Profiling differences in chemical composition of brain structures using Raman spectroscopy.

Raman spectroscopy enables non-invasive investigation of chemical composition of biological tissues. Due to similar chemical composition, the analysis of Raman spectra of brain structures and assignment of their spectral features to chemical constituents presents a particular challenge. In this study we demonstrate that standard and independent component analysis of Raman spectra is capable of assessment of differences in chemical composition between functionally related gray and white matter structures. Our results show the ability of Raman spectroscopy to successfully depict variation in chemical composition between structurally similar and/or functionally connected brain structures. The observed differences were attributed to variations in content of proteins and lipids in these structures. Independent component analysis enabled separation of contributions of major constituents in spectra and revealed spectral signatures of low-concentration metabolites. This provided finding of discrepancies between structures of striatum as well as between white matter structures. Raman spectroscopy can provide information about variations in contents of major chemical constituents in brain structures, while the application of independent component analysis performed on obtained spectra can help in revealing minute differences between closely related brain structures.

Decrease of serotonin and metabolite in the forebrain and facilitation of lordosis by dorsal raphe nucleus lesions in male rats.

In castrated male rats, a radiofrequency lesion was made in the dorsal raphe nucleus (DRL) and lordosis behavior was observed following treatment with estrogen. After the behavioral test, brains were removed and the contents of 5-HT and 5-HIAA in the forebrain were measured by high pressure liquid chromatography (HPLC). In the results, only 2 of 16 control males without brain surgery showed lordosis, and the mean lordosis quotient (LQ) was extremely low when compared to that in control females. In contrast, all male rats with DRL displayed lordosis and the mean LQ was higher than that of control males without brain surgery but lower than that in control females (P < 0.001). In the DRL males, 5-HT and 5-HIAA contents in the septum (SPT), the preoptic area (POA), the ventromedial hypothalamus (VMH) and the striatum (STM) were lower than those in control male and female groups (P < 0.001). These results suggest that the dorsal raphe nucleus prevents male rats from showing lordosis by serotonergic influence in the forebrain. In addition, HPLC results showed that levels of the 5-HT in the SPT, the POA and the VMH in the female group were higher than those in the control male group (P < 0.05). In female rats, the POA (P < 0.01) and the VMH (P < 0.05) contained larger 5-HT than those in the SPT and the STM, but there were no difference of 5-HT contents in the male rat.

Nitric oxide synthase mRNA levels correlate with gene expression of angiotensin II type-1 but not type-2 receptors, renin or angiotensin converting enzyme in selected brain areas.

Recent data suggest that there is interaction between peripheral angiotensin II and nitric oxide. However, sparse information is available on the mutual interaction of these two compounds in the brain. The potential intercourse of nitric oxide with brain neuropeptides needs to be substantiated by assessing its local production and gene expression of the synthesizing enzymes involved. The aim of the present study was to evaluate whether the gene expression of brain nitric oxide synthase (bNOS) is related to the sites of gene expression of different components of the rat brain renin angiotensin system (renin, angiotensin converting enzyme (ACE) or angiotensin receptors of AT1 and AT2 subtypes). The levels of corresponding mRNAs were measured and correlated in nine structures of adult rat brain (hippocampus, amygdala, septum, thalamus, hypothalamus, cortex, pons, medulla and cerebellum). As was expected, positive correlation was observed between renin and angiotensin-converting enzyme mRNAs. Moreover, a significant correlation was found between brain NO synthase and AT1 receptor mRNAs, but not with mRNA of the AT2 receptor, ACE and renin. Parallel distribution of mRNAs coding for bNOS and AT1 receptors in several rat brain structures suggests a possible interaction between brain angiotensin 11 and nitric oxide, which remains to be definitely demonstrated by other approaches.

Glucocorticoids affect gonadotropin-releasing hormone immunoreactivity in musk shrew brain.

Multiple interactions between the hypothalamic-pituitary-adrenal and the hypothalamic-pituitary-gonadal systems exist. In this study, we asked if glucocorticoid administration affected gonadotropin-releasing hormone (GnRH) immunoreactivity. We found that musk shrews treated with dexamethasone (DEX), a synthetic glucocorticoid, had more GnRH-immunoreactive (ir) cells in the forebrain than did cortisol- or control-treated animals. The effects of DEX were noted rapidly, within 15 min, after administration. These effects were observed in the forebrain as a whole and also in specific subpopulations of GnRH-ir cells located in the medial septum/diagonal band and the hypothalamus.

Dysembryoplastic neuroepithelial tumor-like neoplasm of the septum pellucidum: a lesion often misdiagnosed as glioma: report of 10 cases.

The authors report a series of 10 low-grade neoplasms arising in the midline anteriorly in the region of the septum pellucidum with many of the histologic features of dysembryoplastic neuroepithelial tumor (DNT). The patients (five female, five male) ranged in age from 6 to 35 years (mean age, 21.5 years). The most common presenting symptoms were headache, nausea and vomiting, and visual disturbances. Radiographically, the tumors extended into the lateral ventricles from the septal region and obstructed the foramen of Monro. Varying degrees of hydrocephalus were present. The lesions were lobular, well-delineated, hypointense to brain on T1-weighted magnetic resonance imaging, and hyperintense on T2-weighted images. They were uniformly nonenhancing or showed only minimal peripheral enhancement. The tumors, in aggregate, had the histologic features of DNT. These included a mucin-rich background, oligodendrocyte-like cells, "floating neurons," and a "specific glioneuronal element." Seven patients underwent gross total resection and two underwent subtotal resection. No patients received adjuvant chemotherapy or radiotherapy. On follow-up (n = 6; median, 14 months), all tumors had either not recurred or were radiologically stable. On the basis of both neuroimaging and histopathology, DNT-like lesions should be considered in the differential diagnosis of midline intraventricular tumors in children and young adults. Distinction from more aggressive neoplasms is essential because these tumors appear to behave in a benign fashion.

Localization of the VIP2 receptor protein on GnRH neurons in the female rat.

In mammals, the timing and occurrence of the preovulatory LH surge critically depends on the proper functioning of the suprachiasmatic nucleus (SCN). Recent evidence suggests that vasoactive intestinal polypeptide (VIP) conveys time of day information from the SCN to GnRH neurons. However, it is not completely clear whether this action is exerted directly at the level of the GnRH neuron. To determine if GnRH neurons are direct targets for VIP, triple-label immunofluorescence was utilized to simultaneously localize GnRH, VIP and VIP2 receptor protein. The present results demonstrate that about 40% of all GnRH neurons analyzed contain VIP2 receptor immunoreactivity and that VIP-containing processes were seen in close apposition to a significant number of VIP2 receptor-positive GnRH neurons. GnRH neurons that exhibit immunoreactivity for the VIP2 receptor are located predominantly in the OVLT region and the rostral preoptic area. In the median eminence, where the majority of GnRH neurons terminate, VIP2 receptor immunoreactivity was absent. In summary, these findings indicate that VIP can communicate directly with GnRH neurons.

Maternal dietary choline availability alters mitosis, apoptosis and the localization of TOAD-64 protein in the developing fetal rat septum.

Maternal changes in dietary choline availability alter brain biochemistry and hippocampal development in the offspring resulting in lifelong behavioral changes in the offspring. In order to better understand the relationship between maternal diet, brain cytoarchitecture and behavior, we investigated the effects of choline availability on cell proliferation, apoptosis and differentiation in the fetal rat brain septum. Timed-pregnant rats on day E12 were fed AIN-76 diet with varying levels of dietary choline for 6 days. We found that choline deficiency (CD) significantly decreased the rate of mitosis in the progenitor neuroepithelium adjacent to the septum. In addition, we found an increased number of apoptotic cells in the septum of CD animals compared to controls (3.5+/-0.5 vs. 1.7+/-0.5 apoptotic cells per section; p<0.05). However, CD had no effect on apoptosis in the indusium griseum (IG), a region of cortex dorsal to the septum. Using an unbiased image analysis method and a monoclonal antibody we found a decreased expression of the TOAD-64 kDa protein, a marker of commitment to neuronal differentiation during fetal development, in the dorsal lateral septum of CD animals. CD also decreased the expression of TOAD-64 kDa protein in the IG and cortical plate adjacent to the septum. These results show that dietary choline availability during pregnancy alters the timing of mitosis, apoptosis and the early commitment to neuronal differentiation by progenitor cells in regions of the fetal brain septum, as well as hippocampus, two brain regions known to be associated with learning and memory.

Expression of the novel galanin receptor subtype GALR2 in the adult rat CNS: distinct distribution from GALR1.

Recent molecular cloning studies by our laboratory and others have identified the existence of a novel rat galanin receptor subtype, GALR2. In the present study, we examined the regional and cellular distribution of GALR2 mRNA in the rat central nervous system (CNS) by in situ hybridization. For comparative purposes, adjacent sections were probed for GALR1 mRNA expression. Our findings indicate that dorsal root ganglia express by far the highest levels of GALR2 mRNA in the rat CNS. Hybridization signal is mainly concentrated over small and intermediate primary sensory neurons. In spinal cord, the large alpha motoneurons of the ventral horn are moderately labeled and several small, but less intensely labeled, cells are scattered throughout the gray matter. In brain sections, the highest levels of GALR2 mRNA are detected in granule cells of the dentate gyrus, in the mammillary nuclei, and in the cerebellar cortex. Moderate levels of GALR2 mRNA are observed in the olfactory bulb, olfactory tubercle, piriform and retrospinal cortices, hypothalamus (namely the preoptic area, arcuate nucleus, and dorsal hypothalamic area), substantia nigra pars compacta, and sensory trigeminal nucleus. Moderate to weak hybridization signal is also present in several other hypothalamic nuclei, specific layers of the neocortex, periaqueductal gray, and several nuclei within the pons and medulla, including locus coeruleus, lateral parabrachial, motor trigeminal, pontine reticular, hypoglossal, vestibular complex, ambiguus, and facial and lateral reticular nuclei. This novel pattern of GALR2 distribution within the rat CNS differs considerably from that of GALR1, suggesting that specific physiologic effects of galanin may be ascribed to the GALR2 galanin receptor subtype.

Localization of orphanin FQ (nociceptin) peptide and messenger RNA in the central nervous system of the rat.

Orphanin FQ (OFQ) is the endogenous agonist of the opioid receptor-like receptor (ORL-1). It and its precursor, prepro-OFQ, exhibit structural features suggestive of the opioid peptides. A cDNA encoding the OFQ precursor sequence in the rat recently has been cloned, and the authors recently generated a polyclonal antibody directed against the OFQ peptide. In the present study, the authors used in situ hybridization and immunohistochemistry to examine the distribution of OFQ peptide and mRNA in the central nervous system of the adult rat. OFQ immunoreactivity and prepro-OFQ mRNA expression correlated virtually in all brain areas studied. In the forebrain, OFQ peptide and mRNA were prominent in the neocortex endopiriform nucleus, claustrum, lateral septum, ventral forebrain, hypothalamus, mammillary bodies, central and medial nuclei of the amygdala, hippocampal formation, paratenial and reticular nuclei of the thalamus, medial habenula, and zona incerta. No OFQ was observed in the pineal or pituitary glands. In the brainstem, OFQ was prominent in the ventral tegmental area, substantia nigra, nucleus of the posterior commissure, central gray, nucleus of Darkschewitsch, peripeduncular nucleus, interpeduncular nucleus, tegmental nuclei, locus coeruleus, raphe complex, lateral parabrachial nucleus, inferior olivary complex, vestibular nuclear complex, prepositus hypoglossus, solitary nucleus, nucleus ambiguous, caudal spinal trigeminal nucleus, and reticular formation. In the spinal cord, OFQ was observed throughout the dorsal and ventral horns. The wide distribution of this peptide provides support for its role in a multitude of functions, including not only nociception but also motor and balance control, special sensory processing, and various autonomic and physiologic processes.

Overexpression of tumour necrosis factor alpha in the brain of transgenic mice differentially alters nerve growth factor levels and choline acetyltransferase activity.

Tumour necrosis factor alpha (TNF-alpha) is a pleiotrophic cytokine synthesized primarily by macrophages and monocytes, which exerts a variety of biological activities during inflammatory responses, immune reactions, and wound healing. Within the central nervous system (CNS), the basal levels of TNF-alpha are almost undetectable, but increase after neurological insults. Using transgenic mice expressing high levels of TNF-alpha in the CNS, we investigated the effect of this cytokine on the levels of brain nerve growth factor (NGF), a neurotrophin playing a crucial role in the development, maintenance and regeneration of basal forebrain cholinergic neurons. The immunoenzymatic assay and in situ hybridization revealed that the constitutive expression of NGF decreased in the hippocampus, increased in the hypothalamus, while remained unchanged in the cortex. Moreover, septal cholinergic neurons which receive trophic support from NGF produced in the hippocampus display loss of choline acetyltransferase immunoreactivity, suggesting that the reduced availability of NGF may influence negatively the synthesis of brain cholinergic neurons. These observations indicate that the basal level of brain NGF can be influenced negatively or positively by local expression of TNF-alpha and that this cytokine, through dose-dependent regulation of NGF synthesis and release, may be involved in neurodegenerative events associated with aging.

The inter-relationship between gonadal steroids and POMC peptides, beta-endorphin and alpha-MSH, in the control of sexual behavior in the female rat.

Estradiol benzoate (10 microg EB) given to ovariectomized-adrenalectomized rats induced sexual receptivity in half the animals and increased alpha-MSH in the preoptic area, ventromedial nucleus (VMN) and arcuate nucleus (ARC), in all the animals, although levels were significantly higher in the VMN and ARC of the receptive (R) subgroup. EB also raised levels of beta-endorphin in the VMN and ARC in the R rats only. POMC expression was not altered. EB did not affect alphaMSH in extra-hypothalamic areas, but addition of progesterone, raised levels in the septum, amygdala, hippocampus and caudate putamen. Only in the VMN, ARC and septum were the steroid-induced increases correlated with onset of sexual behavior.

Electron microscopic evidence for a cholinergic innervation of GABAergic parvalbumin-immunoreactive neurons in the rat medial septum.

The presence of interconnections between cholinergic and parvalbumin (PARV)-containing gamma aminobutyric acid (GABA)ergic septohippocampal projection neurons is still a matter of debate. To search for contacts of cholinergic collateral axon terminals in the septal-diagonal band region the immunotoxin 192IgG-saporin was applied, which was proved to selectively destroy cholinergic basal forebrain neurons. Seven and 10 days after administration of the immunotoxin, choline acetyltransferase immunoreactivity had disappeared, and numerous neuronal somata and dendrites as well as axonal terminals revealed characteristics of electron-lucent degeneration. Electron-dense degeneration was never observed in dendrites and synaptic boutons. Degenerating terminals were found in contact with PARV-immunopositive and PARV-negative neurons. Because only cholinergic cells were degenerating, the terminals should be collaterals from cholinergic neurons. In addition to such contacts, PARV-immunoreactive boutons were seen in contact with PARV-positive and PARV-negative cells, but were not identified at degenerating postsynaptic profiles. As suggested in other studies, cholinergic boutons contacting GABAergic PARV-containing septal projection cells may influence hippocampal theta activity. Furthermore, multiple synaptic connections of both neuronal populations forming the septohippocampal pathway may contribute to their high rate of survival after fimbria-fornix transection.

Cocaine- and amphetamine-regulated transcript peptide immunohistochemical localization in the rat brain.

Cocaine- and amphetamine-regulated transcript (CART) is a brain-enriched mRNA with a protein product(s) that is a candidate brain neurotransmitter. We have developed antisera to CART peptide fragment 106-129 and have demonstrated specific immunoreactivity (IR) at the light microscopic level throughout the brain, spinal cord, and retina. All brain nuclei previously shown to express CART mRNA are now shown to contain CART peptide IR. Although it is premature to define CART peptide(s) as a neurotransmitter(s), the localization found here suggests an involvement of CART in many processes. CART peptide staining in the nucleus accumbens and basolateral amygdala continue to suggest a role in drug-induced reward and reinforcement. Staining in the olfactory bulbs, the cortical barrels, the retina and its projection areas, the thalamic nuclei, the lateral and dorsal horns of the spinal cord, and the nuclei of the solitary tract are compatible with a major role for CART in sensory processing and autonomic regulation. CART peptides appear to colocalize with some classical neurotransmitters and appear to occur in peripheral neurons as well.

Ontogeny of region-specific sex differences in androgen receptor messenger ribonucleic acid expression in the rat forebrain.

Testosterone and its metabolites are the principal gonadal hormones responsible for sexual differentiation of the brain. However, the relative roles of the androgen receptor (AR) vs. the estrogen receptor in specific aspects of this process remain unclear due to the intracellular metabolism of testosterone to active androgenic and estrogenic compounds. In this study, we used an 35S-labeled riboprobe and in situ hybridization to analyze steady state, relative levels of AR messenger RNA (mRNA) expression in the developing bed nucleus of the stria terminalis, medial preoptic area, and lateral septum, as well as the ventromedial and arcuate nuclei of the hypothalamus. Each area was examined on embryonic day 20 and postnatal days 0, 4, 10, and 20 to produce a developmental profile of AR mRNA expression. AR mRNA hybridization was present on embryonic day 20 in all areas analyzed. In addition, AR mRNA expression increased throughout the perinatal period in all areas examined in both males and females. However, between postnatal days 4 and 10, sharp increases in AR mRNA expression in the principal portion of the bed nucleus of the stria terminalis and the medial preoptic area occurred in the male that were not paralleled in the female. Subsequently, males exhibited higher levels of AR mRNA than females in these areas by postnatal day 10. There was no sex difference in AR mRNA content in the lateral septum, ventromedial nucleus, or arcuate nucleus at any age. These results suggest that sex differences in AR mRNA expression during development may lead to an early sex difference in sensitivity to the potential masculinizing effects of androgen.

Neurochemical characterization of AMPA receptor-containing neurons in the mediolateral septal area of the rat.

The lateral septum receives a massive innervation by excitatory amino acid-containing limbic cortical and hypothalamic afferents, and previous studies have described a wide distribution of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor-containing neurons in this area. The aim of this study was to determine whether different subtypes of AMPA receptors are expressed in the same neurons. Furthermore, considering the fact that a population of lateral septal cells, the "somatospiny neurons," are GABAergic calbindin-containing cells, the coexistence of each subtype of AMPA receptor with calbindin was also investigated. Colocalization experiments were performed on adjacent vibratome sections of the lateral septal area for GluR1 and GluR2/3 AMPA-receptor subunits, GluR1 and calbindin, GluR2/3 and calbindin, as well as GluR1 plus calbindin and GluR2/3 plus calbindin, using the "mirror" colocalization technique. The results are summarized as follows: (1) GluR1 is present in the soma and most intensively expressed in dendrites and somatic and dendritic spines; while GluR2/3 is associated with the soma and proximal dendrites of the neurons. (2) Forty-one percent of the AMPA receptor-containing neurons cocontain GluR1 and GluR2/3. (3) Thirty-eight percent of GluR1- and 28% of GluR2/3-labeled cells express calbindin. (4) Sixty-two percent of the calbindin-immunoreactive neurons contain GluR1 and 51% of them express GluR2/3. (5) Half of the neurons expressing both GluR1 and GluR2/3 also contain calbindin. (6) The distribution of GluR1 plus GluR2/3-containing, GluR1 plus calbindin-containing, and GluR2/3 plus calbindin-containing neurons in the lateral septum are homogeneous. This study indicates the existence of multiple populations of AMPA receptor- and calbindin-containing neurons in the lateral septal area.

Development of cholinergic and GABAergic neurons in the rat medial septum: effect of target removal in early postnatal development.

During normal development of the nervous system, the target fields influence the survival and differentiation of projection neurons, but the factors regulating this interaction remain obscure. In the present study, we have raised the question whether the target region is essential for the postnatal development and maintenance of two different types of central projection neurons, cholinergic and GABAergic septohippocampal cells. In early postnatal rats (P5, P10), the hippocampus was eliminated by unilateral intrahippocampal injections of the excitotoxin N-methyl-D-aspartate. After a long survival time (at P70), we have immunostained serial sections of the septal region with antibodies against choline acetyltransferase (ChAT), the acetylcholine-synthesizing enzyme, or the calcium-binding protein parvalbumin (PARV) which is known to be contained in GABAergic septohippocampal neurons. In the medial septum ipsilateral to the lesioned side, about 60% of ChAT-immunoreactive neurons and 62% of PARV-immunoreactive neurons were found in adulthood even after complete elimination of the hippocampus. Some immunoreactive cells appeared heavily shrunken, but electron microscopic analysis revealed ultrastructural characteristics typical for medial septal neurons obtained from controls. Our results indicate that target elimination during development affected both types of projection cells, although only the cholinergic cells are known to be responsive to target-derived factors.

Parvalbumin-containing cells of the angular portion of the vertical limb terminate on calbindin-immunoreactive neurons located at the border between the lateral and medial septum of the rat.

In the septal complex, both parvalbumin and calbindin neurons cocontain GABA. In the same area, a large number of GABA-GABA synaptic connections can be observed. In order to further characterize their neurochemical nature, as well as the extrinsic and/or intrinsic origin of these GABA terminals, the following experiments were performed: (1) correlated light- and electron-microscopic double immunostaining for calbindin and parvalbumin on septal sections of control rats: (2) light microscopic parvalbumin immunostaining of septal sections after surgical isolation (5 days) of the septum from its telencephalic or (3) hypothalamic afferents; and (4) parvalbumin immunostaining of sections prepared from the entire brain 2 days following horseradish peroxidase injection into the border between the lateral and medial septum. The results demonstrated that: (1) in a well-circumscribed, vertically longitudinal area located between the lateral and medial septum, 0.1-0.6 mm anterior to the bregma, a group of calbindin-containing, nonsomatospiny neurons are surrounded by parvalbumin-immunoreactive baskets; (2) these basket-forming axon terminals establish symmetric synaptic contacts with their targets; and (3) their cells of origin are not in the medial septum, but in the angular portion of the vertical limb. These observations indicate that a portion of the septal complex GABA-GABA synaptic connections represent functional interaction between two different types of GABAergic neurons. The presynaptic GABAergic neurons contain parvalbumin, and the postsynaptic GABAergic cells are immunoreactive for calbindin. Furthermore, a population of the medial septum/diagonal band parvalbumin neurons project only to the hippocampus, while others, which may also send axons to the hippocampus, terminate on lateral septum calbindin cells as well.

Expression of m1-m4 muscarinic acetylcholine receptor immunoreactivity in septohippocampal neurons and other identified hippocampal afferents.

Muscarinic cholinergic transmission plays an important role in modulating hippocampal activity and many higher brain functions. Many of the modulatory effects of acetylcholine on hippocampal function result from direct effects in the hippocampus or from actions on the hippocampal afferent neurons. At each site, the differential expression of a family of five distinct but related receptor subtypes governs the nature of the response. The aim of the present study was to identify the subtypes expressed in the hippocampal afferent neurons by combining retrograde tracing with immunocytochemistry. The retrograde tracer, wheat germ agglutinin conjugated to horseradish peroxidase, was injected into the hippocampus unilaterally to label afferent neurons, and was combined with muscarinic (m) acetylcholine (ACh) receptors (mAChRs) with immunocytochemistry to identify the m1-m4 subtypes expressed. The retrogradely labeled cells in the basal forebrain that contribute to the septohippocampal pathway were found to express m2, m3, and, to a lesser extent, m1. Commissural/associational pathway neurons, which were identified by retrogradely labeled cells in the ipsi- and contralateral dentate gyrus, expressed m1, m3, and m4. The retrogradely labeled cells in the entorhinal cortex of the perforant pathway expressed predominantly m1 and m3, with fewer neurons expressing m2 and m4. Raphe-hippocampal cells were found to express m1. Thus, this study provides evidence for the diversity of mAChR subtypes expressed in neurons that project to the hippocampus. The complex modulation by acetylcholine of hippocampal function, therefore, is governed not only by the variety of mAChRs expressed in the hippocampus but also by their differential expression in extrinsic hippocampal afferents.

Distribution of NADPHdiaphorase and calbindin-D28k neurons in the lateral septal area of the guinea pig, with special reference to the enkephalinergic hypothalamo-septal tract.

Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPHd) histochemical techniques were used to identify neurons synthesizing nitric oxide in the lateral septum of the guinea pig. Double immunocytochemical procedures were used to detect neurons immunoreactive for calbindin-D28k and enkephalinergic fibers which project to the lateral septum. The present data demonstrate that (1) the neurons containing NADPH diaphorase and the neurons immunoreactive for calbindin-D28k are observed in discrete regions of the lateral septum; (2) these populations overlap in various areas of the lateral septum including its dorsal and mediolateral parts; (3) NADPH diaphorase and calbindin-D28k are colocalized in neurons located in the overlapping areas; (4) neurons identified by the presence of calbindin-D28k, NADPH diaphorase or both substances, are surrounded by enkephalinergic fibers. These observations indicate the chemical heterogeneity of the lateral septum and suggest that the enkephalinergic hypothalamo-septal tract does not preferentially contact a subpopulation of neurons.

Distribution of follistatin messenger ribonucleic acid in the rat brain: implications for a role in the regulation of central reproductive functions.

Follistatin (FS), which binds to the inhibin/activin beta A- or beta B-subunit is localized with and modulates the biological actions of activin in many systems. However, in contrast to the wide distribution of the activin beta-subunit proteins and messenger RNAs (mRNA) in the brain, demonstration of FS mRNA signal has been limited to the olfactory tubercle and layer II of the frontal cortex. We have hypothesized a more extensive distribution of central FS gene expression and localization in regions coinciding with inhibin/activin beta-subunits and possible activin-mediated effects. In the present study, we examined the central distribution of FS mRNA expression in the normal adult male rat. With in situ hybridization analysis, using a 33P-labeled RNA probe specific for rat FS, gene expression is shown to be widely distributed throughout the brain. Abundant FS mRNA expression is localized in several areas of the olfactory bulb as well as the frontal cortex, a few thalamic nuclei, and in septal regions. Moderate FS mRNA is observed in the caudate putamen and various hypothalamic areas including the paraventricular, ventromedial, dorsomedial, and arcuate nuclei. Several brain stem regions are also found to express FS mRNA, including the medial vestibular and solitary tract nuclei. Notably, FS mRNA, including the medial vestibular and solitary septal/diagonal band region is localized in patterns that are highly correlative with those of GnRH gene expression and hence may serve to regulate possible activin-mediated effects in these areas. FS mRNA is also expressed in areas associated with the activin-oxytocin pathway (solitary tract nucleus and paraventricular nucleus) and is therefore in a position to modulate the role of activin in the solitary tract nucleus-paraventricular nucleus pathway (afferent system mediating the milk-ejection reflex). The results suggest that FS is centrally localized in sites compatible with a role in the regulation of central reproductive functions.