Transcriptional activity of human brain estrogen receptor-α splice variants: evidence for cell type-specific regulation.

Estrogen receptor α (ERα) isoforms with complex types of alternative splicing are naturally present in the human brain and may affect canonical receptor signaling. In the present study we investigated transcriptional activity of common ERα splice variants from this group with different molecular defects: MB1 (intron retention), TADDI (small deletion between exons 3 and 4 with an insert), the Δ (deletion) 3(⁎)-7(*)/819 (complete skipping of exons 4, 5 and 6 and partial deletion of exons 3 and 7) and the Δ3-6 (lacking exons 3, 4, 5 and 6) in HeLa and M17 cells upon stimulation with (17ß)estradiol or insulin-like growth factor 1 (IGF-1). In HeLa cells, all these splice variants showed the dominant negative function that was more pronounced for the TADDI. In M17 cells the dominant negative variants appeared to be the MB1 and the Δ3-6, whereas TADDI turned out to be a clearly dominant positive variant. In M17 cells mRNA levels of Δ3-6 and Δ3(*)-7(*)/819 variants increased following (17ß)estradiol administration. In Hela cells (17ß)estradiol up-regulated the IGF-1 receptor mRNA levels in cultures transfected with MB1, TADDI and Δ3(*)-7(*)/819. Our data demonstrate that ERα splice variants show differential levels of the transcriptional activity in a cell type-specific way and that IGF-1 signaling pathways are differentially employed in a cell-type specific manner depending on the level of the discrete ERα splice variants expressed. Functional properties of various ERα splice variants and their cell type-specificity should, thus, be considered as potential confounders of estrogen therapy effects on the brain.

Neural stem cells improve neuronal survival in cultured postmortem brain tissue from aged and Alzheimer patients.

Neurodegenerative diseases are progressive and incurable and are becoming ever more prevalent. To study whether neural stem cell can reactivate or rescue functions of impaired neurons in the human aging and neurodegenerating brain, we co-cultured postmortem slices from Alzheimer patients and control participants with rat embryonic day 14 (E14) neural stem cells. Viability staining based on the exclusion of ethidium bromide by intact plasma membranes showed that there were strikingly more viable cells and fewer dead cells in slices co-cultured with neural stem cells than in untreated slices. The presence of Alzheimer pathology in the brain slices did not influence this effect, although the slices from Alzheimer patients, in general, contained fewer viable cells. Co-culturing with rat E14 fibroblasts did not improve the viability of neurons in the human brain slices. Since the human slices and neural stem cells were separated by a membrane during co-culturing our data show for the first time that neural stem cells release diffusible factors that may improve the survival of aged and degenerating neurons in human brains.

Mature astrocytes in the adult human neocortex express the early neuronal marker doublecortin.

Doublecortin (DCX) is a microtubule-associated protein expressed by migrating neuroblasts and is considered to be a reliable marker of neurogenesis. DCX has been used to study the relation between neurogenesis in adult human brain and neurological and neurodegenerative disease processes in the search for putative therapeutic strategies. Using autopsy and surgically resected tissue from a total of 60 patients, we present evidence that DCX is present in several cellular compartments of differentiated astrocytes in the adult human neocortex. One of these compartments consisted of peripheral processes forming punctate envelopes around mature neuronal cell bodies. Markers of glial activation, such as GFAP and HLA, were not associated with DCX immunoreactivity, however, the presence of cytoarchitectural alterations tended to correlate with reduced DCX staining of astrocytic somata. Interestingly, local Alzheimer pathology that showed no relation with cytoarchitectural abnormalities appeared to correlate negatively with the expression of DCX in the astrocytic somata. In combination with the literature our data support the view that DCX in the adult human neocortex may have a function in glia-to-neuron communication. Furthermore, our results indicate that in the adult human neocortex DCX is neither a reliable nor a selective marker of neurogenesis.

Post-mortem brain tissue cultures from elderly control subjects and patients with a neurodegenerative disease.

Aging may be viewed as a progressive loss of normal biological function. Due to complex genetic and environmental interactions, the sequence of functional impairment shows a high degree of individual variability. In humans life style and health care have an additional influence on the aging process. To study aging and age-related disorders of the human nervous system, brain tissue that has undergone aging and pathological alterations can provide valuable study material. Recently, we have shown that adult human postmortem brain tissue can be cultured and experimentally manipulated. This approach permits the study of cellular aspects of human neuronal aging and neurodegenerative processes and complements those existing research methods such as in vivo imaging (MRI, PET, etc.) and fixed or frozen postmortem brain tissue examination.

Decreased hippocampal metabolic activity in Alzheimer patients is not reflected in the immunoreactivity of cytochrome oxidase subunits.

In the present study we have compared histochemically determined cytochrome oxidase activity with the levels of immunocytochemically stained cytochrome oxidase subunits (CO II and CO IV) and ATP synthase in the human hippocampus in relation with Alzheimer's disease. Cytochrome oxidase activity was significantly reduced in all hippocampal areas of Alzheimer patients. The protein levels of subunits II and IV were not different between control subjects and Alzheimer patients. Additionally, it was observed that the active cytochrome oxidase is evenly distributed over both cell bodies and neuropil, while a relatively large pool of inactive enzyme or precursors is limited to the neuronal somata. Further, in Alzheimer patients the CO IV immunoreactivity decreased with age, whereas in control subjects it increased with age. Our results suggest that the assembly of cytochrome oxidase or the processing of its subunits may be impaired.

Membrane properties and morphology of vasopressin neurons in slices of rat suprachiasmatic nucleus.

Vasopressin (VP) neurons in the suprachiasmatic nucleus (SCN) are thought to be closely linked to neural mechanisms for circadian timekeeping. To gain insight into the cellular-physiological principles that govern spike-driven VP release and to examine whether VP cells can be electrophysiologically and morphologically identified by a unique combination of features, we recorded membrane properties by whole cell patch-clamp methods and stained the cells with biocytin. In current-clamp mode, VP neurons recorded during subjective daytime expressed a clear time-dependent inward rectification but no pronounced low-threshold Ca2+ potential after hyperpolarizing current pulses. Their spontaneous firing rate varied between 0.6 and 13.4 Hz and was generally tonic and irregular. Spike afterhyperpolarizations (AHPs) were steeply rising and monophasic. Spikes were preceded by depolarizing ramps mediated by a slow component of Na+ current. Spike trains evoked by depolarizing current pulses displayed frequency adaptation and were usually followed by an AHP lasting 0.5-2.0 s. Spontaneous postsynaptic potentials were present in a majority of cells. Voltage-clamp recordings revealed a Ba2+-sensitive K+ current that exerts a tonic, hyperpolarizing influence on the membrane potential. This set of membrane properties was not significantly different from other cells in the dorsomedial region and is characteristic for cluster I cells, which were described previously and are widely encountered throughout the SCN. None of the cells could be classified as belonging to cluster II or III, which were indeed found mainly outside the dorsomedial region. Morphologically, single VP neurons were characterized by compact, mono- or bipolar dendritic branching patterns and numerous varicosities throughout the dendrites. They generally possessed few axon collaterals, most of which remained inside the boundaries of the SCN but were occasionally seen to project to SCN target areas. In conclusion, VP neurons in the SCN express several active membrane poperties, including time-dependent inward rectification, frequency adaptation in spike trains, monophasic spike AHPs, and Ba2+-sensitive K+ current. VP release is proposed to be governed by tonic and irregular patterns of spontaneous firing. The electrophysiological and cytological properties of VP neurons are representative for a majority of SCN cells and define them as a subset of previously defined cluster I cells.

Immunocytochemical evidence for a diurnal rhythm of neurons showing colocalization of VIP with GRP in the rat suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN), which functions as a biological clock, contains several neuropeptides such as vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI), and gastrin-releasing peptide (GRP). Studies from several laboratories have provided evidence for the coexistence of VIP with PHI and GRP, but reliable data about the proportions of colocalization and a possible diurnal rhythmicity are lacking. In the present study, we therefore aimed at studying these aspects. To this end, rats were killed by perfusion fixation during the middle of the day (Zeitgeber time [ZT] 7) and during the second part of the night (ZT 19). Coronal Vibratome sections through the SCN were double-immunolabeled for the presence of VIP and PHI or for VIP and GRP. Analysis of the sections was done by semi-quantitative confocal laser scanning fluorescence microscopy. It turned out that, in keeping with previous literature data, VIP and PHI always coexist at the cellular level. This was seen in all possible ratios, both during the day and at night. Part of these VIP/PHI-containing neurons (21%) and part of the GRP-containing neurons (33%) showed colocalization during the middle of the day. During the second part of the night, these percentages increased significantly to 28% and 40%, respectively. This increase in percentages was due to a significant, nocturnal increase of the number of profiles showing colocalization, in contrast to the number of profiles exclusively immunoreactive for VIP or GRP.

Electrophysiological and morphological heterogeneity of neurons in slices of rat suprachiasmatic nucleus.

1. Whole cell patch clamp recordings of neurons in slices of the suprachiasmatic nucleus (SCN) were made in order to assess their electrophysiological and morphological heterogeneity. This assessment was accomplished by (i) quantification of intrinsic membrane properties recorded in current clamp mode, (ii) studying frequency distributions of these properties, (iii) grouping of cells based on visual inspection of data records, and (iv) use of cluster analysis methods. 2. Marked heterogeneity was found in the resting membrane potential, input resistance, time constant, rate of frequency adaptation, size of rebound depolarization (low-threshold Ca2+ potential) and regularity of firing. The frequency distribution of these membrane properties deviated significantly from a normal distribution. Other parameters, including spike amplitude and width, amplitude and rising slope of the spike after-hyperpolarization (AHP) and amplitude of the spike train AHP, showed considerable variability as well but generally obeyed a normal distribution. 3. Visual inspection of the data led to partitioning of cells into three clusters, viz. cluster I characterized by monophasic spike AHPs and irregular firing in the frequency range from 1.5 to 5.0 Hz; cluster II with biphasic spike AHPs and regular firing in the same range; and cluster III with large rebound depolarizations and biphasic spike AHPs. In a post hoc analysis, these clusters also appeared to differ in other membrane properties. This grouping was confirmed by hierarchical tree clustering and multidimensional scaling. 4. The light microscopic properties of recorded neurons were studied by biocytin labelling. Neurons had monopolar, bipolar or multipolar branching patterns and were often varicose. Axons sometimes originated from distal dendritic segments and usually branched into multiple collaterals. Many cells with extra-SCN projections also possessed intranuclear axon collaterals. We found no morphological differences between clusters except that cluster III neurons possessed more axon collaterals than cluster I or II cells. 5. These results suggest that SCN neurons are heterogeneous in some basic as well as active membrane properties and can be partitioned into at least three clusters. Cluster I and II cells fire spontaneously in a regular and irregular mode, respectively, and sustain prolonged spike trains. In contrast, cluster III cells have low firing rates but may adopt a burst-like firing mode when receiving appropriate input. While all clusters transmit output to target cells within and outside SCN, cluster III cells in particular are suggested to affect excitability of large numbers of SCN neurons by their extensive local network of axon collaterals.

Evidence from confocal fluorescence microscopy for a dense, reciprocal innervation between AVP-, somatostatin-, VIP/PHI-, GRP-, and VIP/PHI/GRP-immunoreactive neurons in the rat suprachiasmatic nucleus.

The rat suprachiasmatic nucleus (SCN) consists of several classes of neurons which can be identified by their transmitter content. Knowledge of putative interaction between these different cell types is essential in order to understand the possibilities of information processing within the SCN. The aim of the present study was therefore to obtain more information about the mutual innervation between the main cell classes in the rat SCN, viz. those containing the neuropeptides arginine vasopressin (AVP), vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI), gastrin-releasing peptide (GRP) and somatostatin respectively. For this purpose, vibratome sections were double-immunolabelled for seven different peptide combinations and subsequently analysed by high-resolution confocal laser scanning fluorescence microscopy. Attention was focused on axosomatic appositions, the occurrence and frequency of which were quantitatively estimated. Our analysis of double-immunolabelled sections demonstrated that some of the VIP- and some of the GRP-immunoreactive nerve cells and endings showed colocalization. Assuming, on the basis of literature data, that VIP and PHI are always colocalized at the cellular level, the five main cell classes in the SCN appeared to be interconnected, at least axosomatically, in the following reciprocal way: AVP <--> VIP/PHI, AVP <--> GRP, AVP <--> somatostatin, somatostatin <--> VIP/PHI, somatostatin <--> GRP, VIP/PHI <--> GRP, VIP/PHI/GRP <--> GRP, VIP/PHI/GRP <--> VIP/ PHI. In addition to this heterologous axosomatic innervation, these cell groups also showed substantial homologous innervation. Supported by electron microscope data from the literature showing the existence of axodendritic synapses for some of these peptide combinations, our findings strongly suggest that the rat SCN comprises a complex synaptic network with strong interactive capabilities, which is probably a requisite for its biological clock function.

Lack of translation of normal 7B2 mRNA levels in hypothalamic mutant vasopressin cells of the homozygous Brattleboro rat.

The homozygous Brattleboro rat (di/di) synthesizes a vasopressin (VP) precursor with an aberrant C-terminus, which causes a hypothalamic form of diabetes insipidus. The neuroendocrine polypeptide 7B2 is present in VP and oxytocin (OT) neurons of the supraoptic and paraventricular nucleus of the hypothalamus in wild type rats. However, in the di/di rat 7B2 immunoreactivity is absent in the VP cell population, whereas 7B2 levels within the OT cells are unaffected. Remarkably, there is no obvious difference in 7B2 transcript levels between VP and OT neurons in the di/di rat hypothalamus. This study shows that the presence of mRNA does not automatically result in the subsequent synthesis of its protein. Cellular mechanisms underlying this discrepancy are discussed.

Differences in colocalization between Fos and PHI, GRP, VIP and VP in neurons of the rat suprachiasmatic nucleus after a light stimulus during the phase delay versus the phase advance period of the night.

Two groups of four rats each received a 15-minute light stimulus during the first part of the night (ZT14) and the second part (ZT19), respectively. After 45-60 minutes, the animals were killed by perfusion fixation. Adjacent Vibratome sections through the suprachiasmatic nucleus (SCN) were double-immunostained for the presence of peptide histidine isoleucine (PHI), gastrin releasing peptide (GRP) or vasoactive intestinal peptide (VIP) with Fos by using fluorophore-conjugated secondary antibodies. A few sections were triple-immunostained for PHI, GRP or VIP with vasopressin (VP) and Fos. Sections were analyzed with a confocal laser scanning microscope. It turned out that the ZT19 light stimulus induced 4.2 times more nuclear profiles in the SCN immunoreactive for Fos than the light stimulus given at ZT14. The SCN of control animals did not show any Fos immunoreactivity. After the ZT14 light stimulus, approximately 33% of the Fos profiles showed colocalization with a perikaryal profile immunoreactive for PHI, GRP or VIP, whereas at ZT19, this percentage had doubled to approximately 65%. After the light stimulus at ZT14, the relatively low Fos induction was numerically and proportionally most prominent in the PHI-immunoreactive perikarya. As compared with ZT14, the increase of Fos after the ZT19 light stimulus was most pronounced in the GRP-immunoreactive perikarya (21x) followed by VIP (15x) and PHI (5x). This outcome suggests that at least three different cell groups characterized by, respectively, PHI alone, GRP, and VIP fully or partly colocalized with PHI, play a prominent role during light-induced phase shifts: the PHI neurons during light-induced phase delays, the GRP and VIP/(PHI) neurons during light-induced phase advances.

AMPA receptors in cerebellar granule cells during development in culture.

The survival and maturation of differentiating cerebellar granule cells in culture are known to be promoted by excitatory amino acids (EAAs) which, however, compromise the survival of mature cells. In contrast to the trophic effect, the toxic effect of alpha-amino-3-hydroxy-5-methyl-4-isoxasolepropiate (AMPA) could only be elicited when the desensitisation of AMPA receptors was blocked, cyclothiazide being used in this study. Nevertheless, even under these conditions, toxicity induced by AMPA in contrast to kainate was, at 9 DIV, only half of the maximal toxicity attained by 13-16 DIV. Since cellular responses to AMPA depend so dramatically on the maturational stage of granule cells, we examined here whether this characteristic is related to developmental changes in AMPA receptor properties, which may result from changes in the subunit composition of the receptor. In contrast to toxicity, AMPA-induced 45Ca2+ influx (determined in the presence of cyclothiazide and the NMDA receptor blocker MK-801) reached a maximum already at 9 DIV. This also applied to a fraction of the 45Ca2+ uptake which persisted either after Cd2+ application or under Na(+)-free conditions and therefore presumably was mediated directly through AMPA receptor channels. Quantitative analysis of Western blots showed that the amounts of GluR4 and to a lesser extent GluR2/3/4c are substantial already at 2 DIV, remaining fairly constant until 9 DIV, followed by an increase by 16 DIV. However GluR1, which is hardly detectable in granule cells in vivo and is also low early in vitro, increased almost linearly with cultivation time.(ABSTRACT TRUNCATED AT 250 WORDS)

C-Fos expression in the rat brain after pharmacological stimulation of the rat "mediodorsal" thalamus by means of microdialysis.

In order to visualize target cells of thalamic projections in the rat brain we examined the induction of c-fos messenger RNA and Fos-like immunoreactivity following stimulation of the "mediodorsal" thalamus (midline, mediodorsal and intralaminar nuclei) in freely moving rats. The thalamic neurons were activated through disinhibition by perfusion of the GABAA antagonist bicuculline-methyl chloride via a microdialysis cannula placed in the mediodorsal nucleus of the thalamus. The rats were allowed a recovery period of at least 20 h after surgery before being coupled to the perfusion pump. Cannula implantation with or without 4 h of Ringer perfusion caused hardly any detectable c-fos expression in the brain, but 20 min of bicuculline (0.1 mM) perfusion induced high levels of c-fos messenger RNA and Fos protein expression in the area adjacent to the dialysis membrane, indicating activated thalamic neurons. In situ hybridization as well as immunohistochemical analysis of the frontal cortical areas and limbic structures showed a rapid, specific and transient c-fos expression in the medial and lateral prefrontal cortex, nucleus accumbens, mediodorsal striatum, claustrum, nucleus reticularis of the thalamus and amygdala. The overall spatial distribution of the c-fos response was comparable to the innervation patterns of thalamic efferents known from anatomical tracing experiments. The rats were perfused with Ringer while asleep, but they woke up during treatment with bicuculline and displayed an increase in general behavioural activity, which could be correlated to the amount of bicuculline measurable in the dialysate. Pathological behaviours, such as epilepsy, were not noticeable during bicuculline treatment. These results show that it is possible to selectively activate defined anatomical pathways by pharmacological application of drugs using microdialysis in unanesthetized unrestrained animals and to visualize the transsynaptically activated target neurons of these projections. We conclude that this novel experimental approach is indeed suitable for studying functional anatomical pathways.

The monoclonal antibody Alz-50, used to reveal cytoskeletal changes in Alzheimer's disease, also reacts with a large subpopulation of somatostatin neurons in the normal human hypothalamus and adjoining areas.

The monoclonal antibody Alz-50 is directed against Alzheimer's disease-related modified tau proteins and reveals cytoskeletal changes, i.e. neurofibrillary tangles and dystrophic neurites. The present study shows that, in the hypothalamus of non-demented control subjects, this same antibody gives a distinctive staining pattern of a subpopulation of somatostatin neurons and beaded fibres. Furthermore, Alz-50 occasionally recognizes somatostatin-containing cell bodies and dystrophic neurite-like fibers in the (neuritic) senile plaques of AD patients. These observations have direct consequences for the interpretation of Alz-50 staining in diagnostic usage and for the assessment of Alzheimer's disease-like changes induced by beta-amyloid in experimental animal brains. On dot spotting, Alz-50 was found to bind to a number of fragments from the somatostatin precursor, of which somatostatin 15-28 stained best. Preadsorption of Alz-50 by somatostatin 15-28, as well as other specificity tests, failed, however, to provide any clue to the nature of the unknown compound(s) stained in the control hypothalamus.

Localization of corticotropin-releasing hormone (CRH) neurons in the paraventricular nucleus of the human hypothalamus; age-dependent colocalization with vasopressin.

Immunocytochemical staining, using a monoclonal antibody against corticotropin-releasing hormone, was performed on hypothalami of 13 human subjects between 23 and 91 years of age who had not suffered from a primary neurological or psychiatric disease. Corticotropin-releasing hormone (CRH) immunoreactivity was present in neurons of the paraventricular nucleus (PVN) and in their fibers running to the median eminence. The CRH-positive neurons were scattered throughout the PVN, but in the rostral part relatively few cells were present. There were large individual differences in the number and staining intensity of CRH neurons in the PVN and in the staining intensity of the median eminence. These differences seemed not to be attributed to age, sex, postmortem delay, fixation time or hour of death. In the rat, too, no relationship was found between a postmortem delay of up to 24 h and CRH staining intensity of the median eminence. Since the distribution of CRH-immunoreactive neurons in the human PVN strongly overlap with vasopressin, colocalization of these peptides was investigated in a double label study and indeed found in subjects ranging between 43 and 91 years of age. However, cells staining for only one of the peptides were also observed. The vasopressin cells had a mean cellular profile area which was 2.3 times as large as the CRH cells and 2.2 times as large as the CRH and vasopressin containing neurons. In younger subjects (23-37 years of age) no colocalization of the two peptides was seen. The age-dependent colocalization of CRH with vasopressin is interpreted as a sign of increased activation of the CRH neurons with age.

Fc-mediated nonspecific staining of the porcine brain with rabbit antisera in immunocytochemistry is prevented by pre-incubation of the sera with proteins A and G.

Nonspecific staining was detected in immunocytochemical procedures on the porcine hypothalamus with rabbit antisera, irrespective of the antigen specificity of the sera, in magnocellular neurons of the paraventricular (PVN) and supraoptic nuclei (SON), and in the vasopressin- and oxytocin-containing nucleus (VON). The present study was designed to test the hypothesis that this staining is mediated by the Fc portion of rabbit immunoglobulins. Rabbit antisera against neuropeptides localized predominantly outside the PVN, SON, and VON were employed in combination with different detection methods. The intensity of the nonspecific staining varied depending on the antiserum and persisted after pre-absorption of the antisera with their homologous peptides. Nonspecific staining and antigen-specific staining were differentially affected by the method of tissue fixation. The nonspecific staining could be prevented by preincubation of the antisera with proteins A and G, which left the antigen-specific staining intact, whereas additional preabsorption with homologous peptide abolished all staining. These observations suggest that the Fc region of IgGs is indeed involved in the nonspecific staining. On press-blots of homogenates from SON tissue subjected to isoelectric focusing, one band in the low-pH region was found with all antisera. Pre-incubation of the antisera with protein A abolished the staining of this band but did not affect staining of antigen-specific bands. Pre-incubation with proteins A and G is proposed as a routine control to check for nonspecific staining mediated by the Fc region of IgGs in immunocytochemical procedures, particularly those that employ rabbit sera in porcine brain.

Vasopressin and angiotensin II are absent but spontaneously reappear in solitary hypothalamic neurons of the homozygous Brattleboro rat.

The homozygous Brattleboro rat (di/di) synthesizes a vasopressin (VP) precursor with a different C-terminus, which is not packaged in granules. In addition, the expression of a coexisting peptide, angiotensin II (Ang II), is disturbed. During postnatal life a small but increasing number of solitary post-mitotic hypothalamic neurons of the di/di rat undergoes a switch to a genuine heterozygous phenotype. Here we report the reappearance of Ang II in these heterozygous cells, which suggests that for the expression of Ang II a normal VP precursor is required. Based upon the present study and literature data it is proposed that at the level of the endoplasmic reticulum a compartmentalization of the synthesis of various peptide precursor occurs.

Increased vasopressin immunoreactivity in the rat brain after a postmortem interval of 6 hours.

Enhanced immunocytochemical staining of vasopressin-containing neurons was observed after incubation of rat brain slices in Ringer medium for 6 h at room temperature, as compared to brain tissue fixed immediately after death. Hypothalamic vasopressin neurons in the supraoptic nucleus, the paraventricular nucleus and the suprachiasmatic nucleus stained more intensely after a postmortem interval of 6 h. Extrahypothalamic vasopressin neurons (VP) in the bed nucleus of the stria terminalis, the medial amygdala and the locus coeruleus proved to be stained as well. Extrahypothalamic VP neurons in the locus coeruleus could, until now, only be visualized after in vivo pretreatment with colchicine. In addition, staining was observed at two new sites, the dorsal raphe nucleus and the lateral septum. Staining of VP was corroborated by application of different antibodies directed against the intact vasopressin molecule as well as by antibodies directed against the other parts of the vasopressin precursor molecule, i.e. neurophysin and glycopeptide. The specificity of the VP-staining was validated by using pre-immune serum and using Brattleboro rat brain tissue, resulting in a negative staining in both cases. Furthermore, homogenated punches of the suprachiasmatic nucleus were submitted to iso-electric focussing on polyacrylamide gel, followed by press blotting and subsequent immunocytochemical staining for vasopressin. Iso-electric foccussing enabled us to characterize and quantify peptides in the suprachiasmatic nucleus. The vasopressin content increased 6 h postmortem, while c-terminal glycopeptide and neurophysin levels remained stable. Similar results were observed in the suprachiasmatic nucleus from decapitated rats whose brains were left intact in the skull for 6 h at room temperature.

Immunochemical detection of peptides and proteins on press-blots after direct tissue gel isoelectric focusing.

A sensitive method is described for the detection of tissue peptides and proteins. They are separated by tissue isoelectric focusing using thin large-pore polyacrylamide gels, containing detergent and dimethylformamide, and are fixed with either glutaraldehyde or formaldehyde in gelatin-coated nitrocellulose membranes using press-blotting. The fixed peptide and protein antigens are visualized by immunoperoxidase staining. The spectrum of fixed tissue constituents may also be used to test antiserum reactivity and specificity in immunocytochemical staining procedures. Isoelectric focusing of 2 microL homogenates of the neurointermediate lobe of the pituitary allowed the immunodetection of peptides and proteins of various sizes and the determination of isoelectric points. However, direct application onto gels of small pieces of frozen tissue sections, sliced in a cryostat, appeared to be more efficient. By direct tissue isoelectric focusing of brain tissue, peptides were effectively eluted and separated from sections up to 100 microns thickness. This allowed the detection of small peptides with a detection limit of approximately 10 pg/section.

Mesotocin and vasotocin in the brain of the lizard Gekko gecko. An immunocytochemical study.

The distribution of mesotocin and vasotocin was studied in the brain of the lizard Gekko gecko with antisera specific for either peptide. Both mesotocinergic and vasotocinergic perikarya are found in the paraventricular and supraoptic nuclei of the hypothalamus, whereas vasotocinergic neurons are exclusively present in the bed nucleus of the stria terminalis and in a cell group of the rhombencephalon. The distributional pattern of the mesotocinergic fibers corresponds closely to that of the vasotocinergic fibers. However, throughout the entire brain the mesotocinergic innervation is less dense than the vasotocinergic innervation. No sex differences are present in the mesotocinergic fiber system.