Rhodiola sacra aqueous extract (RSAE) improves biochemical and sperm characteristics in cryopreserved boar semen.

Although Rhodiola sacra aqueous extract (RSAE) has been used in many studies as an antioxidant, its effects on semen characteristics and its antioxidant properties during cryopreservation of boar sperm have never been evaluated. Semen was collected from five Duroc boars (2-4-year-old) twice weekly and frozen-thawed in extender with RSEA. Motion characteristics were assessed with a computer-aided semen analysis (CASA) system, whereas other sperm quality end points were assessed by routine methods. The effective concentration of RSEA in extender ranged from 4 to 8mg/L and the effect of RSEA on sperm quality was better in glycerol-free extender than extender containing glycerol (P<0.05). In frozen-thawed boar semen, there was a direct correlation (P<0.05) between RSEA concentration and glutathione (GSH) concentrations, mitochondrial activity, and hypoosmotic swelling test (HOST), and an inverse correlation (r=-0.982, P<0.05) between RSEA concentration and malondialdehyde (all end points were significantly higher at 6mg/L than in the control group). In summary: (i) the effective concentration of RSEA in extender ranged from 4 to 8mg/L; (ii) the effect of RSEA on sperm quality was better in extender without glycerol; and (iii) there was a significant correlation between RSEA concentrations and concentrations of GSH and MAD in frozen-thawed boar semen (antioxidant effects of RSEA were concentration-dependent). Further studies are needed to define the active ingredient in RSEA that protects boar sperm against ROS.

Nociceptive and thermoreceptive lamina I neurons are anatomically distinct.

Pain and temperature stimuli activate neurons of lamina I within the dorsal horn of the spinal cord, and although these neurons can be classified into three basic morphological types and three major physiological classes, earlier studies did not establish a structure/function correlation between their morphology and their physiological responses. We recorded and intracellularly labeled 38 cat lamina I neurons. All 12 fusiform cells were nociceptive-specific, responsive only to pinch and/or heat. All 11 pyramidal cells were thermoreceptive-specific, responsive only to innocuous cooling. Of ten multipolar cells, six were polymodal, responsive to heat, pinch and cold, and four were nociceptive-specific. Five unclassified cells had features consistent with this pattern. These results support the view that central pain and temperature pathways contain anatomically discrete sets of modality-selective neurons.

A conserved p38 mitogen-activated protein kinase pathway regulates Drosophila immunity gene expression.

Accumulating evidence suggests that the insect and mammalian innate immune response is mediated by homologous regulatory components. Proinflammatory cytokines and bacterial lipopolysaccharide stimulate mammalian immunity by activating transcription factors such as NF-kappaB and AP-1. One of the responses evoked by these stimuli is the initiation of a kinase cascade that leads to the phosphorylation of p38 mitogen-activated protein (MAP) kinase on Thr and Tyr within the motif Thr-Gly-Tyr, which is located within subdomain VIII. We have investigated the possible involvement of the p38 MAP kinase pathway in the Drosophila immune response. Two genes that are highly homologous to the mammalian p38 MAP kinase were molecularly cloned and characterized. Furthermore, genes that encode two novel Drosophila MAP kinase kinases, D-MKK3 and D-MKK4, were identified. D-MKK3 is an efficient activator of both Drosophila p38 MAP kinases, while D-MKK4 is an activator of D-JNK but not D-p38. These data establish that Drosophila indeed possesses a conserved p38 MAP kinase signaling pathway. We have examined the role of the D-p38 MAP kinases in the regulation of insect immunity. The results revealed that one of the functions of D-p38 is to attenuate antimicrobial peptide gene expression following exposure to lipopolysaccharide.

Morphological classes of spinothalamic lamina I neurons in the cat.

We examined the morphology and distribution of retrogradely labeled spinothalamic tract (STT) neurons in lamina I (the marginal zone) of the spinal dorsal horn after large injections of cholera toxin subunit B (CTb) or Fast Blue (FB) into the contralateral thalamus of cats. Based on the shape and orientation of the somata and proximal dendrites in horizontal sections, three distinct cell types were identified: (1) fusiform cells with small, spindle-shaped somata and bipolar, longitudinal dendritic arbors; (2) pyramidal cells with triangular somata and three main dendritic origins with primarily longitudinal arborizations; and (3) multipolar cells with larger, multiangular somata and four or more radiating dendritic arbors directed both longitudinally and mediolaterally. These three morphological types differed significantly in the number of primary dendrites and the size of the somata. Subclasses of multipolar cells were noted. Nearly all cells could be categorized into these three classes consistently in horizontal sections. A small number of cells with transitional shapes or with small, round somata were unclassified. The proportional distributions of these cell types were found to vary over the length of the spinal cord (from the third cervical through the coccygeal segments) in three cats. The overall proportions of cell types were 34% fusiform, 36% pyramidal, 25% multipolar, and 5% unclassified. The proportions of pyramidal and multipolar cells were strikingly higher within the C7-8 and L6-7 segments and lowest in the thoracic segments. In contrast, fusiform cells formed about 20% of the labeled lamina I STT population in the C7-8 and L6-7 segments but more than 60% in thoracic segments. Across all nine cats, the proportions were similar within the cervical (C5-8) and lumbosacral (L5-S1) enlargements, although considerable interanimal variability was noted. These distinct morphological types of lamina I STT cells with differential longitudinal distributions probably have different functional roles. They may correspond to the three main physiological classes of lamina I STT cells.

Electrophysiological and morphological differentiation of chandelier and basket cells in the rat hippocampal formation: a study combining intracellular recording and intracellular staining with biocytin.

Using standard intracellular recording techniques 38 nonpyramidal cells or interneurons have been sampled in hippocampal slices of the rat. Among 38 physiologically identified interneurons, all 27 cells labeled with biocytin were morphologically demonstrated to be nonpyramidal and nongranule cells. The vast majority of these cells showed typical fast spiking discharges, i.e., a shorter duration action potential followed by a brief but prominent after hyperpolarisation potential without frequency adaptation in response to prolonged depolarizing current injection. However, 4 cells clearly exhibited frequency adaptation. Based on their axonal arborizations, the former group included basket interneurons innervating the principle cell body layers and axodendritic interneurons projecting to the molecular layer of the dentate gyrus; whereas the latter 4 cells belonged to chandelier interneurons selectively terminating the axon initial segments of principle cells. These results support the notion that interneurons in the hippocampal formation are heterogeneous with respect to their morphology and electrophysiological characteristics, suggesting that the electrophysiological behavior of hippocampal interneurons may be associated with their functional activities.

Morphological heterogeneity of non-pyramidal neurons in the CA1 region of the rat hippocampus.

Using techniques of combining intracellular recording and intracellular staining with biocytin, 19 neurons have been sampled within or close to the CA1 pyramidal cell layer in hippocampal slices of the rat. All of these cells were physiologically characterized as non-pyramidal or interneurons based on their action potential properties and responses to somatic depolarization. After injection of biocytin into these identified cells, all these cells were morphologically confirmed as non-pyramidal cells. Five cell types were distinguished according to the distribution patterns of their axon trees and locations of the somata. (1) Basket cells (n = 10) with somata located within or close to the pyramidal cell layer had axon arborizations restricted in the same layer. (2) Chandelier cell somata (n = 3) were located in the pyramidal cell layer and their axon arborizations were selectively distributed in the deep stratum oriens (adjacent to the pyramidal cell layer), where axon initial segments of pyramidal cell were located. (3) Three neurons recorded from the deep stratum oriens had axon trees covering both the pyramidal cell layer and the deep stratum oriens (n = 2) or mainly projecting to the distal stratum radiatum and stratum lacunosum-moleculare (n = 1). (4) Two axodendritic cells with somata located in the pyramidal cell layer had axon trees spanning over the stratum oriens and radiatum. (5) One interneuron, like a basket cell, had an axon tree confined in the pyramidal cell layer, but its beaded axon terminals selectively contacted with the somata of the presumed non-pyramidal cells in the pyramidal cell layer, instead of pyramidal cells. These results provide further evidence that CA1 interneurons are heterogeneous with respect to the laminar distributions of their axon terminals in this region. These specific patterns of interneuron axon trees reflect the selectivity of CA1 interneurons in the postsynaptic domains of the target cells, which may be functionally associated with differential neuronal activities.

Fluorogold as a retrograde tracer used in combination with immunohistochemistry.

The fluorescent dye Fluorogold has been recently introduced as a retrograde neuronal tracer that can be used in combination with immunohistochemistry. It has, however, lately been reported that immunohistochemical processes induced rapid photobleaching of Fluorogold. In the present study we prove that it is the water in the sections, and not the immunohistochemical processing itself, that is responsible for the photobleaching. This problem is easily corrected by using a 29:1 or 19:1 glycerine-PBS buffer mounting medium.

Effects of electrical stimulation of the central nucleus of the amygdala and the lateral hypothalamic area on the oval nucleus of the bed nuclei of the stria terminalis and its adjacent areas in the rat.

Our recent studies demonstrated that there are reciprocal connections between the oval nucleus (Ov) of the bed nuclei of the stria terminalis (BST) and the central nucleus of the amygdala (Ce) or the lateral hypothalamic area (LHA). The present experiments found that spontaneous unit discharges of 73 neurons in the Ov and its adjacent areas were modified in 58 neurons (79.45%) by focal brain stimulation in the Ce. After focal LHA stimulation, the spontaneous unit discharges of 23 (53.49%) of 43 Ov neurons, which could be influenced by focal Ce stimulation, were changed. Thirteen Ov neurons were antidromically activated by focal Ce or LHA stimulation. These results indicate that: (1) neuronal activities in the Ov and its adjacent areas can be modulated by Ce or LHA; (2) there are functional reciprocal connections between Ov and Ce or LHA; and (3) the Ce and LHA afferents converge upon some Ov neurons.

Local projections of GABAergic neurons in the dentate gyrus and CA1 region in the rat hippocampal formation.

Using retrograde transport of wheat germ agglutinin conjugated colloidal gold (WGA-gold) combined with immunoreactivity for glutamate decarboxylase (GAD), a specific synthesizing enzyme for gamma-aminobutyric acid (GABA), local projections of GABAergic neurons in the dentate gyrus and CA1 were examined. In the hilus of the dentate gyrus, it was found that GABAergic neurons in the granule cell layer projected to the ipsilateral upper leaf of the molecular layer, with a mediolateral extension of more than 1.2 mm and a rostrocaudal extension of over 0.8 mm. Non-GABAergic neurons in nearly the entire hilar area were found to project to the ipsilateral upper leaf of the molecular layer. In the dorsal CA1 region, GABAergic neurons in the stratum pyramidale and radiatum converged onto the ipsilateral stratum pyramidal/oriens, with a mediolateral extension of over 1 mm and a rostrocaudal extension of over 0.7 mm. These results provide direct evidence that in both the dentate gyrus and CA1, GABAergic interneurons from a fairly large field converge onto a very small target area. This suggests that the output signals from GABAergic neurons in the dentate gyrus and CA1, and non-GABAergic neurons in the dentate gyrus, may propagate beyond the anatomical limits contained in conventional slice preparations of the hippocampal formation.

Convergence of somatosensory and baroreceptive inputs onto parabrachio-subfornical organ neurons in the rat: an electrophysiological study.

Electrophysiological characteristics were described for neurons of the parabrachial nucleus (PBN) which receive baroreceptive and somatosensory inputs in the rat. Following focal electrical stimulation in the ipsilateral caudal nucleus of the tractus solitarii (NTS), the firing rates of these neurons were increased in 94 (55.6%), and decreased in 38 (22.5%). Fifty-three (54.5%) of 97 PBN neurons tested were excited, and 11 (11.3%) inhibited in response to contralateral common peroneal nerve (CPN) stimulation. Of these neurons, 52 were found to respond to both caudal NTS and CPN stimulation. Effects of subfornical organ (SFO) stimulation were also examined in 151 of 169 parabrachial neurons. 13 (7.7%) were activated antidromically and were located in the lateral division of the PBN, while 34 (22.5%) were affected orthodromically. Stimulation of the caudal NTS resulted in both a fall in the heart rate and changes of PBN neuronal firing rates. Similar effects were elicited by activating peripheral baroreceptors by the administration of phenylephrine, an alpha-adrenergic agonist. These results strongly indicate that: (1) the lateral PBN is involved in central cardiovascular control; (2) somatosensory and baroreceptive messages may converge onto some PBN neurons; and (3) some PBN neurons may relay baroreceptive information from the caudal NTS to the SFO.

Responses of neurons in the rat anterior hypothalamic periventricular zone to osmotic stimulation and angiotensin II in vitro.

Extracellular recordings were made in vitro from neurons in slices of the rat periventricular zone surrounding the anterior portion of the third ventricle. Spontaneous unit activity of these neurons was decreased in 39.5% of the neurons tested, increased in 32.9%, and biphasically changed (i.e., excitation-inhibition) in 10.5%, in response to bath application of hypertonic artificial cerebrospinal fluid (aCSF) containing elevated levels of NaCl (330 mOsm). Similar responses were observed following bath application of hypertonic aCSF prepared with additional mannitol. Angiotensin II in the perfusion medium dramatically increased spontaneous unit activity in 73.7% of neurons tested, while, unexpectedly, 5.3% were inhibited. These results suggest that some neurons in the periventricular zone are themselves osmosensitive, and may be involved in the regulation of water balance.

Coexistence of corticotropin releasing factor and neurotensin within oval nucleus neurons in the bed nuclei of the stria terminalis in the rat.

Within the oval nucleus of the bed nuclei of the stria terminalis of the rat, corticotropin releasing factor (CRF)- and neurotensin (NT)-immunoreactive (ir) neurons were densely and evenly distributed. A substantial number of these neurons showed both CRF and NT immunoreactivities.

Neuronal responses of the anterior commissural nucleus to osmotic stimulation and angiotensin II in hypothalamic slices in the rat.

The firing rate and pattern of activity of neurons in the anterior commissural nucleus (ACN), which was rich in oxytocin-containing neurons, were studied electrophysiologically in hypothalamic slices. Extracellular recording showed that most ACN neurons exhibited irregular or regular continuous spontaneous unit activity. Other neurons showed short burst patterns of activity or were silent. The majority of ACN neurons were activated by bath application of angiotensin II, and a substantial number of them showed inhibitory or excitatory responses to hypertonic bathing medium. These results indicate that magnocellular neurosecretory neurons in the ACN may participate in the regulation of water balance.

[Electrophysiological characteristics of morphologically identified chandelier interneurons in rat hippocampal slices].

Using standard intracellular recording and staining techniques, 4 chandelier interneurons were sampled and anatomically confirmed from 39 physiologically identified interneurons in rat hippocampal slices. Compared to pyramidal and granule cells, all 4 chandelier interneurons-(CA1 region, n = 3, dentate gyrus, n = 1) had typical action potentials of interneurons, i. e., a shorter duration of action potential was followed by a deep after hyperpolarization. However, all these 4 cells exhibited a varying degree of spike frequency adaptation in response to depolarizing current injection, which usually do not occur in basket interneurons. Morphologically, axonal terminals of chandelier cells were beaded and exclusively distributed in the stratum oriens of the CA1 and the granule cell layer of the dentate gyrus, where the initial segments of pyramidal and granule cells are located. These results indicate that the chandelier interneurons differ from basket interneurons in the rat hippocampus with in physiology and in morphology.

[Responses of neurons in lateral parabrachial nucleus to antidromic stimulation of subfornical organ and to orthodromic stimulation of nucleus tractus solitarius in the rat].

Single unit discharges were extracellularly recorded from the neurons in the lateral parabrachial nucleus (LPBN) and responses of the recorded units to antidromic stimulation of the subfornical organ (SFO) and to orthodromic stimulation of the nucleus tractus solitarius (NTS) were observed in urethane-anesthetized rats. Following electrical stimulation of the SFO, 9.9% (51/151) of the LPBN units were antidromically activated. After activation of peripheral baroreceptors by raising arterial blood pressure with an intravenous injection of phenylephrine, 40.7% (22/54) of the LPBN units were inhibited and 27.8% (17/54) excited. Following orthodromic stimulation of the depressor area in the NTS, 55.6% (94/169) of the LPBN units showed an increase and 22.5% (38/169) a decrease in firing rates. Among the LPBN neurons antidromically activated by SFO stimulation, 2 units were inhibited by phenylephrine administrated i.v.; of the 8 units tested, when the NTS was stimulated, 6 were excited and 2 inhibited. The results suggest that the LPBN neurons may receive inhibitory or excitatory baroreceptive inputs from the NTS and then relay it directly to SFO.