Ligand Design (in preparation)

Edited by P. Michael Conn

Volume 13. Neuropeptide Analogs, Conjugates, and Fragments (in preparation)

Edited by P. Michael Conn

Volume 14. Paradigms for the Study of Behavior (in preparation)

Edited by P. Michael Conn

Volume 15. Photoreceptor Cells (in preparation)

Edited by Paul A. Hargrave

Volume 16. Neurobiology of Cytokines (Part A) (in preparation)

Edited by Errol B. De Souza

Volume 17. Neurobiology of Cytokines (Part B) (in preparation)

Edited by Errol B. De Souza

1

Steroid Receptors in the Central Nervous System

Louise M. Freeman and S. Marc Breedlove

Publisher Summary

Steroids are lipophilic molecules consisting of four interconnected carbon rings, the so-called cyclopentanoperhydrophenanthrene rings, also known as sterane. Cholesterol serves as the primary precursor to steroid synthesis, and the capacity and proclivity with which an organ will produce a particular steroid depend on the activity of various enzymes required for synthesis of the steroid. Steroids are usually classified either by the organs that primarily secrete them or by the class of action they promote. Most androgenic steroids are also anabolic, that is, they promote muscle growth. Steroids act by binding to specific protein receptors that are synthesized by target cells. Different steroids bind to different receptors, which display considerable homology with each other and with other proteins derived from a super family of genes, all of which have in common the property of altering gene expression. These proteins have a highly variable N-terminal region, a highly conserved central region that binds DNA, and a moderately conserved, ligand-binding C terminus.

Introduction

Steroid hormones exert powerful influences over the structure and function of both the developing and the mature nervous system. In fact, the proposition that steroid hormones could alter the developing and mature nervous system was first indicated on the basis of behavior. Castrated males of most species will eventually stop copulating and testosterone treatment can reinstate the behavior as long as the regimen is maintained. Similarly, sexual receptivity in females is abolished by ovariectomy and, in rats, for example, sequential treatment with estrogen and progesterone will result, a few hours later, in 6–8 hr of receptivity. Phoenix et al. (1) proposed that, in addition to such activational effects, steroids could also organize the developing nervous system, permanently rendering it masculine or feminine. The eventual discovery of sexual dimorphism in the vertebrate central nervous system (CNS) and the discovery that early exposure to testicular steroids was responsible for the development of masculine morphology amply confirmed this organizational hypothesis (2). Although the organizational–activational distinction has been useful to behavioral endocrinologists, the basic cellular action of steroids, change in gene expression, is similar throughout development (3). This change can often manifest itself as an alteration in the behavior of the organism. As such, steroids provide an opportunity to perturb normal neural processes and monitor the resulting changes in behavior, thereby illuminating the structural and/or physiological bases of neural development and function. We will review the methods of monitoring steroid influence, which center, as we will see, on monitoring the receptors for these hormones.

Steroids are lipophilic molecules consisting of four interconnected carbon rings, the so-called cyclopentanoperhydrophenanthrene rings, also known as sterane (Fig. 1). Cholesterol serves as the primary precursor to steroid synthesis, and the capacity and proclivity with which an organ will produce a particular steroid depend on the activity of various enzymes required for synthesis of the steroid. There are several competing systems of nomenclature for the various steroids, but we will rely on the names most commonly associated with each hormone in the neuroscience literature. Steroids are usually classified either by the organs that primarily secrete them or by the class of action they promote (Table I). Most androgenic steroids are also anabolic, that is, they promote muscle growth.

Table I

Partial Listing of Steroids Active in the Nervous System

aNote that thyroid hormones are not true steroids, but resemble steroids and behave much like steroids; for example, they alter gene expression after binding to a member of the steroid receptor superfamily of proteins.

Fig. 1 Structure of some common steroid hormones. Cholesterol serves as the most common source of the basic steroid nucleus of four interconnected carbon rings (sterane). Most of the synthetic steps are facilitated by specific enzymes; thus, production of steroids is controlled by the relative activity of various enzymes and hence particular synthetic pathways. Testosterone can serve as a prohormone to either estradiol or dihydrotestosterone, but the reverse reactions do not occur.

Steroids act by binding to specific protein receptors that are synthesized by target cells. Different steroids bind to different receptors, which display considerable homology with each other and with other proteins derived from a superfamily of genes, all of which have in common the property of altering gene expression (4). These proteins have a highly variable N-terminal region, a highly conserved central region that binds DNA, and a moderately conserved, ligand-binding C terminus. The lipophilic nature of steroids allows them to enter cells freely and bind to receptors that, depending on the receptor, are found in either the cytoplasm or the nucleus. The allosterically conjugated steroid-receptor complex in turn binds to DNA to alter gene transcription. The sequence of nucleotides in the promotor region to which the steroid-receptor complex binds, that is, the hormone-responsive element, differs somewhat for various steroids, but they are by and large quite similar. This has led to considerable speculation about cofactors, dimerization, and complex interactions that would allow different hormones (e.g., progestins and androgens) to exert such disparate biological effects when their receptors appear to bind the same DNA sites.

Investigation of steroid action can monitor (i) steroids themselves, (ii) the enzymatic machinery that produces them, (iii) the protein receptors that empower them to alter gene expression, (iv) alterations in gene expression, or (v) consequent changes in cell morphology and/or function. At this point it should be noted that there is an increasing number of reports of steroid effects that occur too quickly to reflect changes in gene expression; such effects may be the result of steroid action on the plasma membrane (5, 6). This chapter, however, is concerned with steroids in their traditional gene-regulating role and with the monitoring of classical steroid receptors as an index of steroid action. Because relatively few cells in the CNS produce receptors to a given steroid hormone, once a steroid has been shown to alter behavior or some other index of neural function, the mapping of neural centers that possess the appropriate receptor can quickly focus the search for the mechanisms mediating that change by eliminating many neural candidates. We will point out examples of such deductive reasoning as we describe the various methods for monitoring steroid receptors.

Biochemical Assays

In biochemical assays one dissociates the tissue or cells of interest and exposes suspected receptors to radiolabeled steroid ligands. After allowing sufficient time and appropriate conditions for them to bind each other, steroid-receptor complexes are separated from free steroid by one of a variety of techniques in order to infer both the number and the affinity of the protein molecules for the steroid of interest. This approach is complicated by the fact that many other proteins also have a weak affinity for steroids. Thus one must distinguish between the functionally significant, specific binding by the relatively rare receptors from the functionally insignificant, nonspecific binding by the other, abundant proteins. Nonspecific binding is assessed by incubating the labeled ligand in the presence of 100- to 1000-fold unlabeled ligand. Such competition should displace labeled ligand from almost all of the relatively rare, high-affinity receptor sites, but prevent little of the label from binding to the low-affinity, nonspecific sites. One simply subtracts the amount of binding to the labeled steroid in the presence of excess unlabeled ligand (nonspecific binding) from the amount of binding when only labeled steroid is available (total binding) to estimate specific binding (Fig. 2). Among the many ways to separate bound from unbound steroid are Sephadex columns (7) or activated charcoal, each of which detain unbound steroid; glass fiber filters that do not bind free steroid but that will nonspecifically bind protein, and therefore the occupied receptor (8, 9); and DNA cellulose filtration, which exploits the affinity of steroid-receptor complexes for nucleotides (10).

Fig. 2 Schematic depiction of the amount of protein binding to a radiolabeled steroid hormone from a homogenate sample of brain incubated in increasing concentrations of hormone. The upper curve depicts the amount of binding seen when only labeled steroid is added. This binding represents both high-affinity, specific sites (e.g., receptors) and the low-affinity, nonspecific binding of many proteins. The lower curve depicts the amount of labeled steroid bound in the presence of 100-fold cold, that is, unlabeled steroid, and thus represents nonspecific binding only. Subtracting the values of the bottom curve (nonspecific binding) from those of the top (total binding) estimates the specific binding (middle curve). The asymptote in the middle curve indicates the saturation of receptors at a particular concentration of hormone.

As with other allosteric interactions, the steroid-receptor complex has a tendency to dissociate, which can be affected by factors such as pH and temperature. Binding is usually measured at low temperatures, which slow chemical interactions and facilitate their detection. The rate at which the steroid and receptor bind together, the association constant Ka, can be understood as the concentration of complexes [S-R] divided by the product of the concentration of free steroid [S] and free receptors [R]:

Similarly, the dissociation constant Kd is equal to the inverse of Ka, that is, the proportion of the product of concentrations of unbound steroid and receptors divided by the concentration of steroid–receptor complexes:

One can estimate both the Kd and the concentration of receptor by the use of Scatchard analyses, in which one counts the specific binding at a variety of concentrations of labeled steroid, including concentrations beyond saturation of the receptors. Plotting the ratio of specifically bound steroid over free steroid on the y axis versus the concentration of bound steroid on the x axis generates a Scatchard plot (Fig. 3). If the total number of receptor sites is n, then the concentration of unoccupied receptor is the total minus those receptors that are occupied:

Fig. 3 Scatchard plot of steroid hormone-specific binding as calculated by the method depicted in Fig. 2. The abscissa indicates the concentration of specifically bound ligand, while the ordinate indicates the ratio of specifically bound to unbound hormone. The slope of such a plot is an estimate of the affinity of the receptor for the ligand, while the x intercept estimates the total number of binding sites. In this example, tissue from an androgen-competent animal is compared with tissue from an androgen-incompetent (tfm) rat. The affinities of the receptors seem identical, but the tfm rats have far fewer functional receptors ( 35).

Substituting this value for [R] in the previous equation for Kd and algebraically rearranging the terms into the Scatchard format of [S-R]/[S] for the y term and [S-R] for the x term yields the following formula:

Thus, the slope of the Scatchard line should be proportional to Kd, while the x intercept estimates the total number of receptors. A nonlinear Scatchard plot indicates a more complicated binding phenomenon, either more than one class of receptor or some type of cooperativity phenomenon; in that case, the Kd and n estimates are more difficult (11) and must be considered with some caution. Biochemical techniques also allow comparisons of cross-competing steroids.

Some of the complicating factors of examining CNS steroid receptors with biochemical assays include the following.

1. Lack of cellular resolution: The specific site of receptor localization is limited by the amount of nervous tissue that can be practically dissected. Microdissection techniques (12) are a common feature in studies that examine steroid receptor levels in specific brain nuclei (13–15).

2. Attempts to separate homogenized tissue into nuclear and cytoplasmic fractions may be incomplete, leading to incorrect conclusions concerning the location of receptors within a cell. Such an artifact may account for the cytosolic estrogen receptor detected by some when other techniques suggest estrogen receptors reside primarily in the nucleus (16–18).

3. The radiolabeled ligand may cross-react with some other protein. For instance, rat α-fetoprotein (AFP) binds estradiol with high affinity and can distort biochemical studies of estrogen receptors; this problem has been overcome by using either a ligand (moxestrol) or a separation method (DNA–cellulose) that does not bind AFP (19).

4. The ligand could be metabolized to something else before binding a protein.

Steroid Autoradiography

In the classic model, the steroid–receptor complex must interact with DNA to affect target cells. Therefore, one can identify such cells by the accumulation of steroid in their nuclei. This monitoring is typically accomplished by injecting a radiolabeled steroid into systemic circulation, allowing sufficient time for nuclear accumulation (1–2 hr) and placing thin slices of the tissue of interest next to film to detect radioactivity within the nuclei of target but not nontarget cells. Because of the need for fairly high spatial resolution to detect silver grains over the nuclei rather than cytoplasm, most steroid autoradiography is done with a low-energy-emitting isotope, tritium, which will affect only those portions of the film quite close to the label. The need for spatial resolution also favors placing the tissue on microscope slides previously dipped in nuclear track emulsion such as NTB-3 (Kodak, Rochester, NY), which, after sufficient exposure (6–100 weeks), can be developed in the manner of standard black-and-white film.

Because of concerns that the steroid might diffuse away from the site of accumulation, early steroid autoradiography used unfixed tissue rapidly removed from decapitated subjects (20). Tissue was quickly frozen, then sectioned in a cryostat in a darkroom and mounted on emulsion-coated slides, which, at room temperature, quickly thaw the sections and cause them to adhere. Optimal safelights, such as Thomas Instruments duplex (Charlottesville, VA) for Kodak NTB-emulsion can greatly facilitate sectioning. This thaw-mount method of steroid autoradiography in the brain was developed simultaneously by Pfaff (21) and Stumpf (22). Several subsequent studies have successfully examined nuclear accumulation in perfused animals (23, 24). It is possible to use fluorescent retrograde markers to identify particular classes of neurons in autoradiograms. Arnold (25) first managed this feat in the fresh-frozen bird brain using primuline as the marker, but the apparent stability of the labeled steroid during perfusion has made flurogold available as an anatomical marker (26,27). The most common criterion for determining if a cell has accumulated steroid is a density of silver grains over the nucleus three to five times that of the background, but more quantitative criteria, including Poisson models, are also available (28).

Some of the disadvantages of steroid autoradiography are the following.

1. The considerable amount of time that must be allowed for the tissue to expose the film: In some cases a few weeks are sufficient, but in others more than a year of exposure is required before convincing concentration can be seen over nuclei (29). One can obviate this delay by use of high specific activity labels (i.e., six tritium atoms in one molecule) or radiolabels with a shorter half-life. For example, the developing time of estradiol autoradiograms can be reduced from 4 months to 17 days by the use of ¹²⁵I-tagged estradiol (30). Another estradiol analog, 11β-methoxy-16-iodoestradiol (MIE2), is reported to give comparable resolution in only 16 hr (23). However, one must be concerned about how far from the cell nucleus the particle may travel in order to expose the film when higher energy ligands are used.

2. The considerable expense of injecting sufficient radiolabeled steroid to label all target cells within an animal: This consideration makes experiments with large animals especially prohibitive, but may be circumvented via in vitro autoradiography, in which the tissue of interest is removed and exposed to a solution of radiolabeled steroid. This technique has been successful in peripheral tissues with a high concentration of steroid receptors (e.g., the genital tract) (31), but to our knowledge its only successful demonstration in the CNS is the labeling of estrogen receptors in the rat brain by MIE2 (32). This method allowed topographical localization of receptors within 2 hr but did not give cell-by-cell resolution.

3. The concern that radioactivity detected in the nucleus of a cell may not be part of the originally injected ligand, but a metabolite. This is, of course, also a concern in biochemical assays.

4. The demands of working with a sharp microtome blade, radioactive material, and delicate tissue in semidarkness.

5. The concern that steroids may have important, nongenomic actions that may not require nuclear accumulation and therefore may not be monitored by autoradiography.

On the other hand, among the advantages of steroid autoradiography are the cell-by-cell resolution of target neurons and, in conjunction with anatomical markers such as histofluorescence (33) or immunocytochemistry (34), the distinction of various classes of neurons. One also gains an indication of whether nuclear accumulation, a prerequisite of function, has taken place. For example, the single-base pair substitution and single-amino acid substitution in the tfm rat mutation (35) cannot be detected via any available antisera or probes to the transcript, but can be seen in the failure of tfm tissue to accumulate steroid (36). Similarly, autoradiography indicates that some spinal motoneurons in the rat are first able to accumulate androgen between days 7 and 14 of life (37; see Fig. 4), which constrains hypotheses about the sites of androgen action on neuromuscular development. The caveat that the steroid may be metabolized to another hormone has been used to assay such metabolism by intentionally labeling those hydrogen atoms that are cleaved in the conversion of testosterone to estradiol (see Fig. 1) (38).

Fig. 4 Steroid autoradiograms showing the ontogeny of androgen accumulation in developing perineal motoneurons in rats. Note that the density of silver grains over the unstained neuropil is comparable across these autoradiograms, but the density of silver grains over motoneuronal nuclei increases with age. Based on the Poisson criterion, only a minority of these motoneurons evidenced accumulation at 7 days of age (P7), whereas by P14, the adult number of labeled motoneurons was achieved (open arrows point to labeled cells and solid arrows point to unlabeled cells). The density of labeling over the nuclei increases at subsequent ages ( 37). Bar = 40 μm.

Immunocytochemistry

The availability of the rat monoclonal antibody H222 to the estrogen receptor provided one of the first indications that even unbound steroid receptors might normally reside in cell nuclei, and that previous detection of unbound receptors in cytoplasmic fractions may have been an artifact of the biochemical process (39, 40). For immunocytochemistry (ICC) in the CNS, tissue is typically fixed, sectioned, and then exposed to reactions either as free-floating sections (41) or mounted on slides for subsequent reactions. Binding of the primary antibody is visualized with standard ICC methods such as peroxidase–anti-peroxidase (42), immunofluorescence (43), or avidin–biotin complexing (44). Signals can be enhanced by a multiple bridging technique (45) or immunogold–silver staining (46). Standard ICC concerns are relevant when localizing steroid receptors in the CNS. For instance, does omitting the primary antibody or preabsorbing with the appropriate proteins or peptides abolish staining? Does another protein, perhaps another steroid receptor, share the epitope and therefore also bind the antisera?

Immunocytochemistry offers the same spatial resolution as autoradiography at greatly reduced costs and delays. Estrogen receptor-like immunoreactivity detected by H222 has generally agreed well with steroid autoradiography in guinea pig brain (47), and in zebra finch and canary brain (41), but H222 has not consistently succeeded in rat brain [but see also Henry et al. (48)]. Immunocytochemical techniques can be used to double-label neurons and have indicated that individual neurons can respond to more than one steroid. Gahr (49) has combined H222 immunocytochemistry with dihydrotestosterone autoradiography to show that some neurons of the zebra finch brain express both androgen and estrogen receptors. By sequential ICC using different primary antibodies and the use of different chromogen or fluorescent compounds for visualization, Warembourgh et al. (43) have shown that estrogen and progesterone receptors can occupy the same neurons in the guinea pig brain. Another advantage of ICC is that receptors can be studied at the subcellular level in the absence of their natural ligands. For example, in adrenalectomized rats, electron microscopic examination of glucocorticoid receptors visualized by immunogold–silver-enhanced ICC showed them to be located primarily in the cytoplasm, while replacement therapy causes translocation to the nucleus (50). A similar study in ovariectomized rats, however, suggests that most estrogen receptors are located in the nucleus whether occupied or not (51). Available antisera against the rat androgen receptor indicate that this protein normally resides in the nucleus of peripheral tissues (51a, 51b) and of motoneurons, at least in intact males (S. M. Breedlove, unpublished observations).

Monitoring Steroid Receptor Transcripts

Steroid receptors can also be studied in the CNS by examining the RNA message for the receptor with a radiolabeled DNA or RNA probe complementary to the steroid receptor message. Such techniques show that the gene for the steroid receptor protein is being transcribed; it is, of course, possible that translational or posttranslational events prevent the formation of a functional receptor. Nonetheless, studies localizing receptor messages generally show good agreement with studies locating steroid receptors by other means (52–54). One advantage in searching for the receptor message is that mRNA is a relatively stable molecule (55), allowing, for example, the localization of mRNAs from human autopsy tissue (56). Another is that, provided the DNA sequences of the genes are known, probes can be synthesized that distinguish receptor subtypes (57). Two methods by which steroid receptor mRNA can be studied are Northern blots and in situ hybridization. The cloning and sequencing of the genes for steroid receptors have made these methods practical and popular for studying changes in expression of steroid receptor genes.

For a Northern blot, mRNA is extracted from homogenized tissue and separated by gel electrophoresis. The RNAs are then transferred to a nitrocellulose sheet and incubated with a radiolabeled strand of oligonucleotides complementary to the mRNA of interest. The Northern blot technique has been used to locate and quantify levels of androgen receptor (AR) mRNA in the brain, and study the changes in AR mRNA following castration and replacement therapy (58). Northern blots can also demonstrate the specificity of synthetic DNA probes for a particular thyroid receptor subtype (57).

For in situ hybridization, the same essential strategy is applied, but in slices of tissue, allowing cell-by-cell resolution of brain regions that express steroid receptor genes. Comprehensive reviews of methodological concerns are available elsewhere (59–61). In brief, CNS tissue is thinly sliced, mounted, incubated with the labeled cDNA or RNA probe, rinsed, and developed in a manner similar to autoradiography. Common controls include pretreatment with RNase and incubation with the sense-strand probe that does not hybridize to the mRNA. Of particular concern for both Northern blots and in situ hybridization for steroid receptor message is the possibility of the probe hybridizing to transcripts for other members of the steroid receptor family of genes. In situ hybridization maps of steroid receptor expression in the rat brain have been made for estrogen (52–54), androgen (52), progesterone (54), glucocorticoids (62), and thyroid hormones (57). Such maps are in close agreement with the findings of autoradiography and ICC. Although it is difficult to measure absolute levels of mRNA with in situ hybridization, it is possible to compare levels of gene transcription in different regions and under different conditions (63). The shorter half-life of the nucleotide probe means a shorter developing time than in steroid autoradiography. Finally, because of its applicability to human autopsy tissue, in situ hybridization can potentially be used to map steroid receptor sites in the human brain for the first time.

Each of the methods described has its strengths and drawbacks, but they can be used to complement each other. Any comprehensive investigation of steroid action on the CNS should include as many as are practical to gain a complete understanding of the hormone of interest.

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