Plant stress biomarkers from biosimulations: the Transcriptome-To-Metabolome (TTM) technology - effects of drought stress on rice.

Measuring biomarkers from plant tissue samples is challenging and expensive when the desire is to integrate transcriptomics, fluxomics, metabolomics, lipidomics, proteomics, physiomics and phenomics. We present a computational biology method where only the transcriptome needs to be measured and is used to derive a set of parameters for deterministic kinetic models of metabolic pathways. The technology is called Transcriptome-To-Metabolome (TTM) biosimulations, currently under commercial development, but available for non-commercial use by researchers. The simulated results on metabolites of 30 primary and secondary metabolic pathways in rice (Oryza sativa) were used as the biomarkers to predict whether the transcriptome was from a plant that had been under drought conditions. The rice transcriptomes were accessed from public archives and each individual plant was simulated. This unique quality of the TTM technology allows standard analyses on biomarker assessments, i.e. sensitivity, specificity, positive and negative predictive values, accuracy, receiver operator characteristics (ROC) curve and area under the ROC curve (AUC). Two validation methods were also used, the holdout and 10-fold cross validations. Initially 17 metabolites were identified as candidate biomarkers based on either statistical significance on binary phenotype when compared with control samples or recognition from the literature. The top three biomarkers based on AUC were gibberellic acid 12 (0.89), trehalose (0.80) and sn1-palmitate-sn2-oleic-phosphatidylglycerol (0.70). Neither heat map analyses of transcriptomes nor all 300 metabolites clustered the stressed and control groups effectively. The TTM technology allows the emergent properties of the integrated system to generate unique and useful 'Omics' information.

I. Serotonin (5-HT) within dopamine reward circuits signals open-field behavior. II. Basis for 5-HT--DA interaction in cocaine dysfunctional behavior.

Light microscopic immunocytochemical studies, using a sensitive silver intensification procedure, show that dopamine (DA) and serotonin (5-HT) axons terminate on neurons in the nucleus accumbens (NAcc) (A10) terminals and also in dorsal striatum (DSTr) (A9) terminals. The data demonstrate a prominent endogenous anatomic interaction at these distal presynaptic sites between the neurotransmitters 5-HT and DA; the pattern of the 5-HT-DA interaction differs between A10 and A9 terminals. Moreover, in distinction to the variance shown anatomically between 5-HT--DA interactions at distal A9 and A10 sites, the 5-HT--DA interactions at the level of DA somatodendrites, the proximal site, are similar, i.e. 5-HT terminals in the midbrain tegmentum are profuse and have a massive overlap with DA neurons in both ventral tegmental area (VTA) and substantia nigra pars compacta (SNpc). We suggest with reference to the DA neurons of A10 and A9 pathways, inclusive of somatodendrites (sites of proximal presynaptic interactions in the midbrain) and axons (sites of distal presynaptic interactions), that 5-HT--DA interactions in A10 terminals are more likely to exceed those in the DStr arrangement. Furthermore, our neuroanatomic data show that axonally released DA at A10 terminals may originate from proximal 5-HT somatodendrites, i.e. dorsal raphe (DR) or the proximal DA somatodendrites, VTA. In vivo microvoltammetric studies were done with highly sensitive temporal and spatial resolution; the studies demonstrate basal (endogenous) real time 5-HT release at distal A10 and distal A9 terminal fields and real time 5-HT release at proximal A10 VTA somatodendrites. In vivo microvoltammetric studies were performed concurrently and on line with studies of DA release, also at distal A10 and distal A9 terminal fields and at proximal A10 somatodendrites. Serotonin release was detected in a separate voltammetric peak from the DA voltammetric peak. The electrochemical signal for 5-HT release was detected within 10-12 s and that for DA release within 12-15 s, after each biogenic amine diffused through the synaptic environment onto the microelectrode surface. The electrochemical signal for 5-HT and a separate electrochemical signal for DA are detected on the same voltammogram within 22-27 s; each electrochemical signal represents current changes in picoamperes, within seconds of detection time. The amplitude of each electrochemical signal reflects the changes in diffusion of each biogenic amine to the microelectrode surface. Each neurotransmitter has a distinct potential at which oxidation occurs; this results in a recording which has a distinct peak for a specific neurotransmitter. The concentration of each neurotransmitter within the synaptic environment is directly related to the electrochemical signal detected via the Cottrell equation. Voltammograms were recorded every 5 min. At the time that basal 5-HT release and basal DA release were recorded within same animal control, open-field behavioral studies were performed, also concurrently, by infrared photocell beams. The frequency of each behavioral parameter was monitored every 100 ms; the number of behavioral events, were summated every 5 min during the time course of study. Thus, the detection of neurotransmitters occurs in real time, while simultaneously monitoring the animal's behavior by infrared photocell beams. The results from the in vivo microvoltammetric and behavioral data from this study show that basal 5-HT release at distal A10 and A9 terminals dramatically increased with DA release. Moreover, each increase in basal 5-HT release, at both A10 and at A9 terminal fields occurred consistently and at the same time as each increase in open-field locomotion and stereotypy occurred naturally during the animal's exploration in a novel chamber. Thus, the terminology 'synchronous and simultaneous' describes aptly the correlation between 5-HT release at distal A10 and A9 terminal fields and open-field locomo

Nicotine blocks angiotensin II inhibition of LTP in the dentate gyrus.

Angiotensin (ANG)-containing axons, terminals, and receptors have been found in the hippocampus. When angiotensin II (ANG II) is administered to the dentate gyrus, long-term potentiation (LTP) induction, in response to medial perforant path stimulation, is inhibited and it can be blocked by losartan, an ANG II AT1 receptor antagonist. ANG II has been shown to mediate impairment of the retention of an inhibitory shock avoidance response and to be involved in ethanol and diazepam inhibition of dentate gyrus LTP, all of which can be blocked by losartan. Nicotine acetylcholine receptors are found in the hippocampus and nicotine is involved in the enhancement of complex and important psychological functions that are mediated by the hippocampus; therefore, the possibility that nicotine prevents the ANG II inhibition of dentate granule cell LTP was examined. Nicotine pretreatment reduced ANG II inhibition of LTP induction in a dose-dependent manner. Mecamylamine blocked the nicotine antagonism of ANG II-induced LTP inhibition and normal LTP occurred, whereas hexamethonium was ineffective in blocking these central effects of nicotine. Nicotine by itself did not affect normal LTP under these conditions. Nicotinic blocking of the ANG II inhibition of a frequency dependent type of synaptic plasticity provides a function for central nicotinic receptors and a possible mechanism of action a) to explain the enhancement of learning and memory by nicotine, b) an explanation for tobacco smoking while drinking alcohol, and c) a possible basis for the excessive use of tobacco in depression and schizophrenia that supports a possible therapeutic use of nicotine in some mental disorders.

Orexin-A (Hypocretin-1) and leptin enhance LTP in the dentate gyrus of rats in vivo.

Orexin-A (Hypocretin-1) has been localized in the posterior and lateral hypothalamic perifornical region. Orexin containing axon terminals have been found in hypothalamic nuclei and many other parts of the brain; for example, the hippocampus. Two types of orexin receptors have been discovered. Orexin 1 type of receptors have been described and been shown to be widely distributed in the rat brain including the hippocampus. Subsequently Orexin-A was found to impair both water maze performance and hippocampal long term potentiation (LTP). Leptin is expressed in adipose tissue and released into the blood where it affects food intake and can also produce widespread physiological changes mediated via autonomic preganglionic neurons, pituitary gland, and cerebral cortex. Immunoreactivity for leptin receptors has been found in various hypothalamic nuclei including the lateral hypothalamic area as well as the hippocampus especially in the dentate gyrus and CA1. Leptin receptor deficient rats and mice also show impaired LTP in CA1 and poor performance in the water maze. The present study was conducted to determine the effects of 0.0, 30, 60, 90, and 100 nM, orexin-A, and leptin, 0.0, 1.0, 100 nM, 1, and 10 microM, in 1.0 microl of ACSF, applied directly into the dentate gyrus, on LTP in medial perforant path dentate granule cell synapses in urethane anesthetized rats. Orexin-A specifically enhanced LTP at the 90 nM dose; and it was possible to block the enhancement by pretreating the animals with SB-334867, a specific orexin 1 receptor antagonist. Leptin enhanced normal LTP at 1.0 microM but inhibited LTP at lower and higher doses. These results and previous data indicate that the same peptide could possibly have different modulatory post synaptic effects in different hippocampal synapses dependent upon different types of post synaptic receptors.

Immunohistochemical localization of corticotropin releasing factor (CRF) in the hypothalamus of the squirrel monkey, Saimiri sciureus.

Corticotropin releasing factor immunoreactive (CRF-IR) neuronal cell bodies and fibers have been localized in both the paraventricular and supraoptic nuclei of the hypothalamus of the squirrel monkey. The major projection from these nuclei is to the median eminence and neural stem. A few CRF-IR fibers were found in the dorsal pars nervosa primarily adjacent to the pars intermedia. A rostral projection of CRF-IR fibers is associated with the suprachiasmatic nucleus and continues to septal areas. A caudally projecting bundle of fibers was observed entering the midbrain in neuropil adjacent to the aqueduct. The location of CRF-IR components is also compared with those containing vasopressin (AVP).

Angiotensin IV enhances LTP in rat dentate gyrus in vivo.

Angiotensins have been shown to play a significant role in a variety of physiological functions including learning and memory processes. Relatively recent evidence supports the increasing importance of angiotensin IV (Ang IV), in many of these functions previously associated only with Ang II, including learning and memory. An interesting hypothesis generated by these results has been that Ang II is a precursor for the production of a more active peptide fragment, Ang IV. Since Ang II impairs learning and memory, when administered directly or released into the hippocampal dentate gyrus, and inhibits long term potentiation (LTP) in medial perforant path-dentate granule cell synapses, as well; it remained to be seen what effects Ang IV had on LTP in these same synapses. Results of this study show clearly that Ang IV significantly enhances LTP, and the enhancement is both dose and time dependent. The following solutions of Ang IV were administered over a five min period, at the end of baseline and before the first tetanus was applied: 2.39, 4.78, and 9.56 nM. An inverted U-type dose related effect was observed. A complex time related effect was observed with a maximum at 5 min, a return to normal LTP at 30 min and a minimum below normal at 90 min, and a return to normal LTP at 120 min. The effects of the 4.78 nM solution were determined at the following intervals between administration and the first tetanus: 5, 15, 30, 60, 90, and 120 min. The enhancement of LTP can be prevented by pretreatment with Divalinal, an Ang IV antagonist, without any effect on normal LTP. Two solutions of Divalinal were used; 5 nM and 5 microM, and the 5 microM was more effective and completely blocked the enhancement of normal LTP. Results were also obtained with 4.78 nM Nle1-Ang IV (Norleucine), an Ang IV agonist. Norleucine was less effective than Ang IV in the enhancement of normal LTP and displayed a similar time course of activity. Both Ang IV and Norleucine produced a significant suppression of normal LTP at 90 min; that remains to be explained. However, the inhibition by Ang IV was dose dependent and was blocked by Divalinal. The fact that the Ang IV enhancement of normal LTP was blocked by losartan, an Ang II AT1 receptor antagonist, is puzzling since Divalinal had no effect on the inhibition of LTP by Ang II.

Systemic glutamate induces degeneration of a subpopulation of tyrosine hydroxylase-immunoreactive neurons in the rat area postrema.

Neuronal damage in the area postrema (AP) of 12-14-week-old male rats was induced by subcutaneous administration of monosodium glutamate (MSG). An immunocytochemical method was used to visualize catecholaminergic neurons in the AP after MSG-treatment. Some tyrosine hydroxylase-immunoreactive neurons exhibited marked signs of degeneration, while others appeared undamaged. We conclude that catecholamine-synthesizing neurons in the AP are differentially sensitive to the neuroexcitotoxic effect of systemic glutamate.

Immunohistochemical demonstration of serotonin-containing axons in the hypothalamus of the white-footed mouse, Peromyscus leucopus.

The wild white-footed mouse, Peromyscus leucopus, is commonly used for photoperiod studies utilizing physiological, behavioral, and other biological measures indicative of hypothalamic functions. Indoleamines, like melatonin and serotonin, are implicated in regulating these hypothalamic functions. Although neurochemical analyses of hypothalamic serotonin and its receptors have been reported for this species, the relevant neuroanatomy of the serotonin system within mouse hypothalamus has not been studied. A sensitive immunohistochemical method was used to detect serotonin within axons of coronal sections of formaldehyde fixed forebrain from P. leucopus. Large, medium and small diameter serotonin axons were evaluated in most regions, or nuclei, of the hypothalamus rostral to the mammillary region. A fourth type of serotonin axon was observed to have morphology characteristic of terminal arbors. The density of serotonin axons ranged from no staining to very high density similar to other species for which reports exist, i.e., rat, cat, and monkey. The ventromedial hypothalamic nucleus had distinctively lesser density of serotonin axons in this mouse than other species. Evidence of terminal arborization in hypothalamic nuclei and regions was evident. Neuroendocrine, autonomic, and behavioral functions of the hypothalamus are suggested to be regulated by input from serotonin terminals in this wild species of mouse, in correlation with receptor localization as reported by others.

Systemic glutamate induces degeneration of a subpopulation of serotonin-immunoreactive neurons in the area postrema of rats.

Neuronal damage in the area postrema (AP) of adult Sprague-Dawley rats was induced by subcutaneous administration of monosodium glutamate (MSG; 9 mg/g b.wt.). Serotonin-immunoreactive (5-HT-IR) neurons were visualized in the AP 3 h or 7 days after control or MSG treatment. At 3 h post MSG, many 5-HT-IR neurons showed morphological signs of degeneration, such as, cytoplasmic vacuolization, chromatin clumping and dendritic hypertrophy. MSG treatment caused a 30% reduction of detectable AP 5-HT neurons after 7 days. We conclude that a subpopulation of serotonergic neurons in the AP is sensitive to the neuroexcitotoxic effect of systemic glutamate.

Light microscopic immunocytochemical evidence of converging serotonin and dopamine terminals in ventrolateral nucleus accumbens.

The mesencephalic tegmentum contains monoaminergic neurons that project to the nucleus accumbens (NAcc). These monoaminergic neurons consist of the serotonergic (5-HT) neurons of the dorsal and median raphe and the dopaminergic (DA) neurons of the ventral tegmental area (VTA). Recent neurochemical reports describe cocaine-induced alterations in dopamine and serotonin release in NAcc that has coincidental occurrence both spatially and temporally, as shown by in vivo voltammetry. There is a functional role for 5-HT-DA interactions within the NAcc in the underlying mechanism of action of cocaine as well as for 5-HT in A10 DA neurons in the basal or endogenous state whether or not cocaine-relevant reward circuits are involved. Our objective was to study the neuroanatomic localization of tyrosine hydroxylase-containing (TH) and 5-HT-containing axons in the ventrolateral region of the rat NAcc, where codetection of monoamines had been assessed. The significance of this vINAcc is its reciprocal connectivity with VTA, which contains the somatodendritic portions of the mesoacumbens DA neurons. The results showed that, in the vINAcc, the core contained a dense terminal field of TH axons that had an extensive overlap with 5-HT axons in the periphery within the core. Because the in vivo electrochemical codetection of DA and 5-HT assessed in the ventral-most aspect of this overlap zone can be correlated with terminal release, a functional interaction of 5-HT and DA at postsynaptic sites in vINAcc is possible.

Anterograde neuroanatomical tract tracing with central nervous system injections of immunoglobulin G: a light and electron microscopic evaluation.

Normal rabbit serum (NRS) was pressure injected into the forebrain of rats to be tested as an anterograde neuroanatomical tracing substance. Undiluted NRS was stereotaxically injected into the bed nucleus of the stria terminalis (BST) with a 1-microliter Hamilton syringe. Postinjection survival times ranged from 24 h to 14 days. An immunohistochemical method utilizing goat anti-rabbit IgG antibody was used to detect the rabbit IgG within vibratome sections. Visualization of the final reaction product (diaminobenzidine, DAB) was enhanced by a silver/gold postintensification (SGI) method. Rabbit IgG-containing neural structures were examined at both light and electron microscopic (EM) levels. At the injection site neuronal soma, dendrites and axons were filled homogeneously with the SGI-DAB at 24 h, 48 h, 7 days and 14 days indicating local neuronal uptake, storage and transport of rabbit IgG. In the hypothalamus many anterogradely filled axons were present and displayed short collateral branches and terminals. EM examination revealed synaptic terminals containing IgG, without signs of transsynaptic transport after 14 days. Signs of retrograde transport of IgG were never observed. A propensity of neurons to take up, sequester and anterogradely transport immunoglobulin G is indicated.

Serotonin-CRF interaction in the bed nucleus of the stria terminalis: a light microscopic double-label immunocytochemical analysis.

The purpose of the present study was to examine in the bed nucleus of the stria terminalis (BST) of the rat brain the morphological characteristics of interactions between corticotropin-releasing factor (CRF)-producing neuron populations and serotonin (5-HT) axons. A double-label immunocytochemical, light microscopic technique was used to demonstrate axosomatic and axodendritic interactions between 5-HT axons and CRF neurons in the BST. Both the dorsolateral and ventrolateral subpopulations of CRF neurons were targets for the 5-HT afferents. Projections of monoamine neurons to the BST and the CRF neurons that are in the BST are implicated as being major contributors to the neurochemically mediated central regulation of the stress response.

Monoamine innervation of bed nucleus of stria terminalis: an electron microscopic investigation.

Immunocytochemical studies showed distinctive monoamine input to the bed nucleus of the stria terminalis (BST). A comparison of axons immunoreactive (IR) for a catecholamine synthetic enzyme [tyrosine hydroxylase (TH) or dopamine beta-hydroxylase (DBH) or phenylethanolamine-N-methyl transferase (PNMT)] or serotonin (5-HT) was performed. TH-IR axons had a greater density in the lateral BST, but DBH-IR and 5-HT-IR axons had a greater density in the medial BST. PNMT-IR axons were dense in the intermediate BST. TH-IR axons had a greater density than DBH- and PNMT-IR axons in the dorsolateral BST, but DBH-IR axons had the greatest density in the ventrolateral BST. Ultrastructural studies revealed that TH-IR terminals formed synapses with soma, dendrites, spines, and axons in the dorsolateral BST. DBH-IR terminals formed synapses with dendritic shafts and spines, and 5-HT-IR terminals formed synapses with dendrites in the ventrolateral BST. Only some 5-HT-IR axons were myelinated. The medial vs. lateral organization of the noradrenergic and dopaminergic afferents in the BST of the rat brain is now evident and is similar to the human brain. The medial-lateral functional subdivision of the BST is supported by the pattern of dopaminergic, noradrenergic, and serotonergic afferents. This demonstration of epinephrine-producing afferents in the BST is the first detailed description of adrenergic input to the BST and aided the determination that catecholaminergic innervation of the ventrolateral BST is predominantly noradrenergic as has been proposed for many years. However, the additional demonstration of rich dopaminergic innervation of the dorsolateral subnucleus suggests further division of the BST into dorsal and ventral functional subgroups.

Catecholamine-CRF synaptic interaction in a septal bed nucleus: afferents of neurons in the bed nucleus of the stria terminalis.

Projections of catecholamine neurons to the bed nucleus of the stria terminalis (BST), especially its corticotropin releasing factor (CRF)-producing neurons, are implicated as being major contributors to the neurochemically mediated central regulation of the stress response. The purpose of the present study was to examine in the BST of the rat brain the morphological characteristics of interactions between two neuron populations of the brain, catecholaminergic and CRF neurons. A double-label immunocytochemical, light and electron microscopic technique allowed the demonstration of the synaptic interaction between dopamine (DA, i.e., tyrosine hydroxylase-containing) and norepinephrine (NE, i.e., dopamine-beta-hydroxylase-containing) axons and CRF neurons in the BST. DA terminals formed synapses with dendrites and soma of CRF neurons in the dorsolateral BST. NE terminals formed synapses with dendrites of CRF neurons in the ventrolateral BST. In conclusion, catecholamine afferents can directly affect the contribution of CRF neurons of the BST to an animals response to stress.

Lateral hypothalamic stimulation inhibits dentate granule cell LTP: direct connections.

We discovered that angiotensin II (Ang II) applied directly to the dentate gyrus inhibited LTP induction in medial perforant path-dentate granule cell synapses and that the inhibition can be blocked by losartan, an Ang II AT1 receptor specific antagonist. In the first part of this study we found that electrical stimulation of the lateral hypothalamus (LH) inhibits LTP in these synapses and the inhibition can be blocked by pretreating the animals with losartan, indicating that LH angiotensin-containing neurons project to the dentate gyrus. Results of the second part of the study demonstrate clearly that some angiotensin-containing LH neurons project directly to dentate granule cells. LH neurons were identified by retrograde tracers applied to the granule cell layer. Double-labeled neurons containing angiotensin and HRP were sparsely distributed and both fusiform and multipolar LH neurons appeared in a small cluster lateral and ventral to the fornix at the level of the paraventricular nucleus. Large numbers of angiotensin staining neurons were observed in the hypothalamus. Results support our hypothesis that some angiotensin containing LH neurons project directly to the dentate gyrus.

Air righting: role of the NMDA receptor channel and hippocampal LTP.

Air righting results in an animal turning over when it is dropped from a height in an inverted position. In the rat, air righting is a complex set of movements that depends only on an intact labyrinth and the normal vestibular input. Visual modulation of air righting does not develop until adulthood; and the ability to estimate the time to impact requires bilateral visual cues and indicates that air righting is a complex set of perceptually controlled movements and learning. The general purpose of this study was to determine the role of the NMDA receptor-ion channel on air righting and hippocampal LTP. Specifically: to measure the effects of various doses of CPP, an NMDA receptor antagonist, and MK-801, an ion channel blocker, on (a) air righting and (b) hippocampal LTP induction in medial perforant path-granule cell synapses. The following doses were used: CPP-0, 1, 5, and 10 mg/kg i.p.; MK-801-0. 0, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg/kg i.p. Data were analyzed by appropriate ANOVAs and post hoc tests. Results were significant and demonstrate dose-dependent impairment of air righting and inhibition of LTP for both CPP and MK-801, implicating the role of the NMDA receptor and Na(+)/K(+)/Ca(2)+ channel in these effects. Air righting is a complex behavior and appears to be dependent upon NMDA mediated hippocampal LTP.

Ethanol effects on dentate granule cell LTP.

In previous studies we identified a lateral hypothalamic area (LHA) sensitive to ethanol, < 5.0 mM, when the perifornical region of the area is perfused with different concentrations of ethanol. Some of these perifornical neurons contain angiotensin (Ang) and project directly to the dentate gyrus where angiotensin is released and inhibits LTP in medial perforant path-dentate granule cell synapses. The AT1 subtype receptor is involved because pretreatment with losartan, an AT1 antagonist, prevents Ang II, diazepam, and ethanol impairment of LTP as well as their effects on behavior. There is a possibility that these effects were not specific to the LHA; but might be attributable to direct effects of ethanol on postsynaptic granule cells due to diffusion of the ethanol in the extracellular space or by the circulatory system. The purpose of the present study was to determine a dose effect of ethanol on LTP in these same synapses when the dentate gyrus was perfused with several different concentrations of ethanol under the same conditions in urethane anesthetized rats. Ethanol was administered directly into the dentate gyrus by means of a fine stainless steel cannula attached approximately 1.0 mm from the tip of the glass capillary recording electrode. Results show that the threshold for ethanol in the dentate is higher by a factor of ten, > 30 mM and < 50 mM; and that at higher doses ethanol can have a direct effect on the LHA; and possibly toxic due to increasing ethanol in the blood circulatory system.

Bicuculline sensitive depressor response to ethanol infusion into the lateral hypothalamus.

Decreased GABA function in the hypothalamus increases mean arterial pressure (MAP) and heart rate (HR). Since ethanol acts on GABA-A receptors, blocking GABA-A receptors can prevent a decrease of MAP and HR by ethanol in the lateral hypothalamus (LH). Ethanol at 5-30 mM, with or without 25 ng/microl bicuculline, was infused into the LH, and the activity of the site was validated with 100 nmoles of serotonin. Male rats were anesthetized with pentobarbital, and the femoral artery was catheterized to measure MAP and HR. Microinfusion was performed with a 28-gauge cannula placed into the LH. Serotonin increased MAP and HR within 15 sec. Ethanol decreased the MAP by -21.15 +/- 3.92 mmHg and HR by -53.61 +/- 14.95 BPM, at 15 min, which recovered by 15 min after the infusion was terminated. These maximum decreases were produced by 20 mM ethanol giving a U-shaped dose response. The aCSF vehicle had no effect. Bicuculline prevented ethanol-induced changes and had no effect when administered alone. Both serotonin and ethanol have direct effects on LH neurons with cardiovascular function. Ethanol produces this effect through GABA-A receptors.

Demonstration of distinct corticotropin releasing factor--containing neuron populations in the bed nucleus of the stria terminalis. A light and electron microscopic immunocytochemical study in the rat.

Immunocytochemical light and electron microscopic studies revealed two distinct populations of corticotropin releasing factor (CRF) - containing neurons, a dorsolateral and ventrolateral group, located in the bed nucleus of the stria terminalis (BST) of the rat brain. CRF neurons of the dorsolateral group had a smaller diameter and more primary dendrites than those of the ventrolateral group. CRF neurons in the dorsolateral BST had both somatic and dendritic spines, smooth contoured nuclei, and many dense and alveolate vesicles in their cytoplasm. Whereas, CRF neurons in the ventrolateral BST had only dendritic spines, irregularly-shaped indented nuclei and contained only alveolate vesicles in their cytoplasm. The only obvious difference in the type of unidentified afferents that synapsed on the CRF neurons of the BST could be attributed to the presence of the somatic spines on the CRF neurons of the dorsolateral population. Otherwise, the CRF neurons of the BST had a profuse innervation that included axosomatic, axospinous and axodendritic synapses. CRF-containing axons were distributed unevenly throughout the BST. The density of CRF axons was greatest in the lateral subdivisions of the BST, but the ventromedial BST contained many more CRF axons than the dorsomedial BST. The presence of these two CRF neuron populations in the BST suggests functional subdivision beyond previous proposals of a medial and lateral separation of function. Now there is additional morphological evidence to support the proposal of a dorsal and ventral separation of function within the BST.

Tyrosine hydroxylase- and corticotropin releasing factor-immunoreactivity in the olfactory bulb of the opossum (Didelphis virgianina).

The nerve fiber layer of the opossum olfactory bulb, formed by axons originating from bipolar neurons in the olfactory epithelium, and glomeruli are intensely immunoreactive for olfactory marker protein. The surrounding extra-glomerular neuropil contains numerous periglomerular neurons immunoreactive for either tyrosine hydroxylase or corticotropin releasing factor. Dendrites of both types of immunoreactive neurons extend into the intraglomerular neuropil. CRF-immunoreactive neurons are fewer in number than TH-immunoreactive neurons and are observed primarily in the periglomerular region. Occasional, scattered TH-immunoreactive neurons are seen in the deeper layers of the olfactory bulb.