Guinea Pig Brain Sections Research Articles

A Forgotten Principle in Immunocytochemistry: Optimal Dilution.

Immunocytochemical (ICC) techniques are frequently used in basic and clinical research. Here, we focus on the importance of using antisera/antibodies at optimal dilutions to achieve specificity and reduce costs. Unfortunately, the basic principle, the necessity to test method specificity of the staining by a series of increasing dilutions of primary antiserum/antibodies, is only occasionally seen in papers using ICC. Many researchers rely on the company's information or others' published data. In this study, we show examples with monoclonal antibodies used in the peroxidase-based ICC technique in mouse and guinea pig brain sections. We show images of ICC staining of phospho-S129 alpha-synuclein in A53T mice and NeuN in guinea pig brains and demonstrate that optimal staining with them can be achieved at least at two to three orders of magnitude higher dilutions than generally used in the literature. We strongly recommend that when antisera/antibodies are used for the first time in any laboratory, independent of what the manufacturer or vendor recommends or are found in the literature, a dilution curve should be set up to identify the optimal dilution. This practice provides not only the highest specificity but is also an economic approach.

Immunolocalization of MAP-2 in Routinely Formalin-Fixed, Paraffin-Embedded Guinea Pig Brain Sections Using Microwave Irradiation: A Comparison of Different Combinations of Antibody Clones and Antigen Retrieval Buffer Solutions

The present study was designed to evaluate the efficacy of different microwave pretreatment methods to retrieve microtubule-associated protein 2 (MAP-2) immunoreactivity in formalin-fixed, paraffin-embedded guinea pig brain sections. Brain sections, microwave pretreated in boiling sodium citrate, citric acid, Tris hydrochloride, and EDTA buffers of pH 4, 6, and 8, were labeled with four different clones of MAP-2 monoclonal antibodies. No MAP-2 immunoreactivity was observed in control sections processed without microwave pretreatment. Optimal MAP-2 immunoreactivity was observed only when MAP-2 antibody clone AP18 was used in conjunction with citric acid buffer of pH 6.0. Using this combination, brain sections from nerve agent soman-exposed guinea pigs were found to exhibit marked reduction in MAP-2 immunostaining in the hippocampus. These observations suggest that the clone of the antibody in addition to the type and pH of antigen retrieval (AR) solution are important variables to be considered for establishing an optimal AR technique. When studying counterpart antigens of species other than that to which the antibodies were originally raised, different antibody clones must be tested in combination with different microwave-assisted AR (MAR) methods. This MAR method makes it possible to conduct retrospective studies on archival guinea pig brain paraffin blocks to evaluate changes in neuronal MAP-2 expression as a consequence of chemical warfare nerve agent toxicity.

Localization of neuropeptide Y receptor Y5 mRNA in the guinea pig brain by in situ hybridization

Neuropeptide Y (NPY) has prominent stimulatory effects on food intake in virtually all animals that have been studied. In mammals, the effect is primarily mediated by receptors Y1 and Y5, which seem to contribute to different aspects of feeding behavior in guinea pigs and rats/mice. Interestingly, differences in receptor distribution among mammalian species have been reported. To get a broader perspective on the role of Y5, we describe here studies of guinea pig ( Cavia porcellus), a species which due to its phylogenetic position in the mammalian radiation is an interesting complement to previous studies in rat and mouse. Guinea pig brain sections were hybridized with two 35S-labeled oligonucleotides complementary to Y5 mRNA. The highest expression levels of Y5 mRNA were observed in the hippocampus and several hypothalamic and brain stem nuclei implicated in the regulation of feeding, such as the paraventricular, arcuate and ventromedial hypothalamic nuclei. This contrasts with autoradiography studies that detected low Y5-like binding in these areas, a discrepancy observed also in rat and human. Y5 mRNA expression was also seen in the striatum, in great contrast to mouse and rat. Taken together, these data show that Y5 mRNA distribution displays some interesting species differences, but that its expression in feeding centers seems to be essentially conserved among mammals, adding further support for an important role in food intake.

Microwave pretreatment of sections to improve the immunocytochemical detection of progesterone receptors in the guinea pig hypothalamus

The goal of this study was to evaluate the effects of microwave heat on immunocytochemical staining for progesterone receptors (PR) in frozen sections and in paraffin-embedded sections of guinea pig brain using an antibody previously shown to successfully stain only frozen sections of formalin-fixed materials. A simple, reliable and sensitive retrieval method has been adapted based on microwave irradiation of sections in citrate solution prior to incubation with the anti-PR antibody. Different pH values (3.0, 6.0 and 8.5) of the citrate buffer were tested and the pH at 6.0 was found to give the best results. PR-like immunoreactivity was markedly increased in frozen sections exposed to microwave heating compared with untreated tissue. The integrity of the tissue was not affected by this treatment and specific nuclear immunoreactivity for PR was detected in hypothalamic neurons. Staining of paraffin-embedded sections without microwave pretreatment yielded negative results while irradiation of sections enabled the labeling of hypothalamic regions known to contain progesterone binding sites. Immunocytochemical staining did not reveal any significant difference in the number of PR-immunoreactive neurons between frozen tissue compared with paraffin-embedded tissue. The results indicate that microwave irradiation can unveil PR antigenicity in sections of paraffin-embedded brain and enhances immunostaining in sections of frozen tissue.

Dimerization of 8-OH-DPAT increases activity at serotonin 5-HT1A receptors.

[35S]GTPgammaS binding responses can be used to measure differences between the intrinsic activity of ligands at human 5-hydroxytrypamine-(1A) (h 5-HT1A) receptors expressed in recombinant cell lines. The maximal [35S]GTPgammaS binding response to 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) was lower than that to 5-HT in a recombinant C6-glial membrane preparation and dependent on the GDP concentration: it was attenuated by about 60% vs 5-HT by increasing the concentration of GDP from 0.3 to 30 and 300 microM. Whereas dimerization of 8-OH-DPAT almost did not affect its potency at h 5-HT1A receptors (pEC50: 7.45 and 7.40 for 8-OH-DPAT and its dimer at 30 microM GDP), it increased efficacy at h 5-HT1A receptors. The maximal response to the 8-OH-DPAT dimer was systematically greater than the response to 8-OH-DPAT and identical to that to 5-HT; moreover in contrast to the 8-OH-DPAT monomer, the maximal response to the dimer was unaffected by increasing the GDP concentration. An enhanced 135S]GTPgammaS binding response (44 to 63% vs 8-OH-DPAT) was also observed in the hippocampus, lateral septum, dorsal raphe and cingulate cortex of guinea-pig brain sections using autoradiography of 5-HT1A receptor-activated G-proteins. Hence, the 8-OH-DPAT dimer shows increased efficacy at 5-HT1A receptors compared to 8-OH-DPAT. The differential regulation of the maximal agonist responses by GDP suggests that the [35S]GTPgammaS binding responses to these two ligands could be mediated by different G-protein subtypes upon activation of the 5-HT1A receptor.

G-protein activation at 5-HT1A receptors by the 5-ht1F ligand LY334370 in guinea-pig brain sections and recombinant cell lines.

1. G-protein activation by the 5-ht1F receptor agonist 5-(4-fluorobenzoyl)amino-3-(1-methylpiperidin-4-yl)-1H-indole fumarate (LY334370) was investigated by use of autoradiography of receptor-activated G-proteins in guinea-pig brain sections and [35S]-GTPgammaS binding responses in cell lines stably expressing human 5-HT1A (h 5-HT1A) receptors. 2. LY334370 (10 microM) caused little or no stimulation of [35S]-GTPgammaS binding in guinea-pig brain regions enriched in 5-ht1F binding sites (e.g., claustrum, caudate/putamen and thalamic nuclei), as identified by labelling with 10 nM [3H]-sumatriptan plus 10 nM 5-carboxamidotryptamine (5-CT). 3. Application of LY334370 (10 microM) to guinea-pig brain sections resulted in an increase of [35S]-GTPgammaS binding in hippocampus (123+/-17%), lateral septum (58+/-14%), dorsal raphe (57+/-10%), entorhinal (37+/-11%) and cingulate cortex (28+/-10%). This distribution fits with the G-protein activation mediated by 5-HT1A receptors as found with lisuride (10 microM), and labelling of 5-HT1A receptors by 140 pM [125I]-4-(2'-methoxy-phenyl)- -[2'-(n-2"-pyridinyl)-p-iodobenzamido]-ethyl-piperazine (p-MPPI). 4. The LY334370-mediated [35S]-GTPgammaS response was antagonized by the selective, silent 5-HT1A receptor antagonist N-[2-[4-(2-methoxyphenyl)1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohex anecarboxa-mide (WAY100635, 1 microM) in each of the brain structures investigated. The distribution pattern of the [35S]-GTPgammaS binding response and the antagonist profile suggest that the LY334370-induced response in guinea-pig brain is mediated by 5-HT1A receptors. 5. The maximal LY334370-induced [35S]-GTPgammaS binding response (83 to 94%) in membranes of recombinant C6-glial/h 5-HT1A and HeLa/h 5-HT1A cells was close to that of 5-HT, suggesting LY334370 to exert high intrinsic activity at h 5-HT1A receptors. 6. In conclusion, in guinea-pig brain sections and recombinant cell lines the 5-ht1F receptor agonist LY334370 causes G-protein activation that is mediated by 5-HT1A receptors. Caution should be taken when employing this ligand as a putative selective 5-ht1F agonist.

5-Hydroxytryptamine1A and 5-hydroxytryptamine1B receptors stimulate [35S]guanosine-5'-O-(3-thio)triphosphate binding to rodent brain sections as visualized by in vitro autoradiography.

[35S]Guanosine-5'-O-(3-thio)triphosphate ([35S]GTPgammaS) binding to G proteins was measured by in vitro autoradiography in guinea pig and rat brain sections after activation by 5-hydroxytryptamine (5-HT) receptor agonists. 5-Carboxamidotryptamine stimulated binding strongly in hippocampus and lateral septum and weakly in substantia nigra. This effect was blocked in the substantia nigra by the 5-HT1B/1D receptor antagonist GR-127,935 and in the former two regions by the 5-HT1A antagonist NAN-190. 5-HT1B/1D receptor agonists stimulated binding in substantia nigra and in areas containing 5-HT1A receptors. In guinea pig substantia nigra, 5-(nonyloxy)-tryptamine maximally stimulated [35S]GTPgammaS binding by 54%, with an EC50 value of 62 nM; at 100 microM, this agonist increased binding by approximately 200% in hippocampus (with a 2-fold weaker EC50 value). The distribution of [3H]8-OH-DPAT binding sites was identical to that of the [35S]GTPgammaS labeling stimulated by the 5-HT1A agonist (R)-8-hydroxy-2-dipropylaminotetralin [(R)-8-OH-DPAT)]. (R)-8-OH-DPAT, (S)-8-OH-DPAT, and buspirone stimulated [35S]GTPgammaS binding in hippocampus by 340%, 140%, and 78%, with EC50 values of 71, 51, and 132 nM. Enhanced [35S]GTPgammaS binding was not detected in the presence of 5-HT1F, 5-HT2, 5-HT4, and 5-HT7 receptor agonists. Because activation of mu-opioid, muscarinic M2, histamine H3, and cannabinoid receptors was also visualized successfully, these data suggest that only receptors coupled to pertussis toxin-sensitive G proteins can be seen by [35S]GTPgammaS binding autoradiography. This study also shows that different 5-HT receptors coupled to these proteins can show a wide range of [35S]GTPgammaS binding stimulation. Although the functional significance of these variations is unclear, this technique offers advantages over receptor autoradiography because it does not require high affinity radioligands and provides a measure of agonist efficacies in various brain regions.

Detailed mapping of the histamine H 2 receptor and its gene transcripts in guinea-pig brain

Autoradiographic studies of the distribution of the histamine H 2 receptor and its messenger RNAs were performed on serial frontal and a few sagittal sections of guinea-pig brain using [ 125I]iodoaminopotentidine for radioligand binding and a 33P-labelled complementary RNA probe for in situ hybridization, respectively. Both probes were validated by assessing non-specific labelling using non-radioactive competing H 2 receptor ligands and a sense probe for binding sites and gene transcripts, respectively. In some areas, e.g., cerebral cortex, hippocampal complex or cerebellum, such studies were completed by identification of neurons expressing the H 2 receptor messenger RNAs on emulsion-dipped sections. Nissl-stained sections from comparable levels were used to localize brain structures. In many brain areas, the distribution of the H 2 receptor and its messenger RNAs appeared to parallel that known for histaminergic axons. For instance, high levels of both H 2 receptor markers were detected in striatal and limbic areas known to receive abundant histaminergic projections. In contrast, in septum, hypothalamic, pontine and several thalamic nuclei, a comparatively low density of both H 2 receptor markers was detected, suggesting that histamine actions in these areas are mediated by H 1 and/or H 3 receptors. Generally, the distribution of H 2 receptor messenger RNA correlates well with that of [ 125I]iodoaminopotentidine binding sites, although some differences were observed. In a few regions (e.g., substantia nigra, locus coeruleus) high or moderate densities of binding sites were accompanied by a much more restricted expression of H 2 receptor transcripts. Conversely, the mammillary region and the pontine nucleus exhibited higher levels of hybridization than of binding sites. In hippocampus, cerebral and cerebellar cortex there was a selective localization of the H 2 receptor messenger RNA in the granule cells of dentate gyrus, pyramidal cells of the Ammon's horn and cerebral cortex, and Purkinje cells of cerebellum, whereas [ 125I]iodoaminopotentidine binding sites were located in layers where the dendritic trees of these messenger RNA-expressing neurons extend. The same discrepancy between messenger RNAs and binding sites suggests that striatonigral endings are endowed with the H 2 receptor. The histamine H 1 and H 2 receptors both appear to be present in several brain areas, in some cases in a way suggesting their potential co-expression by the same neuronal populations, e.g., in granule and pyramidal cells in the hippocampal formation. This co-expression accounts for synergic responses, e.g., on cAMP generation, previously observed upon co-stimulation of both receptor subtypes. The widespread distribution of the H 2 receptor, namely in thalamic nuclei or in telencephalic areas such as most layers of the cerebral cortex, together with its excitatory role previously established in electrophysiological studies, support its alleged function in mediating the histamine-driven control of arousal mechanisms. In addition, the detection of H 2 receptor expression in brainstem areas from which other monoaminergic pathways involved in the control of states of sleep and wakefulness emanate, e.g., several raphe nuclei, locus coeruleus or substantia innominata, suggests possible interrelationships between all of these systems with highly divergent projections to the thalamus and telencephalon. The present mapping of the H 2 receptor and its gene transcripts should facilitate neurochemical, neurophysiological and behavioural studies aimed at clarifying the role of histaminergic systems in brain.

Autoradiographic characterization of binding sites for [ 3H]NE-100 in guinea pig brain

The receptor binding specificity and neuroanatomical distribution of [ 3H]NE-100 (N, N- dipropyl-2- [4- methoxy-3- (2- phenylethoxy) phenyl] ethylamine monohydrochloride)-labeled sigma receptor in guinea pig brain were examined using quantitative autoradiography. NE-100 potently inhibited [ 3H]NE-100 binding to slide-mounted sections of guinea pig brain with the IC 50 value of 1.09 nM, therefore, NE-100 apparently has high affinity binding sites. Competition studies, under conditions similar to those used to visualize the receptor, yielded the following rank order of potency: NE-100 > haloperidol > DuP734 > (+)pentazocine ⪢ (−)pentazocine. Non-sigma ligands such as phencyclidine (PCP), MK-801 and (−)sulpiride had negligible affinities for [ 3H]NE-100 binding sites. High densities of [ 3H]NE-100 binding sites displaceable by haloperidol were present in the granule layer of the cerebellum, the cingulate cortex, the CA3 region of the hippocampus, the hypothalamus and the pons. The distribution of [ 3H]NE-100 binding sites was consistent with that of [ 3H](+)pentazocine, a sigma 1 ligand. These sigma sites may possibly be related to various aspects of schizophrenia.

Distribution of kappa opioid receptor-like immunoreactivity in guinea pig hippocampal formation

The guinea pig hippocampal formation contains components of a functional kappa opioid system: prodynorphin-derived peptides and at least two distinct kappa receptor subtypes, kappa1 (k1) and kappa2 (k2) receptors 1,2. By autoradiography, k1 binding sites are present predominantly in the molecular layer of the dentate gyrus, while k2 binding appears moderately dense throughout the guinea pig hippocampual formation. However, precise localization of hippocampal kappa receptors has not been reported, although such a high-resolution localization is important to better understand hippocampal opioid systems. The purpose of the present study was to determine the ultrastructural localization of hippocampal kappa opioid receptors. To this end, we developed and characterized a novel antiserum against a peptide fragment of a cloned kappa receptor, and determined the light and electron microscopic localization of this kappa receptor-like immunoreactivity in guinea pig brain sections.

Cloning and characterization of a pharmacologically distinct A 1 adenosine receptor from guinea pig brain

Three full-length cDNA clones encoding the guinea pig A 1 adenosine receptor have been isolated by polymerase chain reaction (PCR) and low-stringency hybridization screening of a guinea pig brain cDNA library. These three cDNAs, though differing in their 5′ untranslated regions, contain the same open reading frame encoding a 326 amino acid residue protein with seven hydrophobic α-helices long enough to form the transmembrane domains, suggesting that it belongs to the G protein-coupled receptor superfamily. This protein is highly homologous to the A 1 adenosine receptors previously cloned from other species. Pharmacological characterization of this receptor transiently expressed in mammalian cells demonstrates that, despite its high homology to A 1 adenosine receptors of other species, the guinea pig A 1 adenosine receptor displays a unique pharmacological profile: high affinity for the A 1-selective antagonist CPX, yet very low affinity for some A 1-selective agonists such as CCPA, CHA and R-PIA. Northern blotting for different guinea pig tissues and in situ hybridization for guinea pig brain sections reveal an abundant and broad distribution of mRNA of this A 1 subtype receptor in the brain.

Gamma-aminobutyric acidA-receptor messenger ribonucleic acid (alpha-1 subunit) detection by in situ hybridization.

The inhibitory neurotransmitter gamma-aminobutyric acid (GABA) is most likely involved in the efferent cochlear neurotransmission. In situ hybridization (ISH) results in specific annealing of a labelled nucleic acid probe to complementary sequences in fixed tissue and allows subsequent visualization of the location of the probe. We used the ISH technique to localize messenger ribonucleic acid (mRNA) sequences of the alpha-1 subunit of the GABAA receptor with an S-35 labeled oligonucleotide probe. Experiments were performed in rat and guinea pig brain sections and surface tissue preparations of the guinea pig cochlea. Positive signals were obtained for the alpha-1 probe in cortical and hippocampal regions of the rat brain and had weaker expression in the guinea pig brain. Alpha-1 subunit mRNA was localized in Purkinje cells and in stellate and basket cells of the stratum moleculare in the rat and guinea pig cerebellum. In surface tissue preparations of the guinea pig cochlea mRNA sequences of the alpha-1 subunit were detectable with high signal expression. Positive signals were seen on both sides of the tunnel of Corti, predominantly in the region of the outer hair cells. The results indicate expression of GABAA-receptor mRNA in cochlear tissue, supporting the importance of GABAA receptors in cochlear neurotransmission.

Autoradiographic comparison of [ 125I]LSD-labeled 5-HT 2A receptor distribution in rat and guinea pig brain

Although the density and distribution of 5-HT 2A(5-hydroxytryptamine-2A) receptors is well established for rat brain, the 5-HT 2A receptor distribution and density in guinea pig brain has not been extensively studied. In the present in vitro study, we have utilized 125I-lysergic acid diethylamide ([ 125I]LSD) to quantify and compare 5-HT 2A receptor density in coronal sections of rat and guinea pig brain. Spiperone (1 μM) and sulpiride (1 μM) were used to displace [ 125I]LSD binding from 5-HT 2A and D 2 binding sites, respectively. Ligand binding was quantified by computer-aided image analysis densitometry (MCID). Similar to the rat, areas of highest specific 5-HT 2A receptor binding (fmol/mg protein) in guinea pig brain included the claustrum and Layer 4 of the cerebral cortex. Significant binding was also found in remaining neocortical layers, islands of Calleja, caudate putamen, olfactory bulb, nucleus accumbens, and choroid plexus. While the rat brain exhibited a high level of specific binding in the tenia tecta and mammillary nuclei, little binding was observed in these regions in the guinea pig. In both rat and guinea pig, low specific binding was found in amygdaloid, thalamic, or cerebellar areas. These studies indicate a general similarity between 5-HT 2A binding site distribution and relative density in guinea pig and rat brain but point to a few brain regions where significant differences exist.

Autoradiographic identification and characterization of σ receptors in guinea pig brain using [ 3H]1(cyclopropylmethyl)-4-(2′-(4≈-fluorophenyl)-2′-oxoethyl) piperidine ([ 3H]DuP 734), a novel σ receptor ligand

The psychotomimetric effects of certain cycloalkyls and benzomorphans that interact with σ receptors has led to the hypothesis that these sites may be important in the etiology of schizophrenia. DuP 734 [1-(cyclopropylmethyl)-4-(2′-(4≈-fluoro-phenyl)-2′-oxoethyl) piperidine HBr] is a novel σ receptor ligand. The receptor binding specificity and neuroanatomical distribution of [ 3H]DuP 734-labeled σ receptors in guinea pig brain were examined using quantitative autoradiography. [ 3H]DuP 734 binding (10 μM haloperidol displaceable) to slide-mounted sections of guinea pig brain was saturable and of high affinity ( K i = 3.9nM). Competition studies, under conditions identical to those used to visualize the receptor, yielded the following rank order of potency: DuP 734 > haloperidol > (+)-pentazocine > (−)-butaclamol > DTG > (+)-SKF 10,047 > (+)-3-PPP > (−)-pentazocine > (+)-butaclamol > U50,488H > (−)-SKF 10,047 > cinanserin > PCP ≫ MK801, sulpiride. High densities of [ 3H]DuP 734 binding sites displaceable by haloperidol were present in the limbic system, in particular the dorsal and ventral bands of Broca as well as the ventral pallidum. Within the hippocampus, the pyramidal layers were sparsely labeled, while higher densities of binding sites were evident in the dentate gyrus. The frontal cortex, the mammillary complex of the hypothalamus, the central gray and red nucleus of the midbrain, the pontine reticular nucleus, the Purkinje cell layer of the cerebellum and dorsal and ventral horns, as well as the central gray matter of the spinal cord, all showed enrichments of [ 3H]DuP 734 binding sites. Lower levels of binding were present in the other regions of the cerebral cortex including parietal, pyriform, occipital, cingulate cortex, as well as the basal ganglia, and negligible specific binding was present in the white matter tracts. The kinetic and pharmacological characteristics and distribution of [ 3H]DuP 734 binding sites in brain are similar to those previously reported for σ receptors.

Autoradiographic visualization and characteristics of [ 125I]bradykinin binding sites in guinea pig brain

The present study was undertaken to localize and characterize bradykinin (BK) binding sites in 10 μm serial sections of guinea pig brain by in vitro quantitative receptor autoradiography. Specific binding of [ 125I-Tyr 8]bradykinin ([ 125I]BK) was localized in the medulla oblongata to the regions of the nucleus of the solitary tract (nTS), the area postrema (AP), the dorsal motor nucleus of the vagus (X) and the caudal subnucleus of the spinal trigeminal nucleus. No significant specific [ 125I]BK binding was seen in other brain regions. The specific binding (85–90% of total binding) was of high affinity and saturable with a K D of73.5±9.9pM and a B max of27.8± 1.9 amol per mm 2 of tissue. In competition studies, the rank order of potencies was:BK>Met-Lys-BK>Lys-BK> >Des-Arg 9-BK. The B 2 receptor antagonist d-Arg 0-Hyp 3-Thi 5,8- d-Phe 7-BK inhibited [ 125I]BK binding with a K i value of3.5±1.5nM while Des-Arg 9-[Leu 8]-BK, a B 1 receptor antagonist did not significantly inhibit [ 125I]BK binding in concentrations up to 10μM. Our finding of specific high affinity [ 125I]BK binding sites in the nTS, AP and the X is important because these brain areas are known to be involved in central cardiovascular regulation. Moreover, our results suggest that the specific [ 125I]BK binding sites in the guinea pig medulla are of the bradykinin B 2 receptor type.

Presence of estrogen receptor immunoreactivity in the oxytocin-containing magnocellular neurons projecting to the neurohypophysis in the guinea-pig

The immunoperoxidase technique was used on adjacent sections of guinea-pig brain to compare precisely the distribution of estrogen receptor-immunoreactive cells and progesterone receptor-immunoreactive cells in the supraoptic nucleus and the paraventricular nucleus. Only estrogen receptor-immunoreactive neurons were found in the supraoptic nucleus. A large number of estrogen receptor-positive cells were observed in the periventricular magnocellular groups throughout the rostrocaudal extent of the paraventricular nucleus, whereas only a few progesterone receptor-immunoreactive cells were scattered in the anterior portion of this region. We used a combination of axonal tracing with double immunocytochemical detection to determine whether estradiol acts directly on the oxytocin-immunoreactive neurons which project to the neurohypophysis. Oxytocin-immunoreactive cells were found in the supraoptic nucleus, ventrally to the optic pathways, in subchiasmatic and retrochiasmatic areas, and in the anterior hypothalamic area. These cells were also retrogradely labeled by Granular Blue when this tracer was injected intravenously. In the paraventricular nucleus, the Granular Blue/oxytocin-positive cells were observed in the periventricular magnocellular groups whereas Granular Blue labeled neurons were found in both Parvocellular and Magnocellular components. We Found that almost all the oxytocin-immunoreactive cells revealed estrogen receptor immunoreactivity. In conclusion, the comparative study of distribution of estrogen receptors and progesterone receptors in the guinea-pig supraoptic and paraventricular nuclei indicates that, in the supraoptic nucleus, only estrogen receptors are present and that, in the paraventricular nucleus, they are far more numerous than progesterone receptors. The present findings demonstrate that the magnocellular cells which contain estrogen receptors are oxytocinergic. In addition, these cells are retrogradely labeled pointing to a neurohypophysial projection. It is likely that estradiol controls the hypothalamo-neurohypophysial oxytocin system by direct action on the magnocellular neurons.

Comparative neuroanatomical distribution of the κ and μ opioid receptors in guinea pig brain sections

The opiate family of receptors is believed to consist of the μ, δ, κ and σ subtypes. Autoradiographic studies have been conducted to define the distribution of the μ and δ receptors. However, due to the lack of a selective ligand definitive studies have not been reported for the κ receptor. Recently such a ligand was discovered and characterized. Results are presented in which the distribution of κ receptors in guinea pig brain slices were determined using autoradiographic techniques and its distribution compared to that of a typical μ ligand. The results demonstrate numerous differences between the two opioid systems. The κ receptors predominate in the prefrontal cortex, the nucleus accumbens, and deep cortex; whereas the μ receptors have a more intense, broader and different distribution.

A detailed mapping of hist amine H 1-receptors in guinea-pig central nervous system established by autoradiography with [ 125I]iodobolpyramine

[ 125I]Iodobolpyramine, a potent and selective histamine H 1-receptor antagonist derived from mepyramine, was used to generate light microscopic autoradiograms on sections of guinea-pig brain and spinal cord. Histamine H 1-receptors were labelled with high sensitivity over a low background as determined using mianserin or other H 1-receptor antagonists as competing agents. An atlas of H 1-receptors was established using five sagittal sections and 39 frontal sections, the latter serially prepared at 50 μm intervals. Labelled areas were identified by comparison with corresponding, classically stained sections and their density was rated according to an arbitrary scale. Autoradiographic grains were detected in a large variety of gray matter areas whereas they were generally absent from white matter areas. In the cerebral cortex, H 1-receptors are present in all areas and layers with a higher density in lamina IV. In the hippocampal formation, H 1-receptors display a laminated pattern of distribution and are the most abundant in the dentate gyrus (hilus and molecular layer) and in several areas of the subiculum and commissural complex. In the amygdaloid complex, the highest densities are found in the medial group of nuclei. In the basal forebrain, the striatum is moderately labelled whereas the nucleus accumbens, islands of Calleja and most septal nuclei are highly labelled. In the thalamus, H 1-receptors are present in high density, particularly in the anterior, median and lateral groups of nuclei. In the hypothalamus the labelling is highly heterogeneous with high densities in, for example, medial preoptic area, dorsomedial, ventromedial and most posterior nuclei, including the tuberomammillary complex in which histaminergic perikarya and short axons are present. In the cerebellum, the molecular layer is densely labelled but H 1-receptors are also detectable in the granular layer and inner nuclei. In mesencephalon and lower brainstem, H 1-receptors are particularly abundant in the nuclei of origin of most cranial nerves, in areas containing the perikarya of the catecholamine and serotonin systems and in various areas associated with vegetative reflexes, e.g. the area postrema. In the spinal cord, the highest density is found in the external layers of the dorsal horn. This highly heterogeneous distribution of H 1-receptors is discussed in the light of known distributions of histaminergic axons, other classes of histamine receptors and responses to local applications of histamine within the CNS.

Species differences in the localization of ‘peripheral’ type cholecystokinin receptors in rodent brain

A comparison of cholecystokinin (CCK) receptor binding to sections of rat, mouse and guinea pig brain has been performed using 125I-Bolton Hunter CCK and the selective peripheral CCK receptor antagonist L-365,031. In both rat and mouse, 125I-Bolton Hunter CCK binding in the region of the interpeduncular nucleus (IPN) was inhibited by L-365,031 indicating that these receptors resemble CCK receptors found in peripheral tissues. In the mouse especially, dense regions of peripheral CCK receptors were detected either side of the IPN. By contrast, in the guinea pig IPN no evidence of L-365,031-sensitive binding could be found. The present reports shows that in different species, regional variations in brain CCK receptor binding occur not only in the case of classical ‘brain’ receptors, but also for the more discretely localised ‘peripheral’ type CCK receptors.

Fiber tracts that contain more opioid binding sites than gray matter does: A quantitative autoradiographic study in the guinea-pig

Opioid binding sites were localized in cryostat sections of guinea-pig brain by in vitro autoradiography using (−)-[ 3H]bremazocine. Quantification of binding sites in gray matter was accomplished using standard samples of tritium mixed in brain gray matter. The binding sites in fiber tracts were quantified using standards made from white matter to compensate for the quenching caused by myelin. Binding in gray matter corroborated previous findings that the moderately high densities of opioid sites in the cerebellum are of the kappa type and that the V and VI laminae of the cerebral cortex, the substantia nigra and olfactory bulb contain high levels of opioid sties in the guinea-pig. Several fiber tracts such as cerebellar white matter and the corpus callosum contained densities of (−)-[ 3H]bremazocine binding sites equal to, or higher than, the most densely labeled gray matter areas. The dorsal hippocampal commissure and the splenium of the corpus callosum contained 2200 fmol sites/mg protein, two and one half times more than the most densely labeled gray matter areas, the external plexiform layer of the olfactory bulb. These sites may be receptors in transport, but their density constitutes a massive volume of receptors for which a physiological role still needs to be more clearly defined.