Out-of-focus background subtraction for fast structured illumination super-resolution microscopy of optically thick samples.

We propose a structured illumination microscopy method to combine super resolution and optical sectioning in three-dimensional (3D) samples that allows the use of two-dimensional (2D) data processing. Indeed, obtaining super-resolution images of thick samples is a difficult task if low spatial frequencies are present in the in-focus section of the sample, as these frequencies have to be distinguished from the out-of-focus background. A rigorous treatment would require a 3D reconstruction of the whole sample using a 3D point spread function and a 3D stack of structured illumination data. The number of raw images required, 15 per optical section in this case, limits the rate at which high-resolution images can be obtained. We show that by a succession of two different treatments of structured illumination data we can estimate the contrast of the illumination pattern and remove the out-of-focus content from the raw images. After this cleaning step, we can obtain super-resolution images of optical sections in thick samples using a two-beam harmonic illumination pattern and a limited number of raw images. This two-step processing makes it possible to obtain super resolved optical sections in thick samples as fast as if the sample was two-dimensional.

Ontogeny of angiotensin II type 1 receptor mRNAs in fetal and neonatal rat brain.

Studies have demonstrated a specific function of the angiotensin II (Ang II) type 1 receptor (AT(1)) in regulation of adult central cardiovascular, fluid, and pituitary hormone release and a predominant role of the renin-angiotensin system in fetal and neonatal cardiovascular homeostasis. The pattern of brain AT(1) mRNA expression during fetal and neonatal development is currently unknown. We used radiolabeled cRNA probes for in situ hybridization histochemistry to determine the ontogenic development of the two AT(1) subtypes (AT(1a) and AT(1b)) mRNA in rat brain, from 11 days of gestation (E11) to 28 days after birth (P28). No AT(1b) mRNA was detected in the developing brain, whereas AT(1a) mRNA was first detected at E19. The age at which AT(1a) mRNA is first detected varied among different brain areas and expression predominates in areas involved in fluid homeostasis, pituitary hormone release, and cardiovascular regulation, where it persists until P28. AT(1a) mRNA expression is present from E19 onward in the median preoptic nucleus, the vascular organ of the lamina terminalis, the paraventricular nucleus, the periaqueductal gray, the nucleus raphe pallidus, the motor facial nucleus, and very weakly in the nucleus of the solitary tract and the ambiguous nucleus, and at E21 in the subfornical organ, the anterior olfactory nucleus and the piriform cortex. AT(1a) mRNA expression is present after birth in many regions, including the preoptic and lateral hypothalamic areas, the area postrema and medullary reticular nuclei. In conclusion, during brain development, expression of AT(1a) mRNA, appears in late gestation at E19, predominantly in forebrain areas involved in fluid homeostasis and cardiovascular regulation. In contrast, AT(1a) mRNA expression is absent or present only in very small amounts until after birth in many medullary nuclei, known to play an important role in cardiovascular modulation. Our results suggest that, in perinatal life, AT(1a) is involved in fluid and perhaps cardiovascular homeostasis and that the role of Ang II in modulating medullary cardiovascular centers matures later in postnatal life.

Physiological role of a novel neuropeptide, apelin, and its receptor in the rat brain.

Apelin, a peptide recently isolated from bovine stomach tissue extracts, has been identified as the endogenous ligand of the human orphan APJ receptor. We established a stable Chinese hamster ovary (CHO) cell line expressing a gene encoding the rat apelin receptor fused to the enhanced green fluorescent protein, to investigate internalization and the pharmacological profile of the apelin receptor. Stimulation of this receptor by the apelin fragments K17F (Lys1-Phe-Arg-Arg-Gln-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe17) and pE13F (pGlu5-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe17) resulted in a dose-dependent inhibition of forskolin-induced cAMP production and promoted its internalization. In contrast, the apelin fragments R10F (Arg8-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe17) and G5F (Gly13-Pro-Met-Pro-Phe17) were inactive. The physiological role of apelin and its receptor was then investigated by showing for the first time in rodent brain: (i) detection of apelin neurons in the supraoptic and paraventricular nuclei by immunohistochemistry with a specific polyclonal anti-apelin K17F antibody; (ii) detection of apelin receptor mRNA in supraoptic vasopressinergic neurons by in situ hybridization and immunohistochemistry; and (iii) a decrease in vasopressin release following intracerebroventricular injection of K17F, or pE13F, but not R10F. Thus, apelin locally synthesized in the supraoptic nucleus could exert a direct inhibitory action on vasopressinergic neuron activity via the apelin receptors synthesized in these cells. Furthermore, central injection of pE13F significantly decreased water intake in dehydrated normotensive rats but did not affect blood pressure. Together, these results suggest that neuronal apelin plays an important role in the central control of body fluid homeostasis.

A functional enhanced green fluorescent protein (EGFP)-tagged angiotensin II at(1a) receptor recruits the endogenous Galphaq/11 protein to the membrane and induces its specific internalization independently of receptor-g protein coupling in HEK-293 cells.

The angiotensin II (Ang II) AT(1A) receptor was tagged at its C terminus with the enhanced green fluorescent protein (EGFP), and the corresponding chimeric cDNA was expressed in HEK-293 cells. This tagged receptor presents wild-type pharmacological and signaling properties and can be immunodetected by Western blotting and immunoprecipitation using EGFP antibodies. Therefore, this EGFP-tagged AT(1A) receptor is the perfect tool for analyzing in parallel the subcellular distributions of the receptor and its interacting G protein and their trafficking using confocal microscopy. Morphological observation of both the fluorescent receptor and its cognate Galphaq/11 protein, identified by indirect immunofluorescence, and the development of a specific software for digital image analysis together allow examination and quantification of the cellular distribution of these proteins before and after the binding of different agonist or antagonist ligands. These observations result in several conclusions: 1) Expression of increasing amounts of the AT(1A) receptor at the cell surface is associated with a progressive recruitment of the cytosolic Galphaq/11 protein at the membrane; 2) Internalization of the EGFP-tagged AT(1A) induced by peptide ligands but not nonpeptide ligands is accompanied by a Galphaq/11 protein intracellular translocation, which presents a similar kinetic pattern but occurs predominantly in a different compartment; and 3) This Galphaq/11 protein cellular translocation is dependent on receptor internalization process, but not G protein coupling and signal transduction mechanisms, as assessed by pharmacological data using agonists and antagonists and the characterization of AT(1A) receptor mutants (D(74)N and Delta329) for which the coupling and internalization functions are modified.

A highly sensitive quantitative cytosensor technique for the identification of receptor ligands in tissue extracts.

Because G-protein-coupled receptors (GPCRs) constitute excellent putative therapeutic targets, functional characterization of orphan GPCRs through identification of their endogenous ligands has great potential for drug discovery. We propose here a novel single cell-based assay for identification of these ligands. This assay involves (a) fluorescent tagging of the GPCR, (b) expression of the tagged receptor in a heterologous expression system, (c) incubation of the transfected cells with fractions purified from tissue extracts, and (d) imaging of ligand-induced receptor internalization by confocal microscopy coupled to digital image quantification. We tested this approach in CHO cells stably expressing the NT1 neurotensin receptor fused to EGFP (enhanced green fluorescent protein), in which neurotensin promoted internalization of the NT1-EGFP receptor in a dose-dependent fashion (EC(50) = 0.98 nM). Similarly, four of 120 consecutive reversed-phase HPLC fractions of frog brain extracts promoted internalization of the NT1-EGFP receptor. The same four fractions selectively contained neurotensin, an endogenous ligand of the NT1 receptor, as detected by radioimmunoassay and inositol phosphate production. The present internalization assay provides a highly specific quantitative cytosensor technique with sensitivity in the nanomolar range that should prove useful for the identification of putative natural and synthetic ligands for GPCRs.

Cloning, pharmacological characterization and brain distribution of the rat apelin receptor.

The peptide apelin, recently isolated from bovine stomach tissue extracts, has been identified as an endogenous ligand of the human putative receptor protein related to the angiotensin receptor AT(1) (APJ). In this article, we report cloning of the rat apelin receptor cDNA. The sequence shares 90% identity with the human APJ receptor and 31% with the rat AT(1A) angiotensin receptor. Subsequently a stable CHO cell line expressing the receptor fused at its C-terminal part with the enhanced green fluorescent protein (EGFP) was established, allowing to verify its cell surface distribution and to determine the affinity of various apelin and angiotensin fragments on the cloned receptor. As shown for the human APJ receptor, the rat apelin receptor expressed in the cell line was negatively coupled to adenylate cyclase. The apelin fragment K17F (Lys(1)-Phe-Arg-Arg-Gln-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe(17)) inhibited forskolin-stimulated cAMP production at sub-nanomolar concentrations whereas angiotensin II and angiotensin III were inactive. N-terminal elongation of K17F with a tyrosine or the N-terminal deletion of the first four amino acids did not modify the inhibitory action of K17F on cAMP production. In contrast, deletion of the first seven amino acids of K17F or substitution of phenylalanine by an alanine residue at the C-terminus completely abolished the activity of the peptide. In situ hybridization analysis of apelin receptor mRNA expression in the adult rat brain showed intense labeling in the hypothalamus, especially in the supraoptic and the paraventricular nuclei. The anterior and intermediate lobes of the pituitary were also highly labeled, as well as the pineal gland. Labeling was also found in extrahypothalamic structures such as the piriform cortex, the nucleus of the lateral olfactory tract, the central grey matter, the pars compacta of the substantia nigra, the dorsal raphe nucleus, the entorhinal cortex, the dentate gyrus and the Ammon's horn. The hypothalamic and hypophyseal distribution of the receptor suggests an involvement of apelin in the control of neuro- and adenohypophyseal hormone release, whereas its presence in the pineal gland and in discrete higher brain structures points out to possible roles in the regulation of circadian rhythms and of water and food intake behavior.

Ontogeny of angiotensin II type 2 receptor mRNA expression in fetal and neonatal rat brain.

Recent studies have provided evidence for a specific role of the angiotensin II type 2 receptor (AT2) in in vitro neuron differentiation, and in AT2 knock-out mice that display central neurological anomalies. The role of AT2 in brain development is currently unknown. By using radiolabeled cRNA probes for in situ hybridization histochemistry, we determined the ontogenic development of AT2 mRNA in fetal and neonatal rat brain, from 11 days of gestation (E11) to 28 days postnatal (P28). Brain AT2 mRNA is first detected in the lateral hypothalamic neuroepithelium at E13. AT2 mRNA is detected beginning at E15 in the subthalamic and hypoglossus nuclei; at E17 in the pedunculopontine nucleus, cerebellum, motor facial nucleus, and the inferior olivary complex; at E19 in the thalamus, bed nucleus of the supraoptic decussation, interstitial nucleus of Cajal, nuclei of the lateral lemniscus, locus coeruleus, and supragenual nucleus; and at E21 in the lateral septal and medial amygdaloid nuclei, medial geniculate body, and the superior colliculus. The substantia nigra and many telencephalic and medullary nuclei express AT2 mRNA only after birth. Certain structures express AT2 mRNA strongly but transiently during embryonic life, such as the differentiating lateral hypothalamic area at E13, the superior olivary complex at E19 and E21, and the red nucleus at E15 and E17. In conclusion, during brain development, expression of AT2 mRNA appears early at E13, is strongly but transiently expressed in certain structures, and is high and persists until brain maturity in nuclei involved in motor functions and sensory integration. Our results support a dual role of AT2 during brain development in early maturation and differentiation, but also in modulation of established functions during perinatal and adult life.

Identification of endocrine cell populations expressing the AT1B subtype of angiotensin II receptors in the anterior pituitary.

Angiotensin II (Ang II) participates in the regulation of anterior pituitary hormone secretion by acting either directly on the anterior pituitary or indirectly on the hypothalamus. When applied directly on pituitary cells, Ang II increases both ACTH and PRL secretion and has also been reported to affect GH secretion. Three distinct subtypes of Ang II receptors (AT1A, AT1B, and AT2) have been identified; they are unequally distributed and differently regulated in various tissues. We have previously demonstrated that only AT1A receptors are present in the hypothalamus while anterior pituitary cells express predominantly the AT1B subtype. Using in situ hybridization in combination with immunohistochemistry, the aim of the present study was to identify the phenotype of the endocrine cell expressing AT1B receptor messenger RNA (mRNA) in the anterior pituitary of adult male Sprague-Dawley rats. Expression of AT1B receptor mRNA was present in 33.9 +/- 1.0% of anterior pituitary cells. AT1B mRNA is predominantly expressed by lactotropes (78.2 +/- 2.1% of AT1B mRNA-expressing cells) and to a lower degree by corticotropes (18.3 +/- 2.1%) and is not detectable in somatotropes, mammosomatotropes, gonadotropes, or thyrotropes. These results indicate that in adult male rats, Ang II, which has been shown to be synthesized in gonadotropes, can directly stimulate PRL and ACTH release from lactotropes and corticotropes through activation of AT1B receptors. As only 53.8 +/- 2.7% of lactotropes and 23.6 +/- 2.8% of corticotropes expressed AT1B mRNA, our findings suggest a functional heterogeneity of both cell types regarding their sensitivity to Ang II.

Distribution of angiotensin type-1 receptor messenger RNA expression in the adult rat brain.

Angiotensin II and angiotensin III in the brain exert their various effects by acting on two pharmacologically well-defined receptors, the type-1 (AT1) and the type-2 (AT2) receptors. Receptor binding autoradiography has revealed the dominant presence of AT1 in brain nuclei involved in cardiovascular, body fluid and neuroendocrine control. The cloning of the AT1 complementary DNA has revealed the existence of two receptor subtypes in rodents, AT1A and AT1B. Using specific riboprobes for in situ hybridization, we have previously shown that the AT1A messenger RNA is predominantly expressed in the rat forebrain; in contrast the AT1B subtype predominates in the anterior pituitary. Using a similar technical approach, the aim of the present study was to establish the precise anatomical localization of cells synthetising the AT1A receptor in the adult rat brain. High AT1A messenger RNA expression was found in the vascular organ of the lamina terminalis, the median preoptic nucleus, the subfornical organ, the hypothalamic periventricular nucleus, the parvocellular parts of the paraventricular nucleus, the nucleus of the solitary tract and the area postrema, in agreement with previous autoradiographic studies, describing a high density of AT1 binding sites in these nuclei. In addition, AT1A messenger RNA expression was detected in several brain areas, where no AT1 binding was reported previously. Thus, we identify strong expression of AT1A messenger RNA expression in scattered cells of the lateral parts of the preoptic region, the lateral hypothalamus and several brainstem nuclei. In none of these structures was the AT1B messenger RNA detectable at the microscopic level. In conclusion, it is suggested that angiotensins may exert their central effects on body fluid and cardiovascular homeostasis mainly via the AT1A receptor subtype.

Expression of angiotensin type-1 (AT1) and type-2 (AT2) receptor mRNAs in the adult rat brain: a functional neuroanatomical review.

The discovery that all components of the renin-angiotensin system (RAS) are present in the central nervous system led investigators to postulate the existence of a local brain RAS. Supporting this, angiotensin immunoreactive neurons have been visualized in the brain. Two major pathways were described: a forebrain pathway which connects circumventricular organs to the median preoptic nucleus, paraventricular nucleus, and supraoptic nucleus, and a second pathway connecting the hypothalamus to the medulla oblongata. Blood-brain barrier deficient circumventricular organs are rich in angiotensin II receptors. By activating these receptors, circulating angiotensin II may act on central cardiovascular centers via angiotensinergic neurons, providing a link between peripheral and central angiotensin II systems. Among the effector peptides of the brain RAS, angiotensin II and angiotensin III have the same affinity for the two pharmacologically well-defined receptors: type 1 (AT1) and type 2 (AT2). When injected in the brain, these peptides increase blood pressure, water intake, and anterior and posterior pituitary hormone release and may modify memory and learning. The cloning of AT1 and AT2 receptor cDNAs has revealed that these receptors belong to the seven transmembrane domain receptor family. In rodents, two AT1 receptor subtypes, AT1A and AT1B, have been isolated. Using specific riboprobes for in situ hybridization histochemistry, recent studies mapped the distribution of AT1A, AT1B, and AT2 receptor mRNAs in the adult rat and found a predominant expression of AT1A and AT2 mRNA in the brain and of AT1B in the pituitary. Very limited overlap was found between the brain expression of AT1A and AT2 mRNAs. In several functional entities of the brain, such as the preoptic region, the hypothalamus, the olivocerebellary system, and the brainstem baroreflex arc, the colocalization of receptor mRNA, binding sites, and angiotensin immunoreactive nerve terminals suggests local synthesis and expression of angiotensin II receptors. In other areas, such as the bed nucleus of the stria terminalis, the median eminence, or certain parts of the nucleus of the solitary tract, angiotensin II receptors are likely of extrinsic origin. The neuronal expression of AT1A and AT2 receptors was demonstrated in the subfornical organ, the hypothalamus, and the lateral septum. By using double label in situ hybridization, AT1A receptor expression was localized in corticotropin releasing hormone but not in vasopressin containing neurons in the hypothalamus. The information is discussed together with functional data concerning the role of brain angiotensins, in an attempt to provide a better understanding of the physiological and functional roles of each receptor subtype.

Aminopeptidase A: distribution in rat brain nuclei and increased activity in spontaneously hypertensive rats.

Aminopeptidase A is a membrane-bound zinc metalloprotease which cleaves angiotensin II into angiotensin III. Using a new specific aminopeptidase A inhibitor, EC33, we evaluated its enzymatic activity in several microdissected brain nuclei involved in the control of cardiovascular functions and in the pituitary. We compared this distribution with that of the angiotensin I-converting enzyme which converts angiotensin I to angiotensin II. Aminopeptidase A activity was heterogenously distributed with a 150-fold difference between the lowest and the highest levels. The pituitary and the circumventricular organs were the richest source of enzyme, followed by the median eminence, the arcuate nucleus, the area postrema, the choroid plexus and the supraotic and paraventricular nuclei. We did not find any close parallel between aminopeptidase A and angiotensin I-converting enzyme distributions. We examined both enzymatic activities in brain nuclei of spontaneously hypertensive rats. Aminopeptidase A activity was higher in the spontaneously hypertensive rats than in age-matched Wistar Kyoto control rats. The difference was up to 2.5-fold in several brain nuclei involved in the blood pressure regulation; in contrast, no differences in angiotensin I-converting enzyme activity were found in the same regions. The close correspondence between the distribution of aminopeptidase A activity and angiotensin receptors and nerve terminals in the brain associated with the observation that aminopeptidase A activity was overactivated in the spontaneously hypertensive rats suggest that this enzyme may contribute, at least in part, to the regulation of cardiovascular functions by its ability to convert angiotensin II to angiotensin III.

Distribution of angiotensin II type-2 receptor (AT2) mRNA expression in the adult rat brain.

Radioactively labeled cRNA probes were used for in situ hybridization histochemistry to establish a detailed map of the sites of expression of the recently cloned angiotensin II, type 2 (AT2) receptor mRNA in the adult rat brain. The distribution of the AT2 receptor mRNA was consistent with that of the AT2 binding sites, which were previously established by autoradiographic binding studies. Thus, high AT2 receptor mRNA expression was observed in the lateral septum, in several thalamic nuclei, in the subthalamic nucleus, in the locus coeruleus, and in the inferior olive. Due to the superior resolution and sensitivity of in situ hybridization, AT2 receptor expression was localized at the cellular level, and some additional brain nuclei expressing AT2 receptor mRNA have been identified. These include the red nucleus, the pedunculopontine tegmental nucleus, the bed nucleus of the supraoptic decussation, the paragenual nucleus, and numerous brainstem nuclei. Several brain nuclei, such as the motor hypoglossal nucleus and the cerebellar nuclei, where AT2 receptor binding had previously been identified in young animals only, showed a high expression of the AT2 receptor mRNA in the adult rat. No correlation was found between the expression of the AT2 and the type 1 (AT1) receptor mRNAs. A combination of the in situ hybridization and glial fibrillary acidic protein (GFAP) immunohistochemistry shows that the AT2 receptor in the lateral septum showed that the AT2 receptor was not detected in GFAP immunoreactive astroglial cells, therefore indicating that AT2 is neuronal rather than glial in this brain region.

Comparative expression of vasopressin and angiotensin type-1 receptor mRNA in rat hypothalamic nuclei: a double in situ hybridization study.

Angiotensin II (Ang) injected intracerebroventricularly stimulates neurohypophyseal vasopressin (AVP) release into the peripheral circulation. As we have shown previously, central actions of Ang II in the rat forebrain are mediated by the AT1A receptor subtype. In the present paper, we attempted to clarify the cellular localization of the AT1A receptor mRNA in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei, in order to reappraise the conflicting data on the nature of the angiotensin II receptor involved in Ang induced vasopressin release. For this purpose, double in situ hybridization was performed using a radioactive AT1A receptor riboprobe and a digoxygenin labeled AVP oligoprobe, and immunohistochemical localization of the glial marker glial fibrillary acidic protein (GFAP) on the same brain slice. The results show neuronal expression of AT1A receptor mRNA mainly in dorsal and medial parvocellular parts of the PVN, its localization in some magnocellular PVN neurons and the absence of its expression in AVP producing neurons either in the PVN or in the SON. Thus, while indirect evidence indicates the involvement of the AT1A receptor subtype in the regulation of CRH and oxytocin release, the stimulation of vasopressinergic neurons is likely due to indirect mechanisms, or to a yet unknown type of angiotensin receptor.

The angiotensin receptor subtype AT1A predominates in rat forebrain areas involved in blood pressure, body fluid homeostasis and neuroendocrine control.

Subtypes of the angiotensin II (Ang II) type-1 (AT1) receptor are probably involved in distinct actions of the peptide, since their distribution in peripheral organs and regulation of their gene expression are different. We investigated the distribution of AT1A and AT1B receptor subtype mRNAs in the rat forebrain and pituitary using sensitive cRNA probes for in situ hybridization. High level of AT1A receptor mRNA expression is observed in the subfornical organ (SFO) and in the anterior hypothalamus, particularly the periventricular tissue surrounding the anterior portion of the 3rd ventricle (AV3V), which contains the organum vasculosum of the lamina terminalis (OVLT), the median preoptic nucleus and the preoptic periventricular nucleus as well as in the hypothalamic periventricular nucleus and in the parvocellular part of the paraventricular nucleus (PVN). Moderate to strong AT1A labeling was found in the anterior olfactory nucleus, the piriform cortex and the nucleus of the lateral olfactory tract. Very low AT1B receptor mRNA expression was found in the SFO and the PVN. In contrast, strong AT1B receptor mRNA expression coincided with low AT1A receptor mRNA expression in the anterior pituitary. Labeling was cytoplasmic at the light microscopic level. We thus suggest that the AT1A receptor is responsible for the central actions of Ang II in the rat forebrain whereas direct actions of Ang II on the anterior pituitary are mediated by the AT1B receptor subtype.

Ontogeny of angiotensin II type 2 (AT2) receptor mRNA in the rat.

To study the distribution of the recently cloned angiotensin II type 2 (AT2) receptor in the rat fetus, a double stranded cDNA was generated by a new and recently described methodology requiring no cloning procedure. The cDNA obtained after reverse transcription (RT) and polymerase chain reaction (PCR) amplification corresponded to 500 base pairs of the gene coding sequence, and included the SP6 and T7 promoters at the 5' and 3' end, respectively. 35S-labeled cRNA sense and antisense probes were synthesized by in vitro transcription and used for in situ hybridization. From 13 to 19 days of gestation the AT2 receptor mRNA expression evolved and extended from a series of paired spots located para-axially, which were not identifiable at this level of observation, to a distribution in various mesenchymes (perichondrium, subepidermal layers), muscle cells (tongue, diaphragm, stomach), and classical target organs for Ang II (adrenal gland, kidney, aorta). During the first days after birth, the AT2 receptor mRNA decreased and remained detectable only in the adrenal gland and kidney. The distribution of the AT2 receptor mRNA appeared strikingly different from that of the AT1A receptor, which was studied in parallel for comparison.

Autoradiographic localization and quantitative determination of specific binding sites of anxiolytic homophthalazines (formerly called 2,3-benzodiazepines) in the striato-pallido-nigral system of rats.

Homophthalazines (2,3-benzodiazepin-derivates, such as tofisopam, nerisopam, girisopam) constitute a drug family with strong anxiolytic and antipsychotic potencies. By autoradiography, all of these drugs showed a specific distribution pattern of binding sites exclusively in brain areas which relate to the striato-pallido-nigral system, while no specific label was found in any other brain areas in the rat. Quantitative analyses of the autoradiograms by computerized densitometry, as well as by a receptor binding assay on 32 microdissected brain areas showed very high concentrations of tritiated homophthalazines in the glubus pallidus, caudate nucleus, putamen and the substantia nigra. Relatively high density of binding sites was measured in the nucleus accumbens, the olfactory tubercle, the entopeduncular nucleus and the subthalamic nucleus. Concentrations measured in the cerebral cortical areas, cerebellum or brainstem nuclei did not differ from the background. No significant differences were found between the homophthalazines investigated in terms of the distribution patterns or density of binding sites.

A novel specific binding site for homophthalazines in the rat brain.

The specific binding sites of a homophthalazine, girisopam, in rat brain have been localized by qualitative and quantitative autoradiography. This substance exerts strong anxiolytic and antipsychotic effects both in rodents and in humans. High labeling was present in all major components of the extrapyramidal system, such as the caudate-putamen, globus pallidus, subthalamic nucleus, substantia nigra, and the extrapyramidal portion of the accumbens nucleus and the olfactory tubercle, while specific labeling was not seen in any other brain areas including the cerebral cortex, thalamus, cerebellum or brainstem areas. This novel distribution of girisopam is consistent with its antipsychotic effect and anxiolytic properties and may provide a morphological basis for further studies to elucidate the mechanisms of action of homophthalazines in the central nervous system.

Coordination between cardiovascular and respiratory control systems during and after cerebral ischemia.

The dissociation of cardiovascular (arterial hypertension) and respiratory (depression) reactions to severe cerebral ischemia seems to be inconsistent with the usual cooperative behavior of the two systems and their role in managing disturbances in the central chemical environment. In the present study the Cushing reaction was elicited by transient increase of the intracranial pressure 4-11 times in each experiment. The pressor response and changes in the vertebral sympathetic nerve discharge (SND) were compared with the respiratory reaction and with changes in the phrenic nerve activity. The reaction in both nerves developed in two phases. In the phrenic nerve, an initial hyperactivity (increased discharge amplitude and frequency) coincided with augmentation of the rhythmic SND (phase 1) and complete nerve depression developed when the SND was desynchronized (phase 2). The transition in both systems correlated in their latencies and the severity of the ischemia needed for their stimulation. Repetition of the ischemic stimuli increased the occurrence of the respiratory-related rhythmicity in the SND and later changed its character from rhythmic amplitude modulation to respiratory-related high-frequency bursting SND coinciding with the inspiration. It is concluded that, despite the apparent dissociation between the cardiovascular and respiratory reactions, there is a parallel response between the neurophysiological correlates of the two systems to increasing severity of cerebral ischemia.