Intrinsic GABAergic neurons in the rat central extended amygdala

The present study examined the distribution, morphology, and connections of gamma-aminobutyric acid-immunoreactive (GABA-IR) neurons in the three principal components of the central extended amygdala: the central amygdaloid nucleus, the bed nucleus of the stria terminalis (BNST) and the sublenticular substantia innominata. In the central nucleus, large numbers of GABA-IR neurons were identified in the lateral, lateral capsular, and ventral subdivisions, though in the medial subdivision, GABA-IR neurons were only present at very caudal levels. Combined immunocytochemistry-Golgi impregnation revealed that GABA-IR neurons in the lateral central nucleus were medium-sized spiny neurons that were morphologically similar to GABAergic neurons in the striatum. Injections of horseradish peroxidase into the bed nucleus of the stria terminalis labeled a major proportion of the GABA-IR neurons in the central nucleus. In the bed nucleus, the majority of GABA-IR neurons were located in the anterolateral subdivision, ventral part of the posterolateral subdivision and the parastrial subdivision. GABA-IR neurons in the anterolateral bed nucleus were of the typical medium-sized spiny type. Injections of horseradish peroxidase into the central nucleus labeled a few GABA-IR neurons in the posterior part of the anterolateral bed nucleus. GABA-IR neurons were identified in the sublenticular substantia innominata and medial shell of the nucleus accumbens and contributed to the continuum of GABA-IR extending from the central nucleus to the bed nucleus. Injections of horseradish peroxidase (HRP) into the central nucleus, but not the BNST, labeled a few GABA-IR neurons in the substantia innominata. The data point to GABA-IR neurons being a characteristic feature of the central extended amygdala and that GABA-IR neurons participate in the long intrinsic connections linking the major components of this structure. Since lesions of the stria terminalis and basolateral amygdaloid nucleus failed to deplete GABA-IR terminals in the central nucleus, the role of GABA in local and short intrinsic connections in the central extended amygdala is discussed. Further, physiological findings implicating the intrinsic GABAergic system of the central extended amygdala in the tonic inhibition of brainstem efferents are reviewed.     

Selective anesthetic inhibition of brain nitric oxide synthase

BACKGROUND: It has been postulated that nitric oxide (NO) is a neurotransmitter involved in consciousness, analgesia, and anesthesia. Halothane has been shown to attenuate NO-mediated cyclic guanosine monophosphate accumulation in neurons, and a variety of anesthetic agents attenuate endothelium-mediated vasodilation, suggesting an interaction of anesthetic agents and the NO-cyclic guanosine monophosphate pathway. However, the exact site of anesthetic inhibitory action in this multistep pathway is unclear. The current study examines effects of volatile and intravenous anesthetic agents on the enzyme nitric oxide synthase (NOS) in brain. METHODS: NOS activity was determined by in vitro conversion of [14C]arginine to [14C]citrulline. Wistar rats were decapitated and cerebellum quickly harvested and homogenized. Brain extracts were then examined for NOS activity in the absence and presence of the volatile anesthetics halothane and isoflurane, and the intravenous agents fentanyl, midazolam, ketamine, and pentobarbital. Dose-response curves of NOS activity versus anesthetic concentration were constructed. Effects of anesthetics on NOS activity were evaluated by analysis of variance. RESULTS: Control activities were 57.5 +/- 4.5 pmol.mg protein-1.min-1 in the volatile anesthetic experiments and 51.5 +/- 6.5 pmol.mg protein-1.min-1 in the intravenous anesthetic experiments. NOS activity was not affected by ketamine (< or = 1 x 10(-4) M), pentobarbital (< or = 5 x 10(-5) M), fentanyl (< or = 1 x 10(-5) M), and midazolam (< or = 1 x 10(-5) M). Halothane decreased NOS activity to 36.7 +/- 2.5 (64% of control, P < 0.01 from control), 23.8 +/- 4.3 (41%, P < 0.01 from control and < 0.05 from 0.5% halothane), 25.2 +/- 3.8 (44%, P < 0.01 from control and < 0.05 from 0.5% halothane), and 19.7 +/- 2.8 (34%, P < 0.01 from control and < 0.05 from 0.5% halothane) pmol.mg protein-1.min-1 at 0.5, 1.0, 2.0, and 3.0% vapor. Isoflurane decreased NOS activity to 48.9 +/- 6.1 (85% of control), 46.0 +/- 3.2 (80%, P < 0.05 from control), 40.3 +/- 5.1 (70%, P < 0.05 from control), and 34.2 +/- 4.0 (60%, P < 0.05 from control and 0.5% and 1.0% isoflurane) pmol.mg protein-1.min-1 at 0.5, 1.0, 1.5, 2.0% vapor, respectively. CONCLUSIONS: Volatile anesthetics inhibit brain NOS activity in an in vitro system, but the intravenous agents examined have no effect at clinically relevant concentrations. This inhibition suggests a protein-anesthetic interaction between halothane, isoflurane, and NOS. In contrast, intravenous agents appear to have no direct effect on NOS activity. Whether intravenous agents alter signal transduction or regulatory pathways that activate NOS is unknown.     

Enkephalinergic innervation of GABAergic neurons in the dorsal raphe nucleus of the rat

The preembedding double immunoreaction method was used to study interrelations of enkephalinergic and GABAergic neuronal elements in the dorsal raphe nucleus of the Wistar albino rat. The enkephalin-like neuronal elements were immunoreacted by the peroxidase-antiperoxidase method and silver-gold intensified, which showed strongly and was specific. The GABA-like immunoreactive neurons were immunoreacted by the peroxidase-antiperoxidase method only. GABA-like neural somata were postsynaptic to both the enkephalin-like immunoreactive and the non-immunoreactive axon terminals. The enkephalin-like immunoreactive axon terminals were also found to synapse GABA-like immunoreactive dendrites. The GABA-like immunoreactive neuronal elements were also found to receive synapses from other non-immunoreactive as well as GABA-like immunoreactive axon terminals. Almost all of the synapses appeared to be asymmetrical. Possible functional activity of interactions among the enkephalinergic, GABAergic, and serotonergic neuronal elements in the dorsal raphe nucleus are discussed.     

Lidocaine toxicity in primary afferent neurons from the rat

Evidence from both clinical studies and animal models suggests that the local anesthetic, lidocaine, is neurotoxic. However, the mechanism of lidocaine-induced toxicity is unknown. To test the hypothesis that toxicity results from a direct action of lidocaine on sensory neurons we performed in vitro histological, electrophysiological and fluorometrical experiments on isolated dorsal root ganglion (DRG) neurons from the adult rat. We observed lidocaine-induced neuronal death after a 4-min exposure of DRG neurons to lidocaine concentrations as low as 30 mM. Consistent with an excitotoxic mechanism of neurotoxicity, lidocaine depolarized DRG neurons at concentrations that induced cell death (EC50 = 14 mM). This depolarization occurred even though voltage-gated sodium currents and action potentials were blocked effectively at much lower concentrations. (EC50 values for lidocaine-induced block of tetrodotoxin-sensitive and -resistant voltage-gated sodium currents were 41 and 101 microM, respectively.) At concentrations similar to those that induced neurotoxicity and depolarization, lidocaine also induced an increase in the concentration of intracellular Ca++ ions ([Ca++]i; EC50 = 21 mM) via Ca++ influx through the plasma membrane as well as release of Ca++ from intracellular stores. Finally, lidocaine-induced neurotoxicity was attenuated significantly when lidocaine was applied in the presence of nominally Ca(++)-free bath solution to DRG neurons preloaded with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Our results indicate: 1) that lidocaine is neurotoxic to sensory neurons; 2) that toxicity results from a direct action on sensory neurons; and 3) that a lidocaine-induced increase in intracellular Ca++ is a mechanism of lidocaine-induced neuronal toxicity.     

Electron microscopic evidence for a cholinergic innervation of GABAergic parvalbumin-immunoreactive neurons in the rat medial septum

Neutrophilic urticaria (NU) is a histologically defined entity, but its clinical and pathogenetic aspects are poorly understood. We investigated 22 NU patients whom we identified by examining 118 biopsies of weals. The patients comprised 11 of 20 with acute urticaria, nine of 49 with chronic urticaria, one of 10 with cold urticaria and one of 10 controls undergoing prick tests. Clinically, NU patients had a shorter mean duration of disease than other urticaria patients and significantly increased erythrocyte sedimentation rate and leucocytosis. Histologically, not only neutrophil counts, but to a lesser extent also eosinophil counts and mononuclear cell infiltrates were significantly increased in lesional skin of NU, and there was more marked vasodilatation and endothelial swelling. On immunohistochemistry, increased tumour necrosis factor alpha and interleukin (IL)-3 expression was noted, compared with other urticarias, whereas IL-8 expression was only minor. These data characterize NU as an acute phase urticarial reaction associated with an intense inflammatory infiltrate and marked upregulation of some mast cell-derived cytokines.     

Electrical membrane properties of trapezoid body neurons in the rat auditory brain stem are preserved in organotypic slice cultures

The medial nucleus of the trapezoid body (MNTB) is a conspicuous structure in the mammalian auditory brain stem. It is a major component of the superior olivary complex and is involved in sound localization. Recently, organotypic slice culture preparations of the superior olivary complex were introduced to investigate the development of inhibitory and excitatory projections (Sanes and Hafidi, 1996; Lohmann et al., 1998). In the present article, we further assessed the organotypicity of our culture system (Lohmann et al., 1998) and examined electrical membrane properties of MNTB neurons expressed under culture conditions. To do so, MNTB neurons from early postnatal rats (P3-5) were studied after 3-6 days in vitro (DIV) by whole-cell patch-clamp recordings. Their mean resting potential was -59 mV, the input resistance averaged 171 Momega, and the average time constant was 3 ms. Four types of voltage-activated conductances were observed in voltage-clamp recordings. All cells expressed a tetrodotoxin (TTX)-sensitive sodium current. Two types of potassium currents could be characterized: a tetraethylammonium (TEA) -sensitive and a 4-aminopyridine (4-AP)-sensitive conductance, both of which are composed of a transient and a sustained component. Finally, an inwardly rectifying current, activated by hyperpolarizing voltage steps, was found. In current-clamp recordings, depolarizing current pulses typically elicited a single action potential. In the presence of 4-AP, however, these current pulses induced a train of action potentials. The duration of action potentials was increased by 4-AP and the afterhyperpolarization was reduced. Hyperpolarizing current injections induced a "sag" in the membrane potential, indicating the influence of an inwardly rectifying current. Our results demonstrate that MNTB neurons in slice cultures have electrical membrane properties comparable to those of their counterparts in acute slices.     

Biphasic response of spinal GABAergic neurons after a lumbar rhizotomy in the adult rat

The expression of gamma-aminobutyric acid (GABA) and of the isoforms of the enzyme involved in its synthesis, glutamic acid decarboxylase (GAD), is modified in several rat brain structures in different injury models. The aim of the present work was to determine whether such plasticity of the GABAergic system also occurred in the deafferented adult rat spinal cord, a model where a major reorganization of neural circuits takes place. GABAergic expression following unilateral dorsal rhizotomy was studied by means of non-radioactive in situ hybridization to detect GAD67 mRNA and by immunohistochemistry to detect GAD67 protein and GABA. Three days following rhizotomy the number of GAD67 mRNA-expressing neurons was decreased in the superficial layers of the deafferented horn, while GABA immunostaining of axonal fibres located in this region was highly increased. Seven days after lesion, on the other hand, many GAD67 mRNA-expression neurons were bilaterally detected in deep dorsal and ventral layers, this expression being correlated with the increased detection of GAD67 immunostained somata and with the reduction of GABA immunostaining of axons. GABA immunostaining was frequently found to be associated with reactive astrocytes that exhibited intense immunostaining for glial fibrillary acidic protein (GFAP) but remained GAD67 negative. These results indicate that degeneration of afferent terminals induces a biphasic response of GABAergic spinal neurons located in the dorsal horn and show that many spinal neurons located in deeper regions re-express GAD67, suggesting a possible participation of the local GABAergic system in the reorganization of disturbed spinal networks.     

Calcium channels in the GABAergic presynaptic nerve terminals projecting to meynert neurons of the rat

Effects of selective Ca2+ channel blockers on GABAergic inhibitory postsynaptic currents (IPSCs) were studied in the acutely dissociated rat nucleus basalis of Meynert (nBM) neurons attached with nerve endings, namely, the "synaptic bouton" preparation, and in the thin slices of nBM, using nystatin perforated and conventional whole-cell patch recording modes, respectively. In the synaptic bouton preparation, nicardipine (3 x 10(-6) M) and omega-conotoxin-MVIIC (3 x 10(-6) M) reduced the frequency of spontaneous postsynaptic currents by 37 and 22%, respectively, whereas omega-conotoxin-GVIA had no effect. After blockade of L- and P/Q-type Ca2+ channels, successive removal of Ca2+ from external solution had no significant effect on the residual spontaneous activities, indicating that N-, R-, and T-type Ca2+ channels are not involved in the spontaneous GABA release. Thapsigargin, but not ryanodine, increased the frequency of spontaneous IPSCs in both the synaptic bouton and slice preparations, suggesting the partial contribution of the intracellular Ca2+ storage site to the spontaneous GABA release. In contrast, omega-conotoxin-GVIA (3 x 10(-6) M) and omega-conotoxin-MVIIC (3 x 10(-6) M) suppressed the evoked IPSCs by 31 and 37%, respectively, but nicardipine produced no significant effect. The residual evoked currents were abolished in Ca2+-free external solution but not in the external solution containing 10(-5) M Ni2+, suggesting the involvement of N-, P/Q-, and R-type Ca2+ channels but not L- and T-type ones in the evoked IPSCs. Neither thapsigargin nor ryanodine had any significant effects on the evoked IPSCs. It was concluded that Ca2+ channel subtypes responsible for spontaneous transmitter release are different from those mediating the transmitter release evoked by nerve stimulation.     

Mechanism of toxicity of precocene II in rat hepatocyte cultures

Precocene II was more toxic in 24 hour cultures than in 72 hour cultures of rat hepatocytes. In 24 hour cultures, there was no observable toxicity at 75 microM precocene II after exposure for 6 hours, but after 24 hours, 65% of the cells were dead. In contrast, although 794 microM killed 50% of the cells in the 72 hour cultures after a 24 hour exposure, 1 mM killed 96% of the cells within 6 hours. In both 24 and 72 hour cultures, cell death was preceded by a rapid, early loss of mitochondrial membrane potential, followed by decreases in glutathione, reduced pyridine nucleotide status, and plasma membrane Na+/K+-ATPase activity. There was also a rapid loss of ATP in the 72 hour cultures but not in the 24 hour cultures; therefore, onset of cell death may be closely linked to loss of ATP. Inhibition of cytochrome P-450 prevented the toxicity, and partially protected against the loss of membrane potential and glutathione, in 24 hour cultures but was ineffective in 72 hour cultures. Therefore, in addition to depletion of glutathione, precocene II appears to damage mitochondria and plasma membrane functions and can do so by more than one pathway.     

Projections of GABAergic and cholinergic basal forebrain and GABAergic preoptic-anterior hypothalamic neurons to the posterior lateral hypothalamus of the rat

Within the basal forebrain, gamma-aminobutyric acid (GABA)-synthesizing neurons are codistributed with acetylcholine-synthesizing neurons (Gritti et al. [1993] J. Comp. Neurol. 329:438-457), which constitute one of the major forebrain sources of subcortical afferents to the cerebral cortex. In the present study, descending projections of the GABAergic and cholinergic neurons were investigated to the lateral posterior hypothalamus (LHp) through which the medial forebrain bundle passes and where another major forebrain source of subcortical afferents is situated. Retrograde transport of cholera toxin b subunit (CT) from the LHp was combined with immunohistochemical staining for glutamic acid decarboxylase (GAD) and choline acetyl transferase (ChAT) using a sequential peroxidase-antiperoxidase (PAP) technique. A relatively large number of GAD+ neurons (estimated at approximately 6,200), which represented > 15% of the total population of GAD+ cells in the basal forebrain (estimated at approximately 39,000), were retrogradely labeled from the LHp. These cells were distributed through the basal forebrain cell groups, where ChAT+ cells are also located, including the medial septum and diagonal band nuclei, the magnocellular preoptic nucleus, and the substantia innominata, with few cells in the globus pallidus. In these same nuclei, a small number of ChAT+ cells were retrogradely labeled (estimated at approximately 800), which represented only a small percentage (< 5%) of the ChAT+ cell population in the basal forebrain (estimated at approximately 18,000). Both the GAD+ and ChAT+ LHp-projecting neurons represented a small subset of their respective populations in the basal forebrain, distinct from the magnocellular, presumed cortically projecting, basal neurons. In addition to the GAD+ cells in the basal forebrain, GAD+ cells in the adjacent preoptic and anterior hypothalamic regions were also retrogradely labeled in significant numbers (estimated at approximately 5,500) and proportion (> 20%) of the total population (estimated at approximately 30,000) from the LHp. The retrogradely labeled GAD+ neurons were distributed in continuity with those in the basal forebrain through the lateral preoptic area, medial preoptic area, bed nucleus of the stria terminals, and anterior and dorsal hypothalamic areas. Of the large number of cells that project to the LHp in the basal forebrain and preoptic-anterior hypothalamic regions (estimated at approximately 66,000), the GAD+ neurons represented a significant proportion (> 15%) and the ChAT+ neurons a very small proportion (approximately 2%). The relative magnitude of the GABAergic projection suggests that it may represent an important inhibitory influence of the descending efferent output from the basal forebrain and preoptic-anterior hypothalamic regions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Facilitation of miniature GABAergic currents by ruthenium red in neonatal rat hippocampal neurons

The whole cell configuration of the patch-clamp technique was used to study the modulation gamma-aminobutyric acid (GABA)-mediated postsynaptic currents by ruthenium red in CA3 hippocampal neurons in slices obtained from postnatal (P) days P6-P10 old rats. In the presence of kynurenic acid (1 mM), ruthenium red (100 microM) completely blocked stimulus-elicited GABA-mediated postsynaptic currents and reduced by 50% the amplitude of the spontaneous ones. Ruthenium red (100 microM) increased the frequency but not the amplitude of miniature GABAergic currents recorded in the presence of tetrodotoxin (1 microM) and kynurenic acid (1 mM), an effect that was prevented by heparin (100 microM). Ruthenium red did not modify the kinetics of miniature postsynaptic currents and the currents induced by exogenous application of GABA (10 microM) in the presence of tetrodotoxin, suggesting that its action was presynaptic in origin. The effects of ruthenium red on quantal GABA release was independent of external calcium. In a nominally Ca2+-free solution the potentiating effect induced by this polyvalent cation on miniature postsynaptic currents was still present. Intracellular calcium stores were not involved in ruthenium red action, because this polyvalent cation was able to facilitate miniature currents also in the presence of thapsigargin (10-20 microM). These results indicate that ruthenium red has a dual action on GABA release from GABAergic interneurons: it reduces the amplitude of spontaneous events and increases the frequency of miniature currents. The former effect is calcium-dependent, whereas the latter is calcium independent.     

Effects of prenatal ethanol exposure on parvalbumin-expressing GABAergic neurons in the adult rat medial septum

Exposure of human fetuses to ethanol often results in the fetal alcohol syndrome. Animal models of fetal alcohol syndrome have been developed and used to examine the consequences of prenatal ethanol exposure on the central nervous system. The objective of this study was to determine the long-term effects of prenatal ethanol exposure on parvalbumin-expressing (PA+) GABAergic neurons of the rat medial septum. Pregnant Long-Evans rats were maintained on 1 of 3 diets from gestational day 0 to 21: an ethanol-containing liquid diet in which ethanol accounted for 35% of the total calories, a similar diet with the isocaloric substitution of sucrose for ethanol, or a lab chow control diet. Offspring were killed on postnatal day 60, and their brains were prepared for parvalbumin immunocytochemistry. Female rats exposed to the ethanol-containing diet during gestation had 42% fewer PA+ neurons in the medial septum and reduced PA+ cell density when compared with female rats exposed to the sucrose diet. Ethanol females also had fewer PA+ neurons per unit volume than sucrose females. Male rats exposed to ethanol did not display a similar reduction in PA+ neurons or density. No effect of prenatal diet was found on the area or volume of the medial septum, nor were cell diameters affected. As such, prenatal exposure to ethanol seems to reduce permanently the number of PA+ neurons in the female rat medical septum without affecting area, volume, or neuronal size. Functional implications and possible relations to the fetal alcohol syndrome are discussed.     

异丙酚对大鼠海马神经元低电压激活钙电流的抑制作用

目的:观察异丙酚对大鼠海马锥体神经元低电压激活钙电流[low-voltage-activated calcium currents,ICa(LVA)]的影响.方法:培养Wistar大鼠海马锥体神经元,采用全细胞膜片钳技术测定ICa(LVA).加用不同浓度(3、10、30、100、300μmol/L)异丙酚后,计算ICa(LVA)抑制率,建立异丙酚的浓度-效应曲线,选择20μmol/L异丙酚作ICa(LVA)的激活及失活曲线.结果:3 μmol/L的异丙酚对ICa(LVA)的电流幅度无影响;10、30、100、300 μmol/L的异丙酚对ICa(LVA)的电流幅度抑制率分别为(12.6±4.1)%、(29.2±5.7)%、(36.6±5.3)%、(31.6±2.6)%.拟合后的浓度-效应曲线的IC50为16.8 μmol/L,Hill系数为0.15.激活曲线的半数最大激活膜电位(V1/2)由(-10±1)mV移动到(-11±2)mV;K分别为12±1和8±1;失活曲线的V1/2分别为(-25±1)mV和(-25±5)mV,K分别为15±1和16±3.20 μmol/L异丙酚均未使ICa(LVA)的激活曲线及稳态失活曲线发生明显移动.结论:异丙酚对ICa(LVA)通道有抑制作用,异丙酚对中枢神经系统的麻醉作用可能与ICa(LVA)抑制有关.     

Isoform-specific effect of apolipoprotein E on cell survival and beta-amyloid-induced toxicity in rat hippocampal pyramidal neuronal cultures

Dermatosparaxis is a recessive disorder of animals (including man) which is caused by mutations in the gene for the enzyme procollagen N-proteinase and is characterised by extreme skin fragility. Partial loss of enzyme activity results in accumulation of pNcollagen (collagen with N-propeptides) and abnormal collagen fibrils in the fragile skin. How the N-propeptides persist in the tissue and how abnormal fibril morphology results in fragile skin is poorly understood. Using biochemical and quantitative mass mapping electron microscopy we showed that the collagen fibrils in the skin of a dermatosparactic calf contained 57% type I pNcollagen and 43% type I collagen and the fibrils were irregularly arranged in bundles and hieroglyphic in cross-section. Image analysis of the fibril cross-sections suggested that the deviation from circularity of dermatosparactic fibrils was caused by N-propeptides of pNcollagen being located at the fibril surface. Comparison of experimental and theoretical axial mass distributions of the fibrils showed that the N-propeptides were located to the overlap zone of the fibril D-period (where D=67 nm, the characteristic axial periodicity of collagen fibrils). Treatment of the dermatosparactic fibrils with N-proteinase did not remove the N-propeptides from the fibrils, although the N-propeptides were efficiently removed by trypsin and chymotrypsin. However, the N-propeptides were efficiently cleaved by the N-proteinase when the pNcollagen molecules were extracted from the fibrils. These results are consistent with close packing of N-propeptides at the fibril surface which prevented cleavage by the N-proteinase. Long-range axial mass determination along the fibril length showed gross non-uniformity with multiple mass bulges. Of note is the skin fragility in dermatosparaxis, and also the appearance of mass bulges along the fibril long axis symptomatic of the fragile skin of mice which lack decorin. Western blot analysis showed that the dermatosparactic fibrils bound elevated levels of the proteoglycan, compared with normal skin fibrils. The results showed that N-propeptides can distort the morphology of fibrils, that they do not inhibit binding of gap-associated macromolecules (such as decorin) and that the normal mechanical properties of skin are strongly dependent on the close association of near-cylindrical fibrils, thereby enabling maximal fibril-fibril interactions.     

Spatiotemporal appearance of developing LHRH neurons in the rat brain

To obtain insight into the development of the heterogeneous intracerebral populations of luteinizing hormone-releasing hormone (LHRH) neurons, their spatiotemporal appearance was examined at different stages in normal rat embryos, in nasal epithelial explants in vitro, and in intrauterine nasal-operated embryos. Following the appearance of nerve cell adhesion molecule in the nasal placode at embryonic day (E) 12.5, LHRH neurons, generated in the nasal placode at E13.5, penetrated the forebrain vesicle (FV) by E14.5-15.5. After E16.5, as the FV elongated to form the olfactory bulb, the migrating neurons traversed posteriorly through the interhemispheric space to penetrate the septopreoptic (S-P) area. By E18.5, LHRH neurons were detected in the preoptic-diagonal band (P-D) area as well as in the S-P region, along with some scattered extrahypothalamic LHRH neurons. To determine the source of these neurons, we separately cultured dissected parts of E12.5 nasal pit epithelium. Neuronal generation was predominantly from the medial wall epithelium (NAP), but some LHRH neurons originated in the roof epithelium. Cocultures of the NAP (E12.5) with the FV, median eminence-arcuate complex, Rathke's pouch, mesencephalon, or medulla oblongata from E14.5 embryos revealed the ability of LHRH cells to penetrate all of these tissues. Uni- or bilateral nasal destruction was conducted at E16.5 or E15.5, respectively, and examined at E18.5 and E21.5. In the operated embryos, most LHRH neurons were present in the P-D system and some in the S-P area. This finding suggests that the neurons generated before E15.5 are primarily predisposed to form the P-D system, whereas those derived afterward form the S-P system.     

Cardiopulmonary and anesthetic effects of propofol in wild turkeys

OBJECTIVE: To determine safety, anesthetic variables, and cardiopulmonary effects of i.v. infusion of propofol for induction and maintenance of anesthesia in wild turkeys. ANIMALS: 10 healthy, adult wild turkeys. PROCEDURE: Anesthesia was induced by i.v. administration of propofol (5 mg/kg of body weight) over 20 seconds and was maintained for 30 minutes by constant i.v. infusion of propofol at a rate of 0.5 mg/kg/min. Heart and respiratory rates, arterial blood pressures, and arterial blood gas tensions were obtained prior to propofol administration (baseline values) and again at 1, 2, 3, 4, 5, 10, 15, 20, 25, and 30 minutes after induction of anesthesia. All birds were intubated immediately after induction of anesthesia, and end-tidal CO2 concentration was determined at the same time intervals. Supplemental oxygen was not provided. RESULTS: Apnea was observed for 10 to 30 seconds after propofol administration, which induced a decrease in heart rate; however, the changes were not significant. Compared with baseline values, respiratory rate was significantly decreased at 4 minutes after administration of propofol and thereafter. Systolic, mean, and diastolic pressures decreased over the infusion period, but the changes were not significant. Mean arterial blood pressure decreased by 30% after 15 minutes of anesthesia; end-tidal CO2 concentration increased from baseline values after 30 minutes; PO2 was significantly decreased at 5 minutes after induction and thereafter; PCO2 was significantly (P < 0.05) increased after 15 minutes of anesthesia; and arterial oxygen saturation was significantly (P < 0.05) decreased at the end of anesthesia. Two male turkeys developed severe transient hypoxemia, 1 at 5 and the other at 15 minutes after induction. Time to standing after discontinuation of propofol infusion was 11 +/- 6 minutes. Recovery was smooth and unremarkable. CONCLUSION: Propofol is an effective agent for i.v. induction and maintenance of anesthesia in wild turkeys, and is useful for short procedures or where the use of inhalational agents is contraindicated.     

NGF mRNA is expressed by GABAergic but not cholinergic neurons in rat basal forebrain

Nerve growth factor (NGF) supports the survival and biosynthetic activities of basal forebrain cholinergic neurons and is expressed by neurons within lateral aspects of this system including the horizontal limb of the diagonal bands and magnocellular preoptic areas. In the present study, colormetric and isotopic in situ hybridization techniques were combined to identify the neurotransmitter phenotype of the NGF-producing cells in these two areas. Adult rat forebrain tissue was processed for the colocalization of mRNA for NGF with mRNA for either choline acetyltransferase, a cholinergic cell marker, or glutamic acid decarboxylase, a GABAergic cell marker. In both regions, many neurons were single-labeled for choline acetyltransferase mRNA, but cells containing both choline acetyltransferase and NGF mRNA were not detected. In these fields, virtually all NGF mRNA-positive neurons contained glutamic acid decarboxylase mRNA. The double-labeled cells comprised a subpopulation of GABAergic neurons; numerous cells labeled with glutamic acid decarboxylase cRNA alone were codistributed with the double-labeled neurons. These data demonstrate that in basal forebrain GABAergic neurons are the principal source of locally produced NGF.     

Effects of dithiocarbamates on toxicity of cadmium in rat primary hepatocyte cultures

The protective effects of N-benzyl-D-glucamine dithiocarbamate (BGD) and N-p-hydroxymethylbenzyl-D-glucamine dithiocarbamate (HBGD) on the toxicity of Cd in the rat primary hepatocyte cultures were studied. Cytotoxicity was assessed by measuring cell viability, extra cellular lactic dehydrogenase (LDH) activity, and intracellular lipid peroxidation and active oxygen species. Primary hepatocyte cultures were treated with 109CdCl2 (5, 10 or 50 microM Cd and 1.7 KBq of 109Cd/well) for 30 min or 4 h. BGD or HBGD was added to the culture medium to make the final concentration of 100 microM and incubated for 4.5 h in 30 min Cd exposure or 1 h in 4 h Cd exposure. Decreases in the hepatocyte viability caused by all Cd exposure concentrations were significantly prevented by treatment with BGD or HBGD. The treatment with the chelating agents for 4.5 h after Cd exposure for 30 min significantly prevented increases in extracellular LDH activity. Increases in the lipid peroxidation in hepatocytes exposed to Cd for 30 min or 4 h were prevented significantly by treatment with BGD or HBGD for 4.5 h or 1 h, respectively. Moreover, the increases in the level of active oxygen species caused by Cd exposure for 30 min were significantly prevented by treatment with the chelating agents for 1.5 h. These findings suggest that BGD and HBGD protect against the cytotoxicity of Cd in rat primary hepatocyte cultures and that the protective effects of chelating agents presumably result from a decrease in the Cd level, the effective sequestration of the reactive Cd ion, and the direct preventive effect on the active oxygen species in the hepatocytes.     

NADPH-diaphorase neurons contacting the cerebrospinal fluid in the ventricles of rat brain

OBJECTIVE: To investigate, using logistic regression analysis and receiver operator characteristic analysis, the biochemical diagnosis of polycystic ovary syndrome (PCOS) and if it could be improved by using an array of hormone measurements chosen to yield optimal and cost-effective discrimination between women with PCOS and healthy women. DESIGN: Retrospective clinical study. SETTING: Outpatient clinic of reproductive endocrinology at Turku University Central Hospital, Turku, Finland. PATIENTS: Fifty-four oligomenorrheic women with PCOS diagnosed by ovarian ultrasonography, and 29 healthy regularly menstruating women with normal ovarian morphology. MAIN OUTCOME MEASURES: Concentrations of LH, FSH, androstenedione (A), T, and sex hormone-binding globulin in serum. RESULTS: Luteinizing hormone, FSH and A, when used in combination, were the hormonal analytes of highest clinical utility. Diagnostic sensitivity, specificity, and overall concordance of 98%, 93%, and 96%, respectively, were attained. Each of the analytes used alone yielded lower degree of discrimination. CONCLUSIONS: Simultaneous use of the levels of LH, FSH, and A in serum can be used effectively for classification between women with PCOS and healthy women.