Synaptic organization of septal projections in the rat medial habenula: a wheat germ agglutinin-horseradish peroxidase and immunohistochemical study.

The synaptic organization of septal inputs to the rat habenular complex of the dorsal diencephalon was examined employing the anterograde tracer wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). The cellular distribution of substance P (SP) and choline acetyltransferase (ChAT) immunoreactivity was also studied at the light and electron microscopic level. Following placements of tracer within the entire septum, labeled axons were observed in the stria medullaris and in the medial and lateral subnuclei of the habenula. Following injections of tracer in the nuclei triangularis and septofimbrialis of the posterior septum, the medial subnucleus was heavily labeled, whereas the lateral subnucleus was devoid of peroxidase activity. The medial subnucleus possessed labeled myelinated axons and terminals that contained clear, spherical vesicles and formed asymmetric contacts with dendritic spines and shafts. Terminals possessing WGA-HRP activity also formed non-synaptic junctions with other labeled or unlabeled terminals. SP and ChAT immunoreactivity in normal and colchicine-treated animals was confined to dendrites and somata within the medial habenula. Terminals containing clear spherical vesicles formed asymmetric synaptic contacts with these immunoreactive somatic and dendritic profiles. Based on the combined anterograde tracing and immunohistochemical data, it is proposed that septal projections provide a direct innervation to habenular neurons that contain ChAT or SP activity. These septal inputs may play an important role in the facilitation of the ChAT- and SP-positive habenular neurons, both of which provide prominent afferent inputs to the interpeduncular nucleus. Thus, neurons of the habenula and interpeduncular nucleus are under the direct and indirect influence of septal neurons within the limbic forebrain circuit.

Critical exposure time for androgen activation of male sexual behavior in rats.

We determined the minimum number of hours per day of testosterone (T) exposure required to activate male sexual behavior, and correlated these changes with the temporal parameters of androgen receptor occupation. For the first part of the study, castrated Long-Evans male rats received two 10 mm T-filled Silastic capsules in open flank pouches for 4, 8, 12, 16, 18, 21, or 24 hours per day over a 10 day period. Tests for male sexual behavior were conducted on days 2-3, 4-5, 7-8, and 9-10 of T treatment. A significantly higher proportion of males receiving 21 or 24 hr of daily T exposure mounted, intromitted and ejaculated compared to groups with daily T exposures of 18 hr or less. In the second part of this study we assessed whether it was necessary to maintain high levels of androgen receptor occupation during the 21-24 hr exposure period in order to activate male sexual behavior. Cell nuclear androgen receptor occupation was measured in HPAS (combined hypothalamus, preoptic area, amygdala and septum) of rats receiving 12, 21, or 24 hr of T exposure. In all three groups, nuclear androgen receptor occupation was high at the time of capsule removal, and fell significantly by 3 hr following T capsule removal. By 6 hr after T capsule removal, androgen receptor binding had fallen to castrate levels. These results demonstrate that, although relatively brief (less than or equal to 18 hr/day) exposures to testosterone can activate mounts and intromissions, significantly more responses are found in males receiving at least 21 hr of T exposure per day.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative radioimmunohistochemical method using [125I]-protein A to measure the content of methionine enkephalin in discrete rat brain areas.

We report a quantitative radioimmunohistochemical method, using [125I]-protein A in combination with a specific antibody to methionine enkephalin (Met-enk), for determination of the content of this peptide in discrete areas of rat brain. After paraformaldehyde fixation, rat brain sections were incubated with a Met-enk polyclonal antibody, followed by incubation with [125I]-protein A. After autoradiography with 3H-sensitive Ultrofilm, optical densities (OD) were quantified by computerized microdensitometry. The OD obtained were compared to a standard curve, constructed after determination by radioimmunoassay of the Met-enk content in corresponding brain areas from adjacent tissue sections. After comparing 15 different brain areas over a ninetyfold range of concentrations, we found a linear relationship between the content of Met-enk, as determined by radioimmunoassay, and the OD generated by autoradiography. The content of Met-enk in other discrete brain areas can be quantified by interpolation of the OD determined by autoradiography in the standard curve. The method allows, for the first time, precise quantification of peptide concentrations in discrete areas and nuclei from thin sections of rat brain. This technique has a more than 100-fold higher sensitivity than classical radioimmunoassays, with the additional advantage of neuroanatomical localization. It also has the potential for application to the quantification of many other antigens present in brain and other tissues.

Fine structures of nerve fibers with corticotropin-releasing factor-like immunoreactivity in the rat lateral septum.

The fine structures of nerve fibers with corticotropin-releasing factor (CRF)-like immunoreactivity in the rat lateral septum were investigated by means preembedding immunoelectron microscopy. A number of CRF axon terminals formed synapses with cell bodies of non-immunoreactive septal neurons. They occasionally had broad terminal bulges whose subregions showed little or no immunoreactivity for CRF. CRF axon terminals were also in synaptic contact with non-immunoreactive dendrites or dendritic spines. Some dendrites with CRF were postsynaptic to non-immunoreactive axon terminals.

An immunocytochemical and biochemical study of the microtubule-associated protein MAP-2 during post-lesion dendritic remodeling in the central nervous system of adult rats.

A monoclonal antibody against the microtubule-associated protein MAP-2 was used to examine the fate of this molecule during post-lesion dendritic remodeling in the hippocampus and septum of adult rats. Qualitative and quantitative immunocytochemical analyses were carried out in the dentate gyrus after unilateral destruction of the entorhinal cortex (EC). An increase in MAP-2 immunoreactivity was detected in dendritic processes located in the outer 2/3 of the ipsilateral molecular layer (ML) 2 days after the lesion. whereas dendritic staining decreased considerably in the inner 1/3 of the same ML. The increase of staining was also detected 4, 6 and 8 days after the lesion; it was accompanied by an increase in the immunoreactivity in the inner 1/3 of the ML. After that period, a progressive decrease in anti-MAP-2 staining toward control levels was detected along the whole extent of the ipsilateral ML. This was concurrent with alterations in dendritic orientation, and a decrease in stained dendrites in the inner 1/3 of the ML. By 30 days post-lesion anti-MAP-2 staining was almost identical to that of the contralateral ML, although the alterations in dendritic morphology were still present in the ipsilateral ML. Changes in MAP-2 levels were also evaluated by densitometry of Western blots or dot immunobinding of hippocampal extracts obtained at different post-lesion intervals. The results obtained revealed a pattern of change in MAP-2 levels identical to that observed with the immunohistochemical stain. A similar, immunocytochemical and biochemical, analysis conducted in the lateral septal nucleus after unilateral transection of the fimbria showed no changes in the distribution and/or content of MAP-2 at any post-lesion interval analyzed (2, 10 and 20 days post-lesion). The present observations show that post-lesion dendritic remodeling is concurrent with modifications in the levels and distribution of MAP-2. These modifications suggest that the dendritic cytoskeleton is dynamically changing in response to perturbation of the synaptic environment. In addition, our results indicate that these changes may only occur in those neurons which have the capability to remodel their post-synaptic surface in response to deafferentation.

Dopamine-GABA interactions in the nucleus accumbens and lateral septum of the rat.

The relationships between dopaminergic afferents and GABAergic neurones were studied at the electron microscopic level in the rat lateral septum and nucleus accumbens by coupling 6-hydroxydopamine degeneration and gamma-aminobutyric acid (GABA) immunocytochemistry. Degenerating fibres were observed in the two regions making synaptic contact with GABA-immunoreactive and non-labelled cell bodies and dendrites. It is concluded that dopaminergic afferents to the septum and the nucleus accumbens contact, among others, a population of GABAergic cells. A similar route of regulation of the basalo-cortical and septo-hippocampal cholinergic pathways by dopaminergic afferents is proposed.

Asymmetries of brain dopamine metabolism related to conditioned paw usage in the rat.

Concentrations of dopamine, serotonin, and some of their metabolites were analyzed by means of high-performance liquid chromatography in brain samples obtained from rats operantly conditioned to use one paw for water-reinforced lever pressing. In the first experiment, the side of paw usage was determined by physical constraint (forced-handedness condition), whereas in the second experiment the side of paw usage was not restricted (paw-preference condition). Differences in dopamine metabolism were detected between brain samples from the hemispheres located ipsi- and contralaterally to the side of paw usage. A higher dopamine metabolism (indicated by higher metabolite/transmitter ratios) was found in the amygdala ipsilateral to the paw used both under the forced-handedness and paw-preference condition. A higher level of dopamine in the contralateral septum was found in rats sacrificed immediately after 15 min of forced-handedness and an ipsilateral increase was found in rats analyzed 2 h after performance of this task. In addition, a higher dopamine metabolism in the ventral striatum, dorsal striatum, and amygdala was found in the forced-handedness and yoked controls groups than in rats analyzed 2 h after lever pressing. In the second experiment, rats in the paw preference group had a lower dopamine metabolism in the ventral and dorsal striatum, septum, and substantia nigra than did their yoked controls. These results show that changes in dopamine metabolism during conditioned lever pressing can be asymmetrical with respect to the side of paw usage, indicating that the dopamine neurons in the two brain hemispheres are asymmetrically involved in such behavioral tasks.

An ultrastructural study of GABA-immunoreactive neurons and terminals in the septum of the rat.

The fine structure and types of contact made by GABAergic elements in the septal nuclei were studied at the electronmicroscopic level by means of peroxidase immunocytochemistry, using anti-GABA antibodies. Observations were made on normal and colchicine-injected rats. GABA-immunoreactivity was distributed within somata, dendrites, axonal varicosities and terminals, and myelinated axons. The peroxidase reaction product was diffuse in the cytoplasm; cytoplasmic organelles were generally devoid of immunoreactivity, while showing a strong reaction on the outer surface of their membrane. GABA-immunoreactive (GABA-I) neurons were small (10 microns on average) to medium (20 microns) in size, with round or multipolar cell bodies. Additionally, labeled large (30 microns) cells were observed within the myelinated fibers of the medial septal nucleus after intraseptal administration of colchicine. No difference in the ultrastructural features and distribution of the immunoreactivity of the 2 kinds of cell was noticed, except for a higher number of synaptic contacts on large neurons of the medial septum. GABA-I cells of the medial and lateral nuclei received synapses on their soma and dendrites, made by both immunonegative and GABA-I terminals. Nonimmunoreactive boutons contacting GABA-I cell bodies were of 2 types: those containing small, clear synaptic vesicles and those that additionally contained large dense vesicles. Synaptic vesicles of GABA-I boutons were rarely labeled internally, but showed varying electron densities. Synapses made by GABA-I boutons on GABA-I or unlabeled somata and dendrites were always of symmetrical type. Synapses made by non-GABA-I boutons on GABA-I cells were either symmetrical or asymmetrical.

Neurotensin immunoreactive structures in the human infant striatum, septum, amygdala and cerebral cortex.

Neurotensin immunoreactive (NT-IR) neuronal perikarya are present in small numbers in the bed nucleus of the stria terminalis, lateral olfactory stria, substantia innominata, caudate nucleus and putamen of the human infant forebrain. Larger numbers of perikarya are present in the amygdala and related structures. NT-IR axons are present in the medial septal area, bed nucleus of the stria terminalis, caudate nucleus, putamen and amygdala. The cerebral cortex contains a rich network of NT axons with an accentuation in layer II. This network appears to be derived from bundles of axons which traverse the deep white matter from the thalamus.

GABAergic neurons in the septum of the lizard, Podarcis hispanica.

With an antibody to gamma-aminobutyric acid (GABA), GABA-immunoreactive somata and boutons were identified in the septum of a lizard brain. GABA-positive somata occur at all rostrocaudal levels and in all septal nuclei. Parallel acetylcholinesterase (AChE) staining revealed a similar distribution of AChE-positive neurons. GABAergic boutons were seen throughout the septal nuclei, and were more densely concentrated in the outer zone of the dorsolateral septum. Axons of the anterior pallial commissure contained GABA- and AChE-reactivity.

Fimbria-fornix lesion increases nerve growth factor content in adult rat septum and hippocampus.

Bilateral complete transection of the fimbria-fornix causes a significant increase in nerve growth factor (NGF) content both in the septum and hippocampus of the adult rat as measured by a sensitive immunoassay 7 days after lesioning. The finding, that elevation of NGF in septum (250%) was much more pronounced than that in the hippocampus (30-50%), cannot be explained by retrograde axonal transport. Degeneration of central cholinergic neurons might be a potential trigger for NGF production in the CNS.

A radioimmunoassay for the phosphoprotein B-50: distribution in rat brain.

A radioimmunoassay (RIA) for the B-50 protein was developed to determine B-50 in total homogenates of rat tissues. A tracer of purified B-50 was prepared at high activity (10-30 microCi/micrograms protein) by phosphorylating B-50 with carrier-free [gamma-32P]ATP, catalyzed by purified protein kinase C. The RIA was performed using affinity-purified anti-B-50 immunoglobulins G in a detergent containing medium and detected B-50 at levels of 0.1-10 ng. Specificity of the antibodies was ascertained by immunoprecipitation of B-50 from a crude mitochondrial membrane fraction from rat brain and by immunoblotting. For the B-50 content in rat brain the following distribution pattern was found: medulla spinalis less than cerebellum less than hippocampus; cerebral cortex less than periaqueductal gray less than septum. The septum contained 80 micrograms/g tissue weight. The level in liver homogenates was below detection. The regional distribution is in fair agreement with the pattern of the endogenous B-50 phosphorylation in rat brain synaptosomal plasma membranes previously reported.

Elevations in plasma angiotensin II with prolonged ethanol treatment in rats.

Chronic alcohol consumption frequently leads to hypertension in humans. While previous reports have implicated the renin-angiotensin system as a potential mediator of this effect, plasma angiotensin II (AII) levels were either not measured or yielded negative results. The present investigation noted significant elevations in circulating AII in rats intubated daily with ethanol (4 g/kg) for 50 days. Animals administered ethanol only once evidenced AII concentrations equivalent with water intubated controls. Radioligand binding assay data indicated no differences in the number or affinity of Sar1,Ile8-AII binding sites in the thalamus, septum-anterior ventral third ventrical region or adrenal gland comparing those groups chronically treated with ethanol to water intubated controls. These results may support a role for the vasoconstrictive hormone AII in the etiology of alcohol-induced hypertension.

Dissociation of the effects of castration and testosterone replacement on sexual behavior and neural metabolism of dopamine in the male rat.

Sexually experienced, adult male rats were either castrated, castrated and implanted SC with a Silastic capsule containing testosterone (T), or sham operated. Coital performance of castrates gradually declined such that 4 weeks after surgery no males in this group ejaculated whereas 89% and 100%, respectively, of the rats in the castrated, T-treated and the sham-operated groups displayed ejaculation. Males in all three groups were decapitated 33-34 days post-operatively after 10 min exposure either to the behavioral test chamber, with an estrous female restrained in one corner behind a wire mesh screen, or to a home cage. Brains were quickly removed and the caudate-putamen, nucleus accumbens, septum, and preoptic area/anterior hypothalamus were frozen and saved for later estimation of dopamine (DA) and two neural metabolites of DA, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). The concentrations of DA, DOPAC, and HVA, as well as the ratio of DOPAC/DA, did not differ significantly in any of the 4 brain regions assayed among males in the three endocrine groups, regardless of whether they were killed after exposure to an estrous female or a home cage. The results fail to support the hypothesis that T-induced alterations in neurotransmission in nigro-striatal, mesolimbic, or incertohypothalamic DA pathways mediate the activational effect of this steroid on masculine sexual behavior.

Estrous cycle variations in cholecystokinin and substance P concentrations in discrete areas of the rat brain.

Cholecystokinin (CCK) and substance P (SP) were measured in discrete areas of the rat brain at different stages of the estrous cycle. Significantly higher levels of CCK were found in the lateral septum during diestrus as compared to proestrus. In the parietal cortex, CCK concentrations were significantly higher in diestrus than in proestrus. In the amygdala, estrous levels of CCK were significantly higher than proestrous levels. SP concentrations were significantly higher in diestrus than in proestrus in the medial and lateral septum, and the medial and lateral preoptic area. In the amygdala and ventral tegmental area, SP concentrations were significantly higher in estrus than in proestrus. These data suggest that certain CCK and SP neuronal systems may play a role in regulating the hypothalamo-pituitary-gonadal axis and/or be involved in steroid-dependent behavior.

Nuclear and cytosolic androgen receptor levels in the limbic brain of neonatal male and female rats.

Nuclear and cytosolic androgen receptors in the limbic brain were measured in neonatal male and female rat pups. There were no sex differences in cytosolic receptor concentrations during the neonatal period in any of the regions studied (hypothalamus, amygdala, preoptic area and septum). Receptor concentrations in all 4 regions increase gradually over the first 10 days of life, with no change in the affinity for 5-alpha-dihydrotestosterone. Nuclear receptor levels in intact pups, measured using an exchange assay, are highest between days 4 and 8 of life. In general, nuclear receptor levels are higher in males than in females; however, this sex difference is most consistently seen in the amygdala. These results are discussed in relation to sex differences in circulating testosterone levels and with respect to the contribution of androgens to the sexual differentiation of behavior.

Early postnatal handling alters glucocorticoid receptor concentrations in selected brain regions.

Norway rat pups were either handled (H) or undisturbed (nonhandled, NH) in the period between birth and weaning on Day 21. Following weaning, half of the animals in each group were housed socially (Soc), and half were housed in isolation (Isol). At 120-150 days of age, all animals were sacrificed, and the following regions were dissected and frozen at -70 degrees C until the time of assay: frontal cortex, hippocampus, hypothalamus, amygdala, septum, and pituitary. [3H]Dexamethasone (3H Dex) binding in each region was examined by an in vitro, cytosol, receptor assay. 3H Dex binding was significantly higher in the hippocampus of both H-Soc and H-Isol than in NH groups. In the frontal cortex, 3H Dex binding was higher in the H-Soc animals than in the H-Isol and NH-Isol animals. There were no significant handling or housing effects found in the amygdala, hypothalamus, septum, or pituitary. Thus, early postnatal handling appears to influence the development of the glucocorticoid receptor system in the hippocampus and frontal cortex. These results are discussed as providing a possible mechanism for some of the previously reported effects of early handling on the development of the pituitary-adrenal response to stress.

Neurotensin immunoreactivity in post-mortem brain is increased in Down's syndrome but not in Alzheimer-type dementia.

Neurotensin immunoreactivity and choline acetyltransferase (ChAT) activity were measured in post-mortem brain from 10 cases of Down's syndrome (7 aged 53-63 years, one aged 27 years, one aged 16 months and one aged 10 months), 6 cases of Alzheimer-type dementia (ATD) and 19 control subjects (13 aged 40-88 years and 6 aged 9-18 months). Neurotensin concentrations in anterior and basal hypothalamus, amygdala, septal area, caudate nucleus and temporal cortex were unaltered in ATD. The concentrations of neurotensin were significantly increased in the caudate nucleus, temporal cortex and frontal cortex in the cases of Down's syndrome aged 53-63 years with the neuropathological features of ATD, and were also increased in the cerebral cortex of the 27-year-old, which did not have the neuropathological features of ATD, and in two infant Down's cases. ChAT activity was reduced in the ATD and the 53-63-year-old cases of Down's syndrome, but not in the 27-year or 10-month-old Down's cases. The increased neurotensin concentrations appear to be a feature of Down's syndrome not related to the presence of plaques and neurofibrillary tangles or to a deficit in ChAT activity.

The development of the glucocorticoid receptor system in the rat limbic brain. I. Ontogeny and autoregulation.

In order to characterize the development of the glucocorticoid receptor system in the brain, we examined [3H]dexamethasone binding in rat pups at various ages. Using an in vitro, cytosol, receptor assay we found evidence for low levels of glucocorticoid receptors perinatally with a subsequent increase in receptor concentrations that began by about the end of the first week of life. We have also shown that receptors during this period have a ligand specificity similar to that of receptors in adult animals. The postnatal increase in receptor levels parallels an increase in circulating corticosterone titers. Thus, receptor and hormone levels increase coincidentally. In adult animals, however, increasing levels of corticosterone are associated with a decrease in receptor levels and vice versa, such that corticosterone is thought to regulate its own receptor (i.e. autoregulation). This suggested an absence of autoregulation during development. We then determined hippocampal receptor concentrations of rats treated for 5 days with corticosterone, or adrenalectomized (ADX) 5 days prior to assay, examining whether up- or down-regulation occurs throughout development. In adults corticosterone treatment decreased (-45%) and long-term adrenalectomy increased (211%) glucocorticoid receptor concentrations. In contrast, at the youngest age tested (Day 10), the effects of manipulations of corticosterone titers on receptor concentrations were negligible. The potential for autoregulation emerged gradually throughout development. Thus, it appears that corticosterone regulation of its own receptors emerges only by about the time of puberty, and that this permits an increase in receptor levels to occur despite the concurrently increasing levels of circulating corticosterone.

Serotonergic innervation of the forebrain in the North American opossum.

The forebrain distribution of axons showing serotonin-like immunoreactivity was studied in the North American opossum. Serotonergic innervation of the hypothalamus was extensive, particularly within the ventromedial nucleus, the periventricular nucleus and the rostral supraoptic nucleus. Serotonergic axons were also present within the fields of Forel and zona incerta, but they tended to avoid parts of the subthalamic nucleus. In the thalamus serotonergic innervation was dense within the midline nuclei (e.g. the central, intermediate dorsal and rhomboid nuclei) and the ventral lateral geniculate nucleus, but relatively sparse in some of the nuclei more readily associated with specific functions (e.g. the ventrobasal nucleus). Serotonergic axons innervate most areas of the rostral and dorsal forebrain. Areas containing the heaviest innervation included the interstitial nucleus of the stria terminalis and the lateral septal nucleus. Serotonergic innervation of the neocortex varied markedly from region to region and within different layers of the same regions. The retrograde transport of True Blue combined with immunofluorescence for localization of serotonin revealed that serotonergic axons within the forebrain arise mainly within the dorsal raphe and superior central nuclei, but that some originate within the midbrain and pontine reticular formation and the locus coeruleus, pars alpha. Neurons of the raphe magnus and obscurus also innervate the forebrain, but few of them are serotonergic. The use of horseradish peroxidase as a retrograde marker provided evidence that raphe projections to the forebrain are topographically organized. Our results suggest that serotonergic projections to the forebrain, like those to the spinal cord, are connectionally heterogeneous.