Transferrin in the rat prostate Dunning tumor.

A major protein of the rat Dunning prostate tumor has been purified. It has physicochemical properties and an amino acid composition similar to that of transferrin. Furthermore, the isolated tumor protein reacts with antiserum to authentic rat transferrin. Immunoperoxidase staining with rabbit anti-rat transferrin localizes transferrin within tumor acinar glands. Rocket immunoelectrophoresis indicates that transferrin constitutes 30 to 40% of tumor fluid protein, but accounts for only approximately 9% of total serum protein. In normal rat prostate cytosols, the level of transferrin is at least 200 times lower than in tumor cytosol. Nevertheless, dorsal and lateral prostate show variable peroxidase staining indicating the presence of immunoreactive transferrin within acinar glands of these normal tissues. While intense staining for transferrin was found in the interstium of all regions of the normal prostate, transferrin was not detected within acinar glands of coagulating gland, ventral prostate, or seminal vesicle. Immunocytochemical localization of albumin indicates a distribution similar to that of transferrin in normal and neoplastic rat prostate. However, unlike transferrin, the albumin content was lower in tumor fluid than in serum. It is suggested that the high level of transferrin in tumor fluid may be due to selective uptake by the tumor from serum.

Protein inhibitor of activated STAT-1 (signal transducer and activator of transcription-1) is a nuclear receptor coregulator expressed in human testis.

An androgen receptor (AR) interacting protein was isolated from a HeLa cell cDNA library by two-hybrid screening in yeast using the AR DNA+ligand binding domains as bait. The protein has sequence identity with human protein inhibitor of activated signal transducer and activator of transcription (PIAS1) and human Gu RNA helicase II binding protein (GBP). Binding of PIAS1 to human AR DNA+ligand binding domains was androgen dependent in the yeast liquid beta-galactosidase assay. Activation of binding by dihydrotestosterone was greater than testosterone > estradiol > progesterone. PIAS1 binding to full-length human AR in a reversed yeast two hybrid system was also androgen dependent. [35S] PIAS1 bound a glutathione S-transferase-AR-DNA binding domain (amino acids 544-634) fusion protein in affinity matrix assays. In transient cotransfection assays using CV1 cells with full-length human AR and a mouse mammary tumor virus luciferase reporter vector, there was an androgen-dependent 3- to 5-fold greater increase in luciferase activity with PIAS1 over that obtained with an equal amount of control antisense cDNA or mutant PIAS1. Constitutive transcriptional activity of the AR N-terminal+DNA binding domain was increased 6-fold by PIAS1. PIAS1 also enhanced glucocorticoid receptor transactivation in response to dexamethasone but inhibited progesterone-induced progesterone receptor transactivation in the same assay system. mRNA for PIAS1 was highly expressed in testis of human, monkey, rat, and mouse. In rat testis the onset of PIAS1 mRNA expression coincided with the initiation of spermatogenesis between 25-30 days of age. Immunostaining of human and mouse testis with PIAS1-specific antiserum demonstrated coexpression of PIAS1 with AR in Sertoli cells and Leydig cells. In addition, PIAS1 was expressed in spermatogenic cells. The results suggest that PIAS1 functions in testis as a nuclear receptor transcriptional coregulator and may have a role in AR initiation and maintenance of spermatogenesis.

Thyroid receptor activator molecule, TRAM-1, is an androgen receptor coactivator.

An androgen receptor (AR) interacting protein was isolated from a HeLa cell complementary DNA library by two-hybrid screening in yeast using the AR DNA and ligand binding domains [amino acids (aa) 481-919] as bait. AR binding of the protein in yeast was dependent on the presence of testosterone or dihydrotestosterone (DHT). The isolated protein is identical to thyroid receptor activator molecule TRAM-1 but lacking aa 1-458. TRAM-1 is a steroid receptor coactivator-3 (SRC-3) subtype. In affinity matrix assays, 35S-labeled TRAM-1 bound the GST-AR ligand binding domain (aa 624-919) and GST-AR N-terminal and DNA binding domains (aa 1-660), but not the GST-AR DNA binding domain (aa 544-634) alone. Coexpression of TRAM-1 increased DHT-dependent AR transactivation 5-fold and constitutive activity of AR (aa 1-660) N-terminal and DNA-binding domains increased 9-fold. Full-length TRAM-1 (aa 1-1424) and the partial (aa 459-1424) were AR and GR coactivators as was SRC-1. In human testis, immunostaining of SRC-3 colocalized with AR in nuclei of Sertoli cells and peritubular myoid cells, indicating it could function as an AR coactivator in these cells. SRC-3 was also present in nuclei of spermatogenic cells where AR was not expressed, suggesting it might also be a coactivator with other nuclear receptors that regulate spermatogenesis.

The alternate N-terminal sequence of rat androgen-binding protein/sex hormone-binding globulin contains a nuclear targeting signal.

Androgen-binding protein/sex hormone-binding globulin (ABP/SHBG) is an extracellular carrier protein of androgens and estrogens. The protein regulates the bioavailability of sex steroids, and expanding evidence suggests that it also acts as a hormone. ABP/SHBG is secreted by Sertoli cells and hepatocytes using a signal peptide. An alternate messenger RNA encodes a nonsecreted form of ABP/SHBG (Alt-ABP/SHBG) that has a unique N-terminal amino acid sequence. In this study, we report that the alternate N-terminal sequence targets Alt-ABP/SHBG to the nucleus instead of the endoplasmic reticulum. The recombinant Alt-ABP/SHBG expressed in COS-7 cells was located in the nucleus, whereas recombinant cellular ABP/SHBG was primarily cytoplasmic. Neither dihydrotestosterone nor estradiol had any detectable effect on the ABP/SHBG or Alt-ABP/SHBG cellular location. Although the function of the nuclear Alt-ABP/SHBG is unknown, it may act as a modulator of androgen receptor- and/or estrogen receptor-mediated gene regulation.

Identification and immunocytochemical localization of a new thyrotropin-releasing hormone precursor in rat brain.

Antisera were raised to a tridecapeptide, Ser-Asp-Val-Thr-Lys-Arg-Gln-His-Pro-Gly-Arg-Arg-Phe, that was synthesized based on the sequence (residues 166-178) of a proposed cDNA for pro-TRH reported by Lechan et al. With this antiserum, immunostaining of Western blots of rat brain extracts revealed two major proteins with mol wt (Mr = 39,000 and 52,000) considerably larger than that of the largest protein (Mr = 29,000) that could be encoded by the cDNA of Lechan et al. Because these observations suggested the possibility of novel TRH precursors, we studied the immunocytochemical distribution of pro-TRH (39-52K) in rat brain. Our anatomical findings were 4-fold. 1) The distributions of 29K pro-TRH and 39-52K pro-TRH are not identical. 2) TRH is found only in regions containing 29K pro-TRH, 39-52K pro-TRH, or both. 3) There are regions that contain both 29K pro-TRH and 39-52K pro-TRH, but no TRH. 4) Regions containing only 39-52K pro-TRH do not contain 29K pro-TRH mRNA as mapped by Segerson et al. From these electrophoretic and anatomical observations, we postulate the existence of at least one and possibly two additional precursors that can be processed to TRH in rat brain.

Identification of C-cells in normal and goitrous rat thyroid tissues using antiserum to rat thyrocalcitonin and the immunoperoxidase bridge technique.

Application of the immunoperoxidase bridge technique to the light microscopic localization of C-cells in rat thyroid tissue is described. Guinea pig antisera to rat thyrocalcitonin (TCT) were produced by the injection of highly purified rat TCT (100-300 MRC U/mg) emulsified in complete Freund's adjuvant. A 1:1000 dilution of the antiserum used in this study gave a strong positive reaction with rat C-cells, and 1 ml of undiluted antiserum provided sufficient material for staining approximately 5000 slides. The substitution of nonimmune guinea pig serum for the anti-rat TCT serum or the prior absorption of anti-rat TCT serum with increasing amounts of highly purified rat TCT both eliminated the staining of thyroid C-cells. Likewise, no staining was observed in tissue sections from rat parathyroid, ovary, pituitary gland, and skeletal muscle. Antiserum to synthetic human TCT also could be used to identify rat thyroid C-cells. The method revealed abundant C-cells in goiters from rats fed a low-iodine diet for more than 1 year. This finding was supported by electron microscopic evaluation of goitrous tissue and by the detection, by radioimmunoassay, of TCT in thyroid tissue and in peripheral blood from goitrous rats.

Immunocytochemical localization of growth hormone-releasing factor in the rat hypothalamus.

The distribution of GRF-immunoreactive structures in the rat hypothalamus was studied after colchicine treatment with peroxidase-antiperoxidase immunocytochemistry in vibratome sections. The majority of the GRF-immunoreactive cell bodies were found in the arcuate nucleus and the medial perifornical region of the lateral hypothalamus. Scattered cells were seen in the lateral basal hypothalamus, the medial and lateral portions of the ventromedial nucleus, and the dorsomedial and paraventricular nuclei. Fibers from the perifornical cell bodies formed a fan-like projection to the median eminence, where a dense accumulation of GRF-containing processes and terminals was found. GRF terminals were located in the central regions of the median eminence. The localization of GRF-immunoreactive structures in the hypothalamus and median eminence reinforces the view that GRF plays a physiological role in the regulation of pituitary function.

Combined retrograde tracing and immunocytochemical identification of luteinizing hormone-releasing hormone- and somatostatin-containing neurons projecting to the median eminence of the rat.

LHRH and somatostatin or somatotropin-release inhibiting factor (SRIF) are produced by neurons whose cell bodies are located in telencephalic and diencephalic regions in the rat. Many, but not all, of these neurons project to the external zone of the median eminence (ME), where the peptides are released from the nerve terminals into hypophysial portal vessels. In the present study, we identified these neurons by in vivo injection of a retrograde tracer, the lectin wheat germ agglutinin (WGA), into the external zone of the ME. Subsequently, colchicine was given into the lateral ventricle 10-24 h after the WGA injection. The animals were killed 24-48 h after the WGA injection. Vibratome sections of the brains were stained for both WGA and LHRH or SRIF with a dual immunocytochemical technique. Approximately 70% of the LHRH neurons in the septum and the anterior hypothalamus and about 70% of the SRIF neurons in the medial preoptic area, the anterior periventricular area, and the paraventricular nucleus were double labeled, indicating that they projected to the ME. None of the SRIF neurons in the ventromedial and arcuate nuclei were labeled with WGA. Double labeled LHRH cells were either smooth and fusiform or spiny. WGA-accumulating LHRH or SRIF perikarya were intermixed with single labeled LHRH or SRIF cells, which apparently did not project to the ME. The results indicate that there are at least two populations of LHRH neurons in the preoptic-septal region and two populations of SRIF neurons in the medial preoptic and anterior periventricular areas and the paraventricular nucleus of the rat brain: one with access to the portal capillaries of the ME and, therefore, functionally related to the regulation of the pituitary, and another without access to portal capillaries, perhaps functionally related to intracerebral neurotransmission or modulation. Moreover, some hypophysiotropic LHRH and SRIF neurons may have axon collaterals reaching multiple targets within the central nervous system.

FSH stimulation of testicular androgen binding protein (ABP): comparison of ABP response and ovarian augmentation.

Production of testicular androgen binding protein (ABP), ceases following hypophysectomy and can be stimulated by FSH. Within 24 h after the administration of FSH, ABP can be measured in caput epididymis supernatant and by 4 days after FSH treatment, the concentration of ABP reaches a plateau. In a 3-day assay, ABP production in immature hypophysectomized rats was stimulated by 31 mug NIH-FSH-P1 per day (0.08 U NIH-FSH-P1 per 3 days) which is comparable to the sensitivity of the ovarian weight augmentation test in hypophysectomized rats. The relative ovarian weight augmenting and ABP stimulating activities of various FSH preparations were in agreement, suggesting that the biological stimulus of the ABP response is, in fact, FSH. The ABP response to FSH could become a useful testicular bioassay for FSH. Such an assay would be more practicle if ABP could be measured by a radioimmunoassay.

Developmental expression of an androgen-regulated epididymal protein.

Acidic epididymal glycoprotein (AEG), an androgen-regulated secretory protein of rat epididymis, was quantitated by RIA in epididymal extracts of rats of increasing age. Although detectable at 1 day of age, significant concentrations of AEG were not measured until 20 days; concentrations increased steadily, so that by 120 days of age, AEG represented 10% of total soluble protein. AEG mRNA was detected by Northern blot analysis of RNA from epididymides of 5-day-old animals and rapidly increased in amount between 20 and 35 days, reaching a maximum at 45 days. Using immunohistochemistry, AEG was localized in epididymal epithelial cells at 1 day of age. The number of cells staining for AEG increased markedly after 15 days. At 120 days, the immunoreactivity was predominantly localized to the lumen of the epithelial duct. To delineate factors that may influence AEG expression in the developing epididymis, we measured concentrations of androgen and androgen receptor mRNA in tissue extracts prepared from animals of various ages. Androgen receptor mRNA was detectable in epididymal extracts isolated from 1- to 90-day-old animals. Epididymal androgen concentrations were high at all ages (range, 6.0-31.2 ng/g tissue). The marked increase in AEG mRNA concentration at 20 days of age was not associated with an increase in either androgen or androgen receptor mRNA concentrations, suggesting that other factors may be necessary for AEG expression.

The androgen-binding protein gene is expressed in male and female rat brain.

Extracellular androgen-binding proteins (ABP) are thought to modulate the regulatory functions of androgens and the trans-acting nuclear androgen receptor. Testicular ABP and plasma sex hormone-binding globulin (SHBG), which is produced in liver, are encoded by the same gene. We have now found that the ABP-SHBG gene is also expressed in male and female rat brain. Immunoreactive ABP was found to be present in neuronal cell bodies throughout the brain as well as in fibers of the hypothalamic median eminence. The highest concentrations of immunoreactive cell bodies were located in the supraoptic and paraventricular nuclei. Likewise, ABP mRNA was present in all brain regions examined. Analysis of cDNA clones representing brain ABP mRNAs revealed amino acid sequence differences in brain and testicular ABPs. The protein encoded by an alternatively processed RNA has sequence characteristics suggesting that the protein could act as a competitior of ABP binding to cell surface receptors. These data and gene-sequencing experiments indicate that a specific ABP gene promoter is used for transcription initiation in brain. ABP may function in brain as an androgen carrier protein; however, in view of the widespread presence of ABP and ABP mRNA in brain, the protein may have a much broader, yet unknown, function.

Influence of estrogens on mouse uterine epidermal growth factor precursor protein and messenger ribonucleic acid.

Estrogens stimulate the in vivo proliferation of epithelial cells of the mouse uterus. The cumulative evidence from several earlier studies suggests that the mitogenic effect of estrogens is mediated indirectly through a polypeptide growth factor. The primary focus of the present investigation was to determine whether an epidermal growth factor (EGF)-related polypeptide originates in the uterus of the immature or adult mouse under normal or altered estrogen status. Hybridization experiments revealed the presence of the 4.7-kilobase prepro-EGF mRNA in uteri of immature CD-1 mice. The level of this mRNA was augmented at least 2-fold in immature mice treated for 4 days with estrogen, but levels remained markedly low compared to those in submaxillary gland or kidney. Two preparations of pooled uterine luminal fluid from estrogen-treated immature mice contained EGF immunoreactivity (1.2 and 1.7 ng/ml) that was stable in response to acid (50 mM acetic acid) and heat. Negligible EGF (less than 20 pg/uterus) was detected in acid extracts of uteri from ovariectomized or cycling adult mice. After injection of 17 beta-estradiol (0.2 or 2.0 micrograms, ip), the levels of acid-extractable uterine EGF in ovariectomized adult mice up to 48 h after treatment were not different from those obtained with vehicle alone. Immunolocalization of EGF in the mouse uterus was demonstrated only after paraffin sections were first briefly treated with pronase. Staining was observed along the borders of luminal and glandular epithelial cells, especially at the apical region of the cells. Some staining was also observed in the myometrium; stromal cells were negative. Synthesis of the reactive material was apparently estrogen independent, since localization was retained in uteri of both ovariectomized and immature mice. Immunoblots of preparations of membranes from uterine homogenates or epithelial cells revealed a band at mol wt of about 130,000, which, along with other findings of the present study, suggests that EGF occurs predominantly as the membrane-bound precursor form in this organ, as has been previously shown for the kidney. Although the biological role of the precursor in the uterus is not known, we speculate that estrogens function in an autocrine circuit by stimulating processing of the membrane-bound EGF precursor. EGF elaborated by this mechanism might conceivably react with known complementary receptors on uterine epithelial cells to stimulate proliferation.

HE2beta and HE2gamma, new members of an epididymis-specific family of androgen-regulated proteins in the human.

HE2 is an epididymis-specific sperm-binding secretory protein. We isolated a family of HE2-related complementary DNAs from a human caput/corpus library. The transcripts code for identical 71-amino acid N-termini and different C-termini, and 5'- and 3'-untranslated regions. Compared with the original HE2, HE2beta and HE2gamma proteins have a 25-amino acid deletion near the C-terminus, and HE2gamma isoforms have a second deletion. These frame-shifting deletions result in C-termini differing in length, amino acid sequence, including number of cysteines, and isoelectric point. Identical sequences and deletion start and stop points indicate the HE2 isoforms are derived from alternative splicing of 8 or more exons of a single gene. Northern hybridization revealed that the 0.9-kb messenger RNA (mRNA) is most abundant in human caput; there is much less of it (20%) in corpus and little (<5%) in cauda. In castrated Macaca mulatta, HE2 mRNA decreased to 10% of sham-operated levels. Testosterone replacement maintained HE2 mRNA 3- to 5-fold higher than castrate levels, indicating its androgen dependence. Immunohistochemical staining revealed that the beta1 form is highly expressed in principal cells of the initial segment and caput. It is secreted into the lumen and binds to the sperm surface in the postacrosomal and neck regions. The beta2 form is expressed in principal cells primarily in efferent ducts.

Primate epididymis-specific proteins: characterization of ESC42, a novel protein containing a trefoil-like motif in monkey and human.

Epididymal secreted proteins promote sperm maturation and fertilizing capacity by interacting with sperm during passage through the epididymis. Here we investigate the molecular basis of sperm maturation by isolating cDNA clones for novel epididymis-specific expressed sequences. Thirty-six novel cDNAs were isolated and sequenced from a subtracted Macaca mulatta epididymis library. The clones encode proteins with a range of motifs characteristic of protein-modifying enzymes, protease inhibitors, hydrophobic ligand-binding and transport proteins, extracellular matrix-interacting proteins, and transcription regulatory factors. The full length coding sequences were obtained for 11 clones representing a range of abundance levels. Expression of each is regionally localized and androgen regulated. The most abundant, ESC42, contains a cysteine-rich region similar to the signature binding domain of the trefoil family of motogenic wound repair proteins. The monkey and human proteins are nearly 90% identical. Immunohistochemical staining revealed that the protein is most abundant in the epithelium of the caput and is also present in the lumen and bound to sperm. The ESC42 gene, located on chromosome 20q11, contains two exons encoding two nearly identical predicted signal peptides and a third exon encoding the rest of the protein.

The immunocytochemical localization of enkephalin in the central nervous system of the rat.

The immunocytochemical localization of enkephalin-like immunoreactivity (ELI) throughout the rat central nervous system (CNS) was investigated. The detection of ELI-containing structures was facilitated through the use of (1) brains from colchicine-treated rats, (2) the proteolytic pretreatment of sections with pronase and (3) the "double-bridge" staining technique. Our findings confirm the presence of ELI in perikarya, neuronal processes and terminals in many areas of the CNS. In addition, the localization of ELI-containing perikarya is reported for the first time in the following areas: the olfactory bulb, the olfactory tubercle, the lateral preoptic nucleus, several nuclei within the amygdaloid nuclear complex, the hippocampus, the neocortex, the cingulate cortex, the posterior mammillary nucleus, the medial nucleus of the optic tract, the brachium of the inferior colliculus, the ventral tegmental nucleus, the locus ceruleus, the sub-ceruleal region, the lateral trapezoid nucleus, the nucleus reticularis lateralis, and lamina VII of the cervical spinal cord. Our results demonstrate ELI in neurons which are heterogeneous in size, some probably functioning as interneurons and others as projection neurons in different areas of the CNS. The location of these neurons within the brain suggests that these pentapeptides serve diverse functions which include, in addition to nociception, the regulation of neuroendocrine, respiratory, auditory, vestibular, and olfactory functions.

Immunohistochemical localization of nitric oxide synthase and soluble guanylyl cyclase in the ventral cochlear nucleus of the rat.

The diffusible messenger nitric oxide (NO) is implicated in auditory processing. It acts in the brain largely through activation of soluble guanylyl cyclase (sGC), a heterodimer comprised of alpha and beta subunits. The authors used immunohistochemistry to study the NO/guanosine 3',5'-cyclic monophosphate (cGMP) pathway in the cochlear nucleus of Sprague-Dawley rats. Central fibers of the cochlear nerve were stained for neuronal nitric oxide synthase (NOS-I) but not for sGCbeta. Within the ventral cochlear nucleus, a large fraction of principal cells were immunopositive for both NOS-I and sGCbeta; these cells could be seen at times receiving contacts from NOS-I-positive fibers. sGC staining of somatic cytoplasm extended into the distal dendritic tree. At variance with this pattern, NOS-I was concentrated mainly in somata. Double-labeling experiments showed that most of the principal neurons expressed both antigens. By contrast, in the granule cell domain, small cells that were immunopositive for NOS-I rarely corresponded to those that were immunopositive for sGC. To assess whether NOS-I and sGC immunoreactivities colocalize with their respective catalytic activities, the authors performed multiple labeling with L-citrulline (a by-product of the formation of NO from L-arginine) and cGMP, respectively. L-citrulline was restricted to NOS-I-positive elements, and the large majority of NOS-expressing neurons were positive for citrulline. Multiple labeling revealed that almost all sGC-positive neurons also accumulated cGMP both in the ventral cochlear nucleus and in the granule cell domain. These data suggest that NO is a signaling molecule in the cochlear nucleus, perhaps functioning in both a paracrine manner and an autocrine manner.

Plasticity of GABA- and glutamate-containing terminals in the mouse thalamic ventrobasal complex deprived of vibrissal afferents: an immunogold-electron microscopic study.

GABA and glutamate immunogold staining demonstrated that nerve cells of the thalamic ventrobasal complex (VB) of mice were positive exclusively for glutamate. None of the neuronal perikarya reacted the GABA antibody. By using alternate thin sections of the normal VB, it was also shown that large "specific" somatosensory and small corticothalamic terminals, both of which contained spherical synaptic vesicles, exhibited only glutamate-like immunoreactivity. A third axonal type, containing flat-ovoid synaptic vesicles, stained only for GABA. Seventy-five days after coagulation of the vibrissal follicles in newborn mice, a characteristic multiplication of GABA positive axon terminals was observed. In addition, it was demonstrated that, similarly to modified cortical endings (Hámori et al., J. Comp. Neurol. 254:166-183, '86), many GABA positive terminals appeared as specific afferent endings, replacing the missing "specific" vibrissal afferents. This finding shows a remarkable plasticity of inhibitory GABA axons during developmental synaptogenesis and provides further evidence that the size, location, and the type of attachment of presynaptic terminals are dependent on their postsynaptic target.

Ontogeny of cholinergic neurons in the mouse forebrain.

The development of cholinergic neurons in the mouse forebrain was studied by immunocytochemistry with a monoclonal antibody to choline acetyltransferase (ChAT), the rate-limiting enzyme for acetylcholine synthesis. Since this antibody stained dividing cells in ventricular germinal zones as well as differentiating neurons, likely routes of migration could be inferred on the basis of the location of immunoreactive (IR) cells at different gestational ages. Germinal zones for cholinergic cells were observed in all ventricular zones of the forebrain with the ventral zones generating the earliest cells by gestational day 13.5 (GD13.5). On GD14, ChAT IR cells were visible in the germinal zones of the eye, olfactory ventricle, anterior horn, and dorsolateral aspect of the lateral ventricle, lateral ganglionic eminence, ventro- and dorsolateral third ventricle, and in the pineal anlage (epiphysis). ChAT IR neurons continued to develop in these and additional germinal zones on GD15, including the medial, dorsal, and dorsomedial walls of the lateral ventricle, and the medial and dorsal ganglionic eminence. On GD16, ChAT IR neurons were located in the prelimbic, pyriform, and parietal cortices and the lamina terminalis, and a cluster of IR cells was observed in the ventricular zone of the caudatopallial angle. On GD17-18, neurons in the anterior olfactory nucleus, olfactory tubercle, horizontal and vertical nucleus of the diagonal band, and medial septal nucleus stained more darkly and were multipolar, whereas immature bipolar neurons appeared to continue their migration into the hippocampus and along major fiber tracts, such as the corpus callosum, external capsule, fornix and anterior commissure. This study provides a comprehensive view of the zones of origin, probable routes of migration, and final destination of cholinergic neurons in the mouse forebrain.

Substance P-containing pyramidal neurons in the cat somatic sensory cortex.

Light and electron microscopic immunocytochemical methods were used to verify the possibility that neocortical pyramidal neurons in the first somatic sensory cortex of cats contain substance P. At the light microscopic level, substance P-positive neurons accounted for about 3% of all cortical neurons, and the vast majority were nonpyramidal cells. However, 10% of substance P-positive neurons had a large conical cell body, a prominent apical dendrite directed toward the pia, and basal dendrites, thus suggesting they are pyramidal neurons. These neurons were in layers III and V. At the electron microscopic level, the majority of immunoreactive axon terminals formed symmetric synapses, but some substance P-positive axon terminals made asymmetric synapses. Labelled dendritic spines were also present. Combined retrograde transport-immunocytochemical experiments were also carried out to study whether substance P-positive neurons are projection neurons. Colloidal gold-labelled wheat germ agglutinin conjugated to enzymatically inactive horseradish peroxidase was injected either in the first somatic sensory cortex or in the dorsal column nuclei. In the somatic sensory cortex contralateral to the injection sites, a few substance P-positive neurons in layers III and V also contained black granules, indicative of retrograde transport. This indicates that some substance P-positive neurons project to cortical and subcortical targets. We have therefore identified a subpopulation of substance P-positive neurons that have most of the features of pyramidal neurons, are the probable source of immunoreactive axon terminals forming asymmetric synapses on dendritic spines, and project to the contralateral somatic sensory cortex and dorsal column nuclei. These characteristics fulfill the criteria required for classifying a cortical neuron as pyramidal.

An efficient enzyme immunoassay for glutamate using glutaraldehyde coupling of the hapten to microtiter plates.

In order to coat microtiter plates for enzyme immunoassays (EIAs), amino acids and other haptens are usually coupled to larger protein molecules. The formation of such conjugates is not always reproducible. This may lead to inconsistent hapten-protein stoichiometries, unfavorable orientation of the hapten on the protein and/or well-to-well variation in the concentration of the available hapten. In the assay described here the excitatory amino acid (EAA) Glu is coupled directly to polystyrene microtiter wells with GA. Each step of the assay was tested for maximum efficiency. The resulting EIA with Glu as a competitor gave excellent reproducibility (coefficient of variation = 5.87%), an EC50 of 2.02 X 10(-5) M and a detection limit of 1.26 X 10(-6) M. This EIA method is generally useful for a variety of antisera to amino acids and small peptides and a wide range of competing substances. It can be used to characterize the conformational requirements for antigen binding, to assay for glutamate or to identify compounds with glutamate-like structure in unknown solutions.