LIM kinase 1 accumulates in presynaptic terminals during synapse maturation.

LIM kinase 1 (LIMK1) is a cytoplasmic protein kinase that is highly expressed in neurons. In transfected cells, LIMK1 binds to the cytoplasmic tail of neuregulins and regulates the breakdown of actin filaments. To identify potential functions of LIMK1 in vivo, we have determined the subcellular distribution of LIMK1 protein within neurons of the rat by using immunomicroscopy. At neuromuscular synapses in the adult hindlimb, LIMK1 was concentrated in the presynaptic terminal. However, little LIMK1 immunoreactivity was detected at neuromuscular synapses before the 2nd week after birth, and most motoneuron terminals were not strongly LIMK1 immunoreactive until the 3rd week after birth. Thus, LIMK1 accumulation at neuromuscular synapses coincided with their maturation. In contrast, SV2, like many other presynaptic terminal proteins, can be readily detected at neuromuscular synapses in the embryo. Similar to its late accumulation at developing synapses, LIMK1 accumulation at regenerating neuromuscular synapses occurred long after these synapses first formed. In the adult ventral spinal cord, LIMK1 was concentrated in a subset of presynaptic terminals. LIMK1 gradually accumulated at spinal cord synapses postnatally, reaching adult levels only after P14. This study is the first to implicate LIMK1 in the function of presynaptic terminals. The concentration of LIMK1 in adult, but not nascent, presynaptic terminals suggests a role for this kinase in regulating the structural or functional characteristics of mature synapses.

Immunohistochemical localization of subtype 4a metabotropic glutamate receptors in the rat and mouse basal ganglia.

Recent studies suggest that metabotropic glutamate receptors (mGluRs) may play a significant role in regulating basal ganglia functions. In this study, we investigated the localization of mGluR4a protein in the mouse and rat basal ganglia. Polyclonal antibodies that specifically react with the metabotropic glutamate receptor subtype mGluR4a were produced and characterized by Western blot analysis. These antibodies recognized a native protein in wild-type mouse brain with a molecular weight similar to the molecular weight of the band from a mGluR4a-transfected cell line. The immunoreactivity was absent in brains of knockout mice deficient in mGluR4. mGluR4a immunoreactivity was most intense in the molecular layer of the cerebellum. We also found a striking mGluR4a immunoreactivity in globus pallidus, and moderate staining in substantia nigra pars reticulata and entopeduncular nucleus. Moderate to low mGluR4a immunoreactivity was present in striatum and other brain regions, including hippocampus, neocortex, and thalamus. Double labeling with mGluR4a antibodies and antibodies to either a dendritic marker or a marker of presynaptic terminals suggest a localization of mGluR4a on presynaptic terminals. Immunocytochemistry at electron microscopy level confirmed these results, revealing that in the globus pallidus, mGluR4a is mainly localized in presynaptic sites in axonal elements. Finally, quinolinic acid lesion of striatal projection neurons decreased mGluR4a immunoreactivity in globus pallidus, suggesting a localization of mGluR4a on striatopallidal terminals. These data support the hypothesis that mGluR4a serves as a presynaptic heteroreceptor in the globus pallidus, where it may play an important role in regulating g-amino-n-butyric acid (GABA) release from striatopallidal terminals.

Muscarinic receptor subtypes in the lateral geniculate nucleus: a light and electron microscopic analysis.

Neural activity in the dorsal lateral geniculate nucleus of the thalamus (DLG) is modulated by an ascending cholinergic projection from the brainstem. The purpose of this study was to identify and localize specific muscarinic receptors for acetylcholine in the DLG. Receptors were identified in rat and cat tissue by means of antibodies to muscarinic receptor subtypes, ml-m4. Brain sections were processed immunohistochemically and examined with light and electron microscopy. Rat DLG stained positively with antibodies to the m1, m2,and m3 receptor subtypes but not with antibodies to the m4 receptor subtype. The m1 and m3 antibodies appeared to label somata and dendrites of thalamocortical cells. The m1 immunostaining was pale, whereas m3-positive neurons exhibited denser labeling with focal concentrations of staining. Strong immunoreactivity to the m2 antibody was widespread in dendrites and somata of cells resembling geniculate interneurons. Most m2-positive synaptic contacts were classified as F2-type terminals, which are the presynaptic dendrites of interneurons. The thalamic reticular nucleus also exhibited robust m2 immunostaining. Cat DLG exhibited immunoreactivity to the m2 and m3 antibodies. The entire DLG stained darkly for the m2 receptor subtype, except for patchy label in the medial interlaminar nucleus and the ventralmost C laminae. The staining for m3 was lighter and was distributed more homogeneously across the DLG. The perigeniculate nucleus also was immunoreactive to the m2 and m3 subtype-specific antibodies. Immunoreactivity in cat to the m1 or m4 receptor antibodies was undetectable. These data provide anatomical evidence for specific muscarinic-mediated actions of acetylcholine on DLG thalamocortical cells and thalamic interneurons.

Distribution and developmental regulation of metabotropic glutamate receptor 7a in rat brain.

To determine the regional and cellular distribution of the metabotropic glutamate receptor mGluR7a, we used rabbit anti-peptide polyclonal-targeted antibodies against the C-terminal domain of mGluR7a. Here we report that immunocytochemistry at the light-microscopic level revealed that mGluR7a is widely distributed throughout the adult rat brain, with a high level of expression in sensory areas, such as piriform cortex, superior colliculus, and dorsal cochlear nucleus. In most brain structures, mGluR7a immunoreactivity is characterized by staining of puncta and fibers. However, in some regions, including the locus ceruleus, cerebellum, and thalamic nuclei, both cell bodies and fibers are immunopositive. The changes in levels of mGluR7a during development were investigated with immunoblotting and immunocytochemical analysis. Immunoblot analysis revealed that the levels of mGluR7a are differentially regulated across brain regions during postnatal development. In cortical regions (hippocampus, neocortex, and olfactory cortex), mGluR7a levels were highest at postnatal day 7 (P7) and P14, then declined in older rats. In contrast, mGluR7a levels were highest at P7 in pons/medulla and cerebellum and decreased markedly between P7 and P14. In these regions, mGluR7a immunoreactivity was at similar low levels at P14 and P21 and in adults. Immunocytochemical analysis revealed that staining for mGluR7a was exceptionally high in fiber tracts in P7 animals relative to adults. Furthermore, the pattern of mGluR7a immunoreactivity in certain brain structures, including cerebellum, piriform cortex, and hippocampus, was significantly different in P7 and adult animals. In summary, these data suggest that mGluR7a is widely distributed throughout the rat brain and that this receptor undergoes a dynamic, regionally specific regulation during postnatal development.

Cloning and localization of exon 5-containing isoforms of the NMDAR1 subunit in human and rat brains.

Nine isoforms of the rat NMDAR1 receptor subunit have been previously identified, of which several have an alternatively spliced N-terminal insert believed to be important in proton sensitivity of the receptor. The cloning of the human homologues of NMDAR1-3b (hNMDA1-1) and NMDAR1-4b (hNMDA1-2), both bearing the insert, is reported here. A monoclonal antibody generated against the N-terminal region of these isoforms showed reactivity with at least two distinct human brain proteins of approximately 115 kDa. This antibody was further characterized by using a series of truncated fusion proteins and splice variants of NMDAR1 demonstrating its specific recognition of an epitope within the 21-amino acid N-terminal insert, encoded by exon 5. Western blot and immunocytochemical studies were performed to examine the expression of the exon 5-containing isoforms of the NMDAR1 subunit in both rat and human brain.

Presenilin-1 protein expression in familial and sporadic Alzheimer's disease.

Mutations of the presenilin PS1 and PS2 genes are closely linked to aggressive forms of early-onset (< 60 years) familial Alzheimer's disease. A highly specific monoclonal antibody was developed to identify and characterize the native PS1 protein. Western blot analyses revealed a predominant 32-kd immunoreactive polypeptide in a variety of samples, including PC12 cells transfected with human PS1 complementary DNA, brain biopsy specimens from demented patients, and postmortem samples of frontal neocortex from early-onset familial Alzheimer's disease cases (PS1 and PS2), late-onset sporadic Alzheimer's disease cases, and cases of other degenerative disorders. This truncated polypeptide contains the N-terminus of PS1 and appeared unchanged across cases. In 2 early-onset cases linked to missense mutations in the PS1 gene, a PS1 immunoreactive protein (approximately 49 kd) accumulated in the frontal cortex. This protein was similar in size to full-length PS1 protein present in transfected cells overexpressing PS1 complementary DNA, and in lymphocytes from an affected individual with a deletion of exon 9 of the PS1 gene, suggesting that mutations of the PS1 gene peturb the endoproteolytic processing of the protein. Immunohistochemical studies of control brains revealed that PS1 is expressed primarily in neurons, with the protein localized in the soma and dendritic processes. In contrast, PS1 showed striking localization to the neuropathology in early-onset familial Alzheimer's disease and sporadic Alzheimers' disease cases. PS1 immunoreactivity was present in the neuritic component of senile plaques as well as in neurofibrillary tangles. Localization of PS1 immunoreactivity in familial and sporadic Alzheimer's disease suggests that genetically heterogeneous forms of the disease share a common pathophysiology involving PS1 protein.

Light and electron microscopic localization of presenilin-1 in primate brain.

Several genes have been implicated in the pathogenesis of early-onset familial Alzheimer's disease. A majority of the autosomal dominant cases are linked to recently identified mutations in the presenilin-1 gene on chromosome 14. The native presenilin-1 protein in primates has not been well characterized, and its precise localization is unknown. We have studied the native presenilin-1 protein in monkey brain and peripheral tissues by using a monoclonal antibody specific for the N-terminal domain of human presenilin-1. Western blots detect polypeptide species of approximately 49 and approximately 32 kDa from COS-7 and PC12 cells transfected with full-length human presenilin-1 cDNA and from in vitro translations of the normal human presenilin-1 mRNA. A 32 kDa polypeptide is detected in monkey peripheral tissues, with the highest expression in testis and lung. In all brain regions the 32 kDa band is the predominant form of presenilin-1, and it is found in particulate subfractions. Light microscopic immunocytochemistry reveals presenilin-1 staining in all brain regions, with the strongest labeling in neurons and neuropil. In addition, weaker immunoreactivity is also present in glia and blood vessels. Neuronal staining shows significant variability, with particularly intense labeling of certain cell types, including large neocortical and hippocampal pyramidal neurons, magnocellular basal forebrain neurons, brainstem motoneurons, and some populations of interneurons. By electron microscopic immunocytochemistry, highly selective presenilin-1 staining is seen on the cytoplasmic surfaces of membranous organelles, which suggest localization to the endoplasmic reticulum-Golgi intermediate compartment, a subdomain of the endoplasmic reticulum, and some coated transport vesicles.

Identification and localization of huntingtin in brain and human lymphoblastoid cell lines with anti-fusion protein antibodies.

The Huntington disease (HD) phenotype is associated with expansion of a trinucleotide repeat in the IT15 gene, which is predicted to encode a 348-kDa protein named huntington. We used polyclonal and monoclonal anti-fusion protein antibodies to identify native huntingtin in rat, monkey, and human. Western blots revealed a protein with the expected molecular weight which is present in the soluble fraction of rat and monkey brain tissues and lymphoblastoid cells from control cases. In lymphoblastoid cell lines from juvenile-onset heterozygote HD cases, both normal and mutant huntingtin are expressed, and increasing repeat expansion leads to lower levels of the mutant protein. Immunocytochemistry indicates that huntingtin is located in neurons throughout the brain, with the highest levels evident in larger neurons. In the human striatum, huntingtin is enriched in a patch-like distribution, potentially corresponding to the first areas affected in HD. Subcellular localization of huntingtin is consistent with a cytosolic protein primarily found in somatodendritic regions. Huntingtin appears to particularly associate with microtubules, although some is also associated with synaptic vesicles. On the basis of the localization of huntingtin in association with microtubules, we speculate that the mutation impairs the cytoskeletal anchoring or transport of mitochondria, vesicles, or other organelles or molecules.

Electron microscopic analysis of D1 and D2 dopamine receptor proteins in the dorsal striatum and their synaptic relationships with motor corticostriatal afferents.

The precise localization of D1 and D2 dopamine receptors within striatal neurons and circuits is crucial information for further understanding dopamine pharmacology. We have used subtype specific polyclonal and monoclonal antibodies against D1 and D2 dopamine receptors to determine their cellular and subcellular distributions, their colocalization, and their differential connectivity with motor cortical afferents labeled either by lesion-induced degeneration or by anterograde transport of biotinylated dextrans. D1 and D2 are primarily expressed in medium-sized neurons and spiny dendrites. Axon terminals containing D1 were rare whereas D2-immunoreactive axon terminals forming symmetrical synapses with dendrites and spines were common. In 2 microns sections, D1 was localized to 53% of neurons, and D2 to 48% of neurons, while mixing D1 and D2 antibodies labeled 78%. By electron microscopy, D1 was localized to 43% of dendrites and 38% of spines while D2 was localized to 38% of dendrites and 48% of spines. Combining D1 and D2 antibodies resulted in the labeling of 88.5% of dendrites and 92.6% of spines. Using different chromogens for D1 and D2, colocalization was not observed. Ipsilateral motor corticostriatal afferents were primarily axospinous and significantly more synapsed with D1 than D2-positive spines (65% vs 47%). Contralateral motor corticostriatal afferents were frequently axodendritic and no difference in their frequency of synapses with D1 and D2 dendrites and spines was observed. These findings demonstrate differential patterns of expression of D1 and D2 receptors in striatal neurons and axon terminals and their differential involvement in motor corticostriatal circuits.

Distribution of m1-m4 muscarinic receptor proteins in the rat striatum: light and electron microscopic immunocytochemistry using subtype-specific antibodies.

Muscarinic ACh receptors mediate complex and clinically important effects in the striatum. To better understand the roles of the different muscarinic receptor subtypes (m1-m4), we have determined the cellular and subcellular distribution of the m1-m4 receptor proteins in the rat neostriatum using subtype-specific antibodies and avidin-biotin-peroxidase immunocytochemistry for light and electron microscopy. m1 receptor protein is expressed in 78% of neurons and is enriched in spiny dendrites and at postsynaptic densities. A small number of m1-immunoreactive axon terminals were observed, all forming asymmetrical synapses. About 2.5% of striatal neurons express m2 receptor protein with reaction product evident, by light microscopy in scattered large oval neurons with enfolded nuclei and long aspiny dendrites. By electron microscopy, m2 immunocytochemistry labeled somata, aspiny dendrites, and many axon terminals. Most axon terminals containing m2 make symmetrical synapses with somata, and dendritic shafts and spines. In addition, many m2-immunoreactive axon terminals formed asymmetrical synapses with spines or dendrites. m3 receptor protein was not evident in somata by light microscopy but was present in a distinct population of small-caliber spiny dendrites as well as in axon terminals forming asymmetrical synapses with spines. m4 receptor protein was heterogeneously distributed in the neostriatum and localized to 44% of striatal cells. m4-positive neurons had the ultrastructural features of medium spiny neurons with reaction product particularly concentrated in spines, often at postsynaptic densities. Axon terminals containing m4 form asymmetrical synapses, primarily with spines. These findings indicate that the muscarinic receptor proteins occur in distinct populations of striatal neurons; that the receptor proteins concentrate postsynaptically at synapses, including many considered to be noncholinergic; that m2 is the predominant muscarinic autoreceptor in the striatum; and that each receptor subtype may be a presynaptic heteroceptor in the striatum modulating extrinsic striatal afferents.

Lack of strain differences in spatial learning among seizure-prone and seizure-resistant mice.

Learning rates were examined in the following inbred mice strains: DBA/2, C3H/He, C57B1/6J, E1, and ddY. DBA/2 mice become susceptible to audiogenic seizures after 2-3 weeks of age and E1 mice have generalized seizures in response to handling after 3 months of age, but the remaining three strains do not develop seizures. In this study, mice from all five strains underwent 32 training trials in a Morris water maze at 7-9 weeks of age. The seizure-prone DBA/2 and E1 mice, along with the nonepileptic ddY and C57B1/6J mice, exhibited learning at similar rates, but the nonepileptic C3H/He mice were unable to learn the water maze task, probably due to visual difficulties. In the C57B1/6J strain only, female mice learned the task significantly faster than males. There was no difference in the learning rate between the E1 strain and its parent ddY strain, or any correlation between spatial learning ability and kindling rates in these strains.

Olfactory bulb kindling in mice susceptible and resistant to ethanol withdrawal.

The relation between kindling and susceptibility to ethanol withdrawal seizures was investigated using withdrawal seizure-prone (WSP) and withdrawal seizure-resistant (WSR) mice. These lines were developed by selective breeding to be prone and resistant, respectively, to handling-induced convulsions after chronic exposure to ethanol. Development of kindled seizures in response to electrical stimulation of the olfactory bulb was investigated in mice aged 2 and 8 months with no exposure to ethanol. Older WSP mice kindled more slowly than older WSR mice, requiring significantly more stimulations to reach the first stage 3 and the first stage 5 seizures. In younger mice, there was no significant difference between the two lines in the rate of kindling. The lower kindling rate in mature WSP mice is in contrast to their higher sensitivity to handling-induced convulsions on withdrawal from ethanol and other agents. This finding suggests that separate genetic factors underlie these two models of mouse seizures.

Uptake of medroxyprogesterone acetate by progestin and androgen target neurons in the brain and pituitary gland of male cynomolgus monkeys.

Medroxyprogesterone acetate (MPA), a synthetic progestin that reduces plasma testosterone levels, has been used in the treatment of male sex offenders. It also reduces the sexual activity of male macaques. To investigate its sites and mechanisms of action, 11 adult male cynomolgus monkeys were castrated and 7 and 21 h later were pretreated with 20 mg progesterone s.c. (Prog, n = 3), or 5 mg dihydrotestosterone propionate s.c. (DHTP, n = 3) or oil vehicle (controls, n = 5). Twenty-four hours after castration, all males were injected i.v. with 5 mCi [3H]-MPA, and killed after 60 min. Left halves of the brains were processed for thaw-mount autoradiography to identify the neurons accumulating radioactivity, and right halves were analyzed by high performance liquid chromatography (HPLC) to measure the uptake of [3H]-MPA in nuclear fractions. In males pretreated with oil, there were labeled neurons in the ventromedial nucleus (n.), arcuate n., medial preoptic n. and anterior hypothalamic area. In progesterone-pretreated males, labeling was reduced by 84-100% compared with controls (p less than 0.001), but in DHTP-pretreated males there was no effect, and labeling was not significantly different from control levels. Nuclear concentrations of [3H]-MPA measured by HPLC in controls were highest in the hypothalamus, amygdala, preoptic area and pituitary gland. Pretreatments with progesterone reduced the nuclear concentrations of [3H]-MPA in hypothalamus, preoptic area and pituitary gland by 82-95% compared with controls (p less than 0.05), but DHTP pretreatments had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Sites in the male primate brain at which testosterone acts as an androgen.

Quantitative autoradiographic analysis was used to identify regions in the brain of the male primate where androgen binding sites may be involved in the actions of testosterone. Three days after castration, adult male rhesus monkeys received a subcutaneous injection of either dihydrotestosterone propionate (DHTP, 20 mg, n = 6), testosterone propionate (TP, 100 mg, n = 2), or oil vehicle (control males, n = 4). Three hours later, 5 mCi [3H]testosterone was administered as an i.v. bolus. At 60 min, brains were rapidly removed and the left halves were used for autoradiography. In control males, highest percentages of labeled neurons (20-84% using a rigorous Poisson criterion) were observed in the ventromedial, arcuate and premammillary nuclei (n.) of the hypothalamus, medial preoptic n., bed n. of stria terminalis, intercalated mammillary n., lateral septal n. and the medial, cortical and accessory basal n. of the amygdala. Pretreatment with DHTP eliminated labeling in androgen target tissues of the genital tract, and reduced the percentages of labeled neurons to 4-22% of control values in the arcuate, lateral septal, premammillary and intercalated mammillary n., indicating that in these regions testosterone acted predominantly at androgen binding sites. However, in the medial preoptic n., the ventromedial hypothalamic n. and the accessory basal amygdaloid n., DHTP pretreatment resulted in much less blocking which, together with other data, suggested that in these sites, testosterone's actions involved aromatization and interaction with estrogen-binding sites.

Identification of radioactivity in cell nuclei from brain, pituitary gland and genital tract of male rhesus monkeys after the administration of [3H]testosterone.

Enzymes are present in the primate brain that convert testosterone into 17 beta-hydroxy-5 alpha-androstan-3-one (dihydrotestosterone), estradiol-17 beta and 4-androstene-3,17-dione. To identify the metabolites of testosterone that accumulate in cell nuclei obtained from different regions of the brain, 9 adult castrated male rhesus monkeys were injected with 5 mCi [3H]testosterone as an intravenous bolus. After 1 h, brains were rapidly removed and the left halves were used for autoradiography while the right halves were dissected to provide 14 samples. Radioactive metabolites in cell nuclei were identified by high-performance liquid chromatography (HPLC) and by repeated recrystallization. In autoradiograms of brain, most of the labeled neurons were in the hypothalamus, preoptic area and amygdala. These three regions also had the highest levels of radioactivity. The major form of this radioactivity was [3H]estradiol-17 beta (Type I tissues) and the major radioactive androgen present was [3H]testosterone. In all other brain regions and pituitary gland, the major form of radioactivity was unchanged [3H]testosterone (Type II tissues). In genital tract structures, [3H]dihydrotestosterone predominated (Type III tissues). These results suggested that, in contrast to its actions on genital tract structures, testosterone acts on neuronal nuclei mainly in unmetabolized form or after conversion to estradiol-17 beta.

The uptake of [3H]testosterone and its metabolites by the brain and pituitary gland of the fetal macaque.

Testosterone is secreted by the fetal testis during gestation, and this is thought to influence certain aspects of the brain's subsequent development. To study this action at the neuronal level, nine macaque fetuses were injected with 250 microCi [3H]testosterone via the umbilical vein at about 120 days gestation. After 60 min, samples of brain and peripheral tissue were studied by autoradiography or HPLC. Purified nuclear pellets were prepared, and radioactivity in ether extracts was fractionated by HPLC and identified by coelution with internal standard steroids. Concentrations of radioactivity were significantly higher (P less than 0.05) in the hypothalamus-preoptic area than in amygdala, hippocampus, midbrain, and cerebral and cerebellar cortexes, and most of the radioactivity (75%) in the hypothalamus-preoptic area coeluted with 17 beta-estradiol. Radioactivity coeluting with 17 beta-estradiol was also detected in nuclear fractions from amygdala (44%). In contrast, 80% of the radioactivity extracted from pituitary gland nuclei coeluted with testosterone. Most of the neurons labeled in autoradiograms were located in the hypothalamus and preoptic area, fewer were found in the amygdala, and labeling in the frontal or motor cortex did not exceed chance levels. Results suggested that aromatization and, consequently, estrogen receptors play a role in the effects of testosterone on the hypothalamus and amygdala of the primate fetus at this stage of development.

Localization and identification of nuclear radioactivity in the pituitary gland and genital tract after administering 3H-testosterone, 3H-dihydrotestosterone, or 3H-estradiol to male rhesus monkeys.

Target cells for testosterone, dihydrotestosterone, and estradiol in the pituitary gland and genital tract of the male primate were localized by thaw-mount autoradiography, and high performance liquid chromatography was used to identify the metabolites of these steroids in cell nuclei. Castrated rhesus monkeys were injected with 3H-testosterone, 3H-dihydrotestosterone, or 3H-estradiol and killed 60 min later. In the anterior pituitary gland, fewer cells were labeled and less radioactivity was taken up by cell nuclei following the administration of either 3H-testosterone (4% of pars distalis cells and 5 dpm/micrograms DNA) or 3H-dihydrotestosterone (5% of cells and 13 dpm/micrograms DNA) than following the administration of 3H-estradiol (43% of cells and 214 dpm/micrograms DNA). Most of the radioactivity in nuclei was in the form of the unmetabolized parent compound (78-94%). In prostate, seminal vesicles, and penis, 3H-dihydrotestosterone was the predominant form of nuclear radioactivity following both 3H-testosterone (67-90%) and 3H-dihydrostestosterone (94-97%) administration, and both androgens labeled epithelial and smooth muscle cells. In contrast, 3H-estradiol was taken up in unchanged form, by cell nuclei of the genital tract and it labeled connective tissue fibroblasts, but not epithelial cells. Thus, the distributions of target cells for androgens and estrogens were clearly different in all these tissues, and the uptake of testosterone resembled that of its androgenic rather than that of its estrogenic metabolite.

Estrogen binding and the actions of testosterone in the brain of the male rhesus monkey.

Autoradiography and high performance liquid chromatography (HPLC) were used to determine where metabolites of testosterone interact with estrogen binding sites in the brain of the male primate. Three days after castration, animals received a subcutaneous injection of either estradiol benzoate (EB, 200 micrograms/kg, n = 4) or oil vehicle (controls, n = 4). Three hours later, 5 mCi [3H]testosterone was administered as an intravenous bolus. At 60 min, brains were rapidly removed, left halves were used for autoradiography and right halves were dissected into 14 samples for HPLC of nuclear and supernatant fractions. In control males, labeled neurons were observed in preoptic area, hypothalamus and amygdala. In EB-pretreated males, the number of labeled neurons was reduced by 35% in the anterior hypothalamus and ventromedial nucleus, and by 65% in the cortical and accessory basal amygdaloid nuclei, but was not significantly reduced in other brain regions. In hypothalamus, preoptic area and amygdala, EB-pretreatment reduced nuclear concentrations of [3H]estradiol to 37-55% of control levels, but reduced neither the nuclear concentrations of [3H]testosterone nor the supernatant concentrations of [3H]estradiol and [3H]testosterone. The data suggest that the actions of testosterone in regions such as the arcuate nucleus and lateral septal nucleus primarily involve unchanged testosterone or dihydrotestosterone, while in regions such as the amygdala, aromatization and interaction with estrogen receptors is involved also.

Sites at which testosterone may act as an estrogen in the brain of the male primate.

Testosterone is converted to estradiol in specific regions of the primate brain and accumulates as such in the nuclei of cells in hypothalamus, preoptic area, and amygdala. To locate more precisely those neurons in which nuclear estrogen receptors were occupied by estrogenic metabolites of testosterone, we injected 8 castrated male rhesus monkeys with [3H]-estradiol. Four were injected with oil for control purposes, and 4 were pretreated for 3 days with 2 mg/day testosterone propionate. This dose raised plasma testosterone levels into the high physiological range for intact males. After 60 min, brains were rapidly removed, the levels of [3H]-estradiol in nuclei were measured in the right halves of the brains by high-performance liquid chromatography, and labeled neurons were located in the left halves by autoradiography. Compared with the 4 control animals, nuclear levels of [3H]-estradiol in testosterone-treated males were reduced by 77% in the hypothalamus (p less than 0.001), by 93% in the preoptic area (p less than 0.001), and by 90% in the amygdala (p less than 0.05). In autoradiograms from testosterone-treated males, the labeling of neurons was reduced by 72-96% in most of the regions in which the control males showed high percentages of labeled cells. However, there were only small reductions in the number of labeled neurons in lateral septum (by 31%) and arcuate nucleus (by 23%). These two regions, therefore, contained estrogen receptors that were not blocked by pretreatment with testosterone. The simplest explanation for these results is that estrogenic metabolites of testosterone prevented the uptake of [3H]-estradiol by prior occupation of estrogen receptor sites. The rather precise neuroanatomical localization of the effects pointed to the existence of two populations of estrogen target neurons in the primate brain depending on the presence or absence of local aromatase activity.

Pre-optic and hypothalamic neurons accumulate [3H]medroxyprogesterone acetate in male cynomolgus monkeys.

Medroxyprogesterone acetate (MPA) is a synthetic progestin that is reported to be effective in the treatment of paraphilic behavior, including paraphilic aggression, in men. The mechanisms and sites of action for its behavioral effects are not known. Thaw-mount autoradiography was used to help identify sites in the brain at which MPA may act in a male primate. Two adult, castrated male cynomolgus monkeys were administered [3H]MPA and killed one hour later. Radioactivity was concentrated in the nuclei of many neurons in the medial preoptic nucleus (n.), anterior hypothalamic area, ventromedial hypothalamic n., and arcuate n. Virtually no labeled cells were observed in the bed n. of the stria terminalis, lateral septal n., or amygdala. Analysis by high performance liquid chromatography of brain samples from the same animals demonstrated that 84% of the extractable radioactivity in cell nuclei from the hypothalamus and preoptic area was in the form of unmetabolized [3H]MPA. The localization of MPA-concentrating neurons in regions of the brain known to be implicated in regulating both sexual behavior and pituitary function suggests that, among other sites of action, MPA may act directly upon the brain.