Sexually dimorphic immunoreactivity of galanin and colocalization with arginine vasotocin in the chicken brain (Gallus gallus domesticus).

The bed nucleus of the stria terminalis medialis (BSTM) of adult chickens (Gallus gallus domesticus) was previously shown to synthesize arginine vasotocin (AVT) in males only and coincides spatially and temporally with steroid activity regulating male reproductive behavior. Galanin has been shown to be a potent modulator of the behavioral and neuroendocrine responses in the mammalian BSTM and in other sexually dimorphic brain regions. In the present study of adult chickens the morphological relationship of AVT and galanin was examined by immunohistochemical analysis of two limbic structures, the BSTM and the lateral septum (SL). The analysis also included the hypothalamic nuclei supraopticus (SON) and paraventricularis (PVN). In males galanin and AVT were both synthesized in the BSTM, while in females neither galanin nor AVT was present. Furthermore, in the males galanin and AVT were colocalized in the majority of neurons within BSTM and in fibers of the SL. In both sexes galanin neurons in the PVN were scattered between the distinct clusters of AVT neurons and there was no colocalization of galanin and AVT in single PVN neurons. Furthermore, AVT immunoreactivity was significantly higher in the SON than in the PVN in both sexes. In the SON, galanin was colocalized with AVT in significantly more neurons in hens than in males (P

The use of heat-induced hydrolysis in immunohistochemistry on angiotensin II (AT1) receptors enhances the immunoreactivity in paraformaldehyde-fixed brain tissue of normotensive Sprague-Dawley rats.

The research on components of the renin-angiotensin system delivered a broad image of angiotensin II-binding sites. Especially, immunohistochemistry (IHC) provided an exact anatomical localization of the AT(1) receptor in the rat brain. Yet, controversial results between in vitro receptor autoradiography and IHC as well as between immunohistochemical studies using various antisera started a vehement discussion concerning specificity and cross-reactivity of these antisera. In particular the magnocellular subdivision of the paraventricular nucleus (PVN) and the supraoptic nucleus (SON) provided controversial results on the localization of AT(1) receptors. Both areas are known for angiotensin II-induced release of vasopressin (VP) and oxytocin (OXT). To evaluate the significance of the appropriate method of antigen retrieval and its relevance for the detection of AT(1) receptors we performed IHC on AT(1) receptors in paraformaldehyde-fixed and paraffin-embedded brain tissue of Sprague-Dawley rats using either the detergent Triton X-100 or microwave oven heating. This study demonstrates that heat-induced hydrolysis enhances the quality and quantity of immunoreactivity (IR) in IHC on AT(1) receptors. In the organum vasculosum lamina terminalis and in the parvocellular subdivisions of the PVN we report a distribution of AT(1)-like-IR similar to that observed with other methods. However, in addition, we provide evidence that distinct AT(1)-like-IR is also localized in few magnocellular neurons of the PVN and in few parvocellular neurons of the dorsal SON but not in magnocellular neurons of the SON. Moreover, parallel IHC indicates that few magnocellular OXT- or VP-releasing neurons of the PVN as well as parvocellular OXT-releasing neurons of the SON do also contain AT(1) receptors.

Nitric oxide of the supraoptic nucleus influences the salivary secretion, sodium renal excretion, urinary volume and arterial blood pressure induced by pilocarpine.

Male Holtzman rats weighting 200-250 g were anesthetized with zoletil 50 mg/Kg (tiletamine chloridrate 125.0 mg and zolazepan chloridrate 125.0 mg) into quadriceps muscle and stainless steel cannulas were implanted into their supraoptic nucleus (SON). We investigated the effects of the injection into the supraoptic nucleus (SON) of FK 409, a nitric oxide donor, and NW-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor (NOS), on the salivary secretion, arterial blood pressure, sodium excretion and urinary volume induced by pilocarpine, which was injected into SON. The drugs were injected in 0.5 microl volume over 30-60 s. Controls was injected with a similar volume of 0.15 M NaCl. FK 409 and L-NAME were injected at doses of 20 microg/0.5 microl and 40 microg/0.5 microl respectively. The amount of saliva secretion was studied over a five-minute period after injection of pilocarpine into SON. Injection of pilocarpine (10, 20, 40, 80, 160 microg/microl) into SON produced a dose-dependent increase in salivary secretion. L-NAME was injected into SON prior to the injection of pilocarpine into SON, producing an increase in salivary secretion due to the effect of pilocarpine. FK 409 injected into SON attenuating the increase in salivary secretion induced by pilocarpine. Mean arterial pressure (MAP) increase after injections of pilocarpine into the SON. L-NAME injected into the SON prior to injection of pilocarpine into SON increased the MAP. FK 409 injected into the SON prior to pilocarpine attenuated the effect of pilocarpine on MAP. Pilocarpine (0.5 micromol/0.5 microl) injected into the SON induced an increase in sodium and urinary excretion. L-NAME injected prior to pilocarpine into the SON increased the urinary sodium excretion and urinary volume induced by pilocarpine. FK 409 injected prior to pilocarpine into the SON decreased the sodium excretion and urinary volume induced by pilocarpine. All these roles of pilocarpine depend on the release of nitric oxide into the SON. In summary the present results show: a) SON is involved in pilocarpine-induced salivation; b) that mechanism involves increase in MAP, sodium excretion and urinary volume.

Oestrogen receptor-beta and neurohypophysial hormones: functional interaction and neuroanatomical localisation.

Oestrogens affect fluid balance, influencing both ingestive behaviour and renal excretion. The renal effects are partly due to altered release of vasopressin and oxytocin. This study was designed to explore the role of oestrogen receptor-beta (ERbeta) in neurohypophysial hormonal function. Following dietary administration, soya isoflavones reach the brain in sufficient concentration to activate ERbeta, but not oestrogen receptor-alpha (ERalpha). ERbeta function was therefore manipulated by feeding rat diets differing in soya isoflavone content. Fluid balance and neurohypophysial hormone release were measured in male rats maintained for 14 days on a soya isoflavone-free diet or one containing 150 microg/g genistein+daidzein. Food and water intake, body weight, urine flow, osmolality and sodium concentrations were determined daily. After 14 days, plasma and urine osmolality and sodium, vasopressin and oxytocin concentrations were determined. There was no significant difference in weight gain between the two groups or in their excretion of sodium and water or plasma sodium and plasma oxytocin. However, plasma vasopressin was significantly lower in the iso-free group. Double-label immunocytochemistry was used to assess colocalisation of ERbeta with the neurohypophysial hormones in male rats. Cell nuclei showing ERbeta immunoreactivity were abundant in the posterior magnocellular paraventricular nucleus (PVNpm) and in the supraoptic nucleus (SON). Vasopressin-immunoreactive neurones were similarly distributed, forming the core of the PVNpm and the ventral portion of the SON; majority were positive for ERbeta. Cells with oxytocin immunoreactivity were located mainly at the periphery of the PVNpm and in the dorsal SON; only approximately a quarter of these cells showed ERbeta immunoreactivity. Thus, the difference in the effects of the soya diet on vasopressin and oxytocin release may be related to the ERbeta-activating properties of this diet and to the preponderance of this receptor in vasopressin as opposed to oxytocin cells.

Functional and anatomic relationship between cholinergic neurons in the median preoptic nucleus and the supraoptic cells.

The median preoptic nucleus (MePO) has been suggested to be an important area in the brain for the regulation of vasopressin (VP) release under the condition of osmotic stimulation. Fos immunoreactivity (Fos-ir), choline acetyltransferase (ChAT) immunoreactivity and retrograde labeling with fluoro-gold were used in this study to determine whether cholinergic neurons in the MePO can be activated by hypertonic NaCl, and to characterize the specific MePO cells that have anatomic projections to the supraoptic nuclei (SON). The results showed that c-fos expression specifically induced by hypertonic NaCl was found in the ChAT cells of the MePO. A retrograde tracing experiment demonstrated that the MePO neurons projecting to the SON were cholinergic. In addition, hypertonic saline-induced Fos-ir was colocalized with the MePO neurons back labeled with fluoro-gold from the SON. Together, these data provide evidence that the MePO cholinergic neurons are activated by osmotic stimulation, and suggest that cholinergic cells in the MePO are functionally important in the control of the SON neurons under the condition of hypertonic stimulation.

Localization of putative glutamatergic/aspartatergic neurons projecting to the supraoptic nucleus area of the rat hypothalamus.

Oxytocin and vasopressin neurosecretory neurons of the supraoptic nucleus receive a rich glutamatergic innervation. The nerve cells of this prominent structure express various ionotropic and metabotropic glutamate receptor subtypes and there is converging evidence that glutamate acts as an excitatory transmitter in the control of release of oxytocin and vasopressin synthesized in this cell group. The location of the glutamatergic neurons projecting to this hypothalamic region is unknown. The aim of the present investigation was to study this question. [(3)H]D-aspartate, which is selectively taken up by high-affinity uptake sites at presynaptic endings that use glutamate as a transmitter, and is transported back to the cell body, was injected into the supraoptic nucleus area. The neurons retrogradely labelled with [(3)H]D-aspartate were detected autoradiographically. Labelled nerve cells were found in several diencephalic and telencephalic structures, but not in the brainstem. Diencephalic cell groups included the supraoptic nucleus itself, its perinuclear area, hypothalamic paraventricular, suprachiasmatic, ventromedial, dorsomedial, ventral premammillary, supramammillary and thalamic paraventricular nuclei. Within the telencephalon, labelled neurons were detected in the septum, amygdala, bed nucleus of the stria terminalis and preoptic area. The findings provide neuromorphological data on the location of putative glutamatergic neurons projecting to the supraoptic nucleus and its perinuclear area. Furthermore, they indicate that local putative glutamatergic neurons as well as several diencephalic and telencephalic structures contribute to the glutamatergic innervation of the cell group and thus are involved in the control of oxytocin and vasopressin release by neurosecretory neurons of the nucleus.

Vasopressin and aggression in cross-fostered California mice (Peromyscus californicus) and white-footed mice (Peromyscus leucopus).

To examine how developmental experiences alter neural pathways associated with adult social behavior, we cross-fostered pups between the more aggressive and monogamous California mouse (Peromyscus californicus) and the less aggressive and polygamous white-footed mouse (P. leucopus). Cross-fostered males became more like their foster parents when tested as adults. Male white-footed mice became more aggressive only in an aggression test in a neutral arena, whereas the territorial California mice became less aggressive in resident-intruder aggression test, as measured by attack latency. Only the species that displayed a change in resident-intruder aggression showed a change in arginine vasopressin (AVP) levels: cross-fostered California mice had significantly lower levels of AVP-immunoreactive (AVP-ir) staining than controls in the bed nucleus of the stria terminalis (BNST) and the supraoptic nucleus (SON) and a nonsignificant trend toward lower levels in the medial amygdala (MA). Neither species showed changes in AVP-ir staining in a control area, the paraventricular nucleus (PVN). The changes in AVP-ir staining in the BNST and SON may not be caused by stress because cross-fostering was not associated with changes in adult plasma concentrations of two steroid hormones, corticosterone and testosterone, that have been associated with stress-related alterations in AVP pathways. These results suggest that manipulating the early parental environment can directly alter both a neurotransmitter system and species-typical patterns of social behavior, but that these effects may vary between species and under different social contexts.

Histological and histochemical study of the magnocellular neuroendocrine system in white rat after parenteral administration of adrenaline.

The effect of a single dose of adrenaline on the neurosecretory action of the hypothalamo-hypophyseal system was investigated in 90 male rats. Increased release and transport of neurosecretory material were observed after a low dose of adrenaline, and the enhancement of the neurosecretory process was noted following a high dose. As shown in numerous reports, there is a link between the neuroendocrine system and the hypothalamic adrenergic system.

Electrophysiological and morphological characteristics of neurons in perinuclear zone of supraoptic nucleus.

Electrophysiological and morphological characteristics of neurons in perinuclear zone of supraoptic nucleus. J. Neurophysiol. 78: 2427-2437, 1997. Neurons in the perinuclear zone (PZ) of the supraoptic nucleus (SON) are thought to serve as interneurons and may mediate changes in neurohypophysial hormone release in response to physiological changes in blood pressure. However, the morphology and electrophysiological characteristics of PZ neurons are unknown. In the present study, PZ neurons from male and female rats were recorded intracellularly to determine some membrane properties, then filled with biocytin or biotinamide for morphological analysis. In general, PZ neurons had faster spikes than magnocellular SON neurons, and the great majority were characterized by a subthreshold depolarizing hump when depolarized from a hyperpolarized (less than -80 mV) membrane potential. In most neurons, this hump was similar to low-threshold spikes described in other CNS regions. Near-threshold, fast action potentials were clustered near the onset of these depolarizations. Conspicuously absent in all PZ neurons was the strong transient and subthreshold outward rectification characteristic of vasopressin and oxytocin neurons of the SON. These results suggest that PZ neurons are electrophysiologically distinct from neurosecretory neurons of the SON. No differences were found between male and female rats in any of the basic properties examined, including input resistance, membrane time constant, spike height, spike width, spike threshold, and the size of the spike afterhyperpolarization. Morphologically, PZ neurons were diverse but were divided into spiny and aspiny groups. Three spiny neurons and one aspiny neuron contributed an axonal projection to the SON characterized by varicosities suggestive of terminals. In the case of the three spiny neurons, the SON projection was clearly a minor collateral projection. The axon arborized in the PZ, but one or more branches were cut at the edge of the explant, indicating a longer projection. In the remaining neurons, no axonal projection to the SON was detected and several had axons leaving the explant. Some portion of the dendritic tree penetrated the SON in several neurons. The morphology of PZ neurons was thus heterogeneous and suggests that, for some cells at least, the projection to the SON may be a minor collateral component of a much wider axonal projection.

Immunohistochemical localization of corticotropin-releasing factor, [arginine8]-vasopressin and oxytocin neurons in the goat hypothalamus.

Distribution patterns of corticotropin-releasing factor (CRF), [arginine8]-vasopressin (AVP) and oxytocin (OXY) neurons were examined immunohistochemically in the female goat hypothalamus. The majority of the CRF immunoreactive (-IR) cells were located in the parvocellular part of the paraventricular nucleus (PVN) with smaller population found in the magnocellular part of the PVN. CRF-IR cells were also found in the suprachiasmatic nucleus, the preoptic area and around the fornix in the caudal part of the hypothalamus. AVP- and OXY-IR cells were similarly distributed in the hypothalamus. The majority of AVP- and OXY-IR cells were observed in the magnocellular part of PVN and the supraoptic nucleus. Smaller numbers of AVP- and OXY-IR cells were found in the parvocellular part of the PVN and lateral hypothalamic area. AVP-IR but not OXY-IR cells were located in the suprachiasmatic nucleus. CRF-IR fibers were concentrated in the external palisade zone of the median eminence (ME) with a few fibers found in the internal palisade zone of the ME, whereas AVP- and OXY-IR fibers were concentrated in the internal palisade zone of the ME with a few fibers found in the external zone. These results support the view that not only CRF but also AVP and OXY are released into the hypophysial portal blood and involved in the control of pituitary endocrine function in ruminant species.

Semithin cryosections as a tool to perform high resolution immunofluorescence and in situ hybridization analysis of the nervous tissue: a study in the supraoptic nucleus.

Immunofluorescence and fluorescence in in situ hybridization represent powerful approaches to correlate biochemical and molecular data with the structural organization of cells and tissues. However, the analysis of tissues by fluorescence microscopy is limited by the fact that most methods currently used to preserve the morphological integrity of sectioned samples at high resolution do not allow access of the labeled probes to the target molecules. Here we have made use of semithin cryosections obtained from rat supraoptic nucleus to perform immunofluorescence with antibodies directed against cytoplasmic and nuclear antigens, as well as fluorescence in situ hybridization with antisense oligonucleotide probes complementary to the poly(A) tail of mRNA and to specific mRNAs. In addition, DNA was visualized by incubation of sections with digoxigenin-labeled nucleotides in the presence of Escherichia coli DNA polymerase I. The high resolution of this DNA staining in combination with immunolabeling for nuclear antigens provides a powerful tool to analyze the structural and functional compartmentalization of neuronal cell nuclei. The major conclusion from this study is that performing fluorescence microscopy on 1 micron-thick cryosections provides an important tool to accurately localize proteins, DNA and RNA within nervous tissue in general and particularly in the model of supraoptic nucleus. Moreover, the cryosectioning technique appears particularly suited to the study of the localization of specific mRNA species in the neuronal cytoplasm and represents a useful approach to addressing the functional significance of mRNA localization in protein targeting.

Vasopressin- and oxytocin-immunoreactive nerve cells in the aging rat hypothalamus.

Immunohistochemistry and morphometry were used to study the age-related changes in the vasopressin (AVP) and oxytocin (OXT) nerve cells in the paraventricular (PVN), supraoptic (SON) and suprachiasmatic (SCN) nuclei of 3-, 11- and 28-month-old rats. The results showed a statistically significant reduction in the mean number of AVP cells in the PVN, SON and SCN, and of OXT cells in the PVN with advancing age. Different age-related changes in the mean size of the immunoreactive cells were found in the three nuclei: a significant and transitory increase in the AVP and OXT cell sizes in the PVN, a tendency towards increasing the AVP and OXT cell sizes in the SON, and a significant and gradual decrease in the AVP cell size in the SCN. The combination of the morphometric data and staining patterns of the AVP and OXT perikarya and fibers in the PVN and SON pointed to an increased transport of AVP and OXT in 11-month-old rats as well as to a decreased production of these peptides in the PVN of 28-month-old rats. Taken together the staining pattern and the morphometric results showed a progressive loss of AVP cells in the SCN in aging.

Antidiuretic effects of ATP induced by microinjection into the hypothalamic supraoptic nucleus in water-loaded and ethanol-anesthetized rats.

The effects of microinjection of purinoceptor agonists into the hypothalamic supraoptic nucleus (SON) on urination were examined in water-loaded and ethanol-anesthetized rats. Adenosine triphosphate (ATP), but neither adenosine diphosphate (ADP), adenosine monophosphate (AMP) nor adenosine, concentration-dependently decreased the urine outflow with concomitant increase in the urine osmotic pressure. The ED50 value for ATP was approx. 60 nmol. The antidiuretic effect of ATP was blocked either by prior injection of theophylline (an antagonist of the P1-type purinoceptor) or by intravenous administration d(CH2)5-D-Tyr(Et)-valine-arginine-vasopressin (VAVP). These results suggest that ATP injected into the SON has antidiuretic effects due to release of AVP through an activation of theophylline-sensitive purinoceptors.

Retinohypothalamic projections and immunocytochemical analysis of the suprachiasmatic region of the desert iguana Dipsosaurus dorsalis.

Two separate and distinct retinal projections to the hypothalamus in the iguanid lizard Dipsosaurus dorsalis were described using horseradish peroxidase and cobalt-filling techniques. Both of the projections were unilateral and completely crossed; one terminated in the supraoptic nucleus and the other in the suprachiasmatic nucleus. Immunocytochemical analysis showed that the supraoptic nucleus contained cell bodies and fibers that cross-react with antibodies raised against arginine vasopressin, while the suprachiasmatic nucleus contained arginine vasopressin-like immunoreactive fibers emanating from cells in the nearby paraventricular nucleus. The suprachiasmatic nucleus contained a dense plexus of fibers that cross-reacted with neuropeptide-Y antibody. Antiserum against vasoactive intestinal polypeptide showed no reactivity in any part of the forebrain, while antiserum against serotonin showed sparse and uniform reactivity throughout the forebrain, including the suprachiasmatic nucleus. These results, together with other data, indicate that the suprachiasmatic nucleus of D. dorsalis is homologous to the suprachiasmatic nuclei of rodents, structures known to contain circadian pacemakers. We suggest that the suprachiasmatic nucleus may play a similar role in the circadian system of D. dorsalis.

Evidence for reciprocal connections between the dorsochiasmatic area and the hypothalamo neurohypophyseal system and some related extrahypothalamic structures.

In the preceding article, a dorsochiasmatic area (DCh) was described that projects to both paraventricular (PVN) and supraoptic (SON) nuclei. The main afferents of the DCh, revealed by local injections of retrograde tracers, are the hypothalamic PVN and SON, lateral septal nuclei (LSV and SHy), bed nuclei of the stria terminalis (BST), anteroventral third ventricle region, particularly the median preoptic nucleus (MnPO), the subfornical organ, medial preoptic areas, arcuate hypothalamic nucleus, ventromedial hypothalamic nuclei, paraventricular thalamic nucleus, and, more caudally, several structures of the posterior hypothalamus and mesencephalon. The relations between DCh and BST, LSV, SHy, or MnPO appeared reciprocal. In view of their reciprocal relationships with the hypothalamo-neurohypophyseal system and some of their related extrahypothalamic structures, the DCh might be involved in the regulation of the vasopressin (AVP) and/or oxytocin (OT) systems, or in reproductive behavior.

Ontogeny of arginine vasopressin-immunoreactive neurons in the hypothalamus of fetal and newborn sheep.

Arginine vasopressin (AVP) is a peptide hormone which is found in neurons within the paraventricular (PVN) and the supraoptic (SON) nuclei of the hypothalamus. In fetal sheep, this neuropeptide is involved in maturational processes and adaptive responses to 'stress'. This study examined the effect of age on the total number and distribution of AVP-containing neurons in the PVN and SON of fetal sheep and newborn lambs by quantitative light-microscopic immunocytochemistry. Serial coronal sections of hypothalami from three groups of animals were studied: fetuses at 104-109 days of gestation (n = 6) comprising the early group, fetuses at 130-139 days of gestation (n = 5) comprising the late group and newborn lambs at 12-20 postnatal days (n = 5) comprising the neonatal group. This period of development was chosen since adaptive mechanisms to stress are operative at or near the time of birth. Hypothalamic dimensions were measured to determine if maturation had an effect on the size of the AVP-containing subregions of the hypothalamus during this period of development. Dimensions included: ventricle height, optic chiasm width, distances from the dorsal margin of the ventricle to the lateral and medial margins of the optic tract, and distance between the medial margins of the optic tracts. As expected, with increase in maturational age, overall dimensions of the AVP-containing subregions increased significantly (p < 0.05). When early- and late-gestation fetuses were compared to newborn lambs, there was a significant increase in the total number of immunoreactive neurons in both the PVN (p < 0.01, Anova) and SON (p < 0.001, Anova) with age. With advancing age, we also observed an increase in the density of AVP neurons in the middle subregion of the PVN and in the midrostral subregion of the SON. These data suggest that, during the late gestational and early postnatal period, de novo synthesis of AVP genes occurs in these hypothalamic nuclei. This study provides a baseline for further investigation to study the effects of stress on these neurons in the developing ovine fetus and newborn lamb.

Infrequent cellular coexistence of NADPH-diaphorase and calretinin in the neurosecretory nuclei and adjacent areas of the rat hypothalamus.

Colocalization of the calcium-binding protein calretinin and NADPH-diaphorase activity at the cellular level was studied in the magnocellular secretory nuclei of the rat hypothalamus using sequential immunocytochemical and histochemical staining of the same sections. A low degree of colocalization of these markers was observed in certain cellular subpopulations within all the areas considered (supra-optic, paraventricular, circular and both fornicals nuclei and in the hypothalamic area located between the supraoptic and paraventricular nuclei). However, since in the paraventricular nucleus both markers were expressed by different neuronal populations, the coexistence was almost non-existent in some subdivisions of this nucleus. This rare coexistence strongly suggests that NADPH-diaphorase and calretinin are related to different functions shared by restricted hypothalamic neuronal populations.

Immunohistochemical localization of calretinin-, calbindin-D28k- and parvalbumin-containing cells in the hypothalamic paraventricular and supraoptic nuclei of the rat.

The localization of three calcium-binding proteins, calretinin, calbindin-D28k and parvalbumin, in the hypothalamic paraventricular and supraoptic nuclei of the rat was immunohistochemically examined on adjacent sections and their distribution patterns were compared. Overlap between the distribution of calretinin-immunoreactive cells and that of calbindin-D28k-immunostained cells was found in the rostrodorsal part of the supraoptic nucleus, and the caudoventral part of this nucleus contained predominantly calbindin-D28k-stained cells. Cells of the medial and lateral magnocellular subdivisions of the paraventricular nucleus were almost devoid of the calcium-binding proteins examined. No parvalbumin-immunostained cells were observed in either nucleus. This study provides a further characterization of cell bodies in the hypothalamic paraventricular and supraoptic nuclei.

Extremely high titre polyclonal antisera against small neurotransmitter molecules: rapid production, characterisation and use in light- and electron-microscopic immunocytochemistry.

We have produced polyclonal antibodies against the small amino acid neurotransmitters, GABA, glutamate, glycine and taurine, with a simple new technique using antigens co-adsorbed with an adjuvant peptide to gold particles, which causes rapid and massive immune responses in all animals that we have studied. These antibodies are all of extremely high titre; they are typically used in immunocytochemistry at dilutions from 1 in 250,000 to 1 in 1,000,000 which represents an increase in titre of at least two orders of magnitude compared to standard antibody production techniques. Such very high dilutions result in minimal background labeling and a high signal-to-noise ratio when applied to sections of aldehyde-fixed, epoxy resin-embedded tissues at both light- and electron-microscopic levels. Each antibody displays minimal cross-reactivity with other neurotransmitter molecules. We suggest that our technique may be broadly applicable for raising antibodies against a wide variety of antigens of interest to neuroscientists, particularly those that normally elicit weak immune responses. The technique may also assist in clonal expansion prior to generation of monoclonal antibodies and may be viable, with modifications, for use in human immunisations.

Local origins of some GABAergic projections to the paraventricular and supraoptic nuclei of the hypothalamus in the rat.

Axonal transport and immunohistochemical methods were used to characterize the organization of glutamic acid decarboxylase-immunoreactive (GAD-ir) projections to the paraventricular (PVH) and supraoptic (SO) nuclei of the hypothalamus in the rat. In line with prior reports, GAD-ir varicosities were found to be densely and quite uniformly distributed throughout the hypothalamus, including the PVH and the SO. Nonetheless, the periventricular part of the PVH was consistently found to contain a disproportionately high density of GAD-ir elements. Small crystalline implants of the retrograde tracer, true blue, into the PVH labeled GAD-ir cells in the anterior perifornical region, portions of the anterior hypothalamic area immediately ventral to the PVH, a region just dorsal to the rostral SO and extending caudomedially over the optic chiasm and tract, and within the anterior one-third of the PVH itself. Because possible uptake of retrograde tracer by local dendritic processes might have yielded false positive filling of nearby GAD-ir cells, anterograde transport, Phaseolus vulgaris-leucoagglutinin, and combined anterograde transport-immunohistochemical methods were used to attempt to confirm these four putative local sources of GAD-ir inputs. Tracer injections in each of the above mentioned regions labeled sparse to moderate axonal projections to the PVH, which ramified preferentially in the parvicellular division of the nucleus. Projections to the magnocellular division of the PVH and the SO were generally sparse and inconsistently observed in this material. A variable, and generally small, proportion of anterogradely labeled axons and terminals in the PVH also displayed GAD-ir. These results suggest that GABAergic projections to visceromotor cell types in the PVH and SO arise, at least in part, from several diffusely distributed local sources. The fact that these afferents were found to terminate preferentially in the parvicellular division of the PVH makes it likely that additional sources of GABAergic projections to the magnocellular neurosecretory system remain to be identified. Peri- and intranuclear GABAergic neurons could provide an intermediary by which documented (and generally inhibitory) limbic system influences on neuroendocrine function are exerted.