Effects of a traditional Chinese herbal medicine, Kanzo-bushi-to, on the resistance of thermally injured mice infected with herpes simplex virus type 1

The protective effect of Kanzo-bushi-to (TJS-038) was investigated on the opportunistic infection of herpes simplex virus type 1 (HSV) in thermally injured mice (TI-Mice). We have previously reported that TI-Mice were approximately 100 times more susceptible to HSV infection than normal mice (N-Mice) and that CD8+ suppressor T (ST)-cells induced by burn injury were involved in causing this increased susceptibility of TI-Mice. Increased susceptibility of TI-Mice to the infection was reversed to the levels observed in N-Mice when TI-Mice were treated intraperitoneally with TJS-038 at a dose of 5 mg/kg 1 and 4 days after thermal injury. The activity of ST-cells was greatly decreased in TI-Mice treated with TJS-038. The generation of Vicia villosa lectin-adherent CD4+ CD28+ TCR-alpha/beta+ contrasuppressor T (Contra-ST)-cells associated with the appearance of ST-cells was expanded and occurred earlier in spleens of TJS-038-treated TI-Mice as compared with that of untreated TI-Mice. The improved resistance of TJS-038-treated TI-Mice to the infection was transferred to untreated TI-Mice by adoptive transfer of Contra-ST-cells prepared from TJS-038-treated TI-Mice. These results suggest that TJS-038 may restore the resistance of TI-Mice to the HSV infection through the expanded generation of Contra-ST-cells.     

Amyloid beta-protein impairs astrocyte-mediated differentiation of hippocampal neurons

Embryonic rat hippocampal neurons were cultured on poly-D-lysine (PDL) or on cortical astrocytes, some of which had been pretreated for 24 h with amyloid beta-protein (beta-AP). Amino acid-induced currents were quantified. Membrane capacitance (Cm), as well as the amplitude and density of amino acid-evoked currents recorded in neurons cultured on untreated astrocytes were all statistically greater than those recorded in neurons grown on PDL. However, compared to untreated astrocytes, those treated with beta-AP led to significantly lower values in neurons for Cm and GABA, kainate- and NMDA-induced currents, while glycine-activated current values were not significantly different. Furthermore, beta-AP treatment abolished spontaneous Cac2+ fluctuations in astrocytes, which may account for their impaired ability to promote the expression of functional transmitter receptors in neurons.     

The pharmacology of amino-acid responses in septal neurons

Septal cholinergic neurons are known to play an important role in cognitive processes including learning and memory through afferent innervation of the hippocampal formation and cerebral cortex. The septum contains not only cholinergic neurons but also various types of neurons including GABA (gamma-aminobutyric acid)-ergic neurons. Although synaptic transmission in the septum is mediated primarily by the activation of excitatory and inhibitory amino-acid receptors, it is possible that a distinct phenotype of neuron is endowed with a different type for each of the amino-acid receptors and thus they play different roles from each other, since it has been demonstrated within the septum that there is a regional distribution of various types of amino-acid receptor subunits, their expression as different combinations within a specific cell may produce receptor channels with disparate functional properties. As a first step towards knowing the various functions of septal cholinergic neurons, we characterized the functional properties of glutamate, GABA (type A; GABAA) and glycine receptor channels on cultured rat septal neurons which were histologically identified to be cholinergic. These were similar to those of receptor channels on other types of neurons, except for the actions of some neuromodulators. The septal N-methyl-D-aspartate receptor channel was distinct in being less sensitive to Mg2+ and in a voltage-dependent action of Zn2+. The septal GABAA receptor channel exhibited a lanthanide site whose activation resulted in a positive allosteric interaction with a binding site of pentobarbital. The septal glycine receptor channel was only positively modulated by Zn2+; this action of Zn2+ was not accompanied by an inhibitory effect. Our data suggest that the amino-acid receptors on septal cholinergic neurons may play a distinct role compared to other types of neurons; this difference depends on the actions of neuromodulators and metal cations. It would be interesting to compare these effects recorded in tissue culture to those observed with septal cholinergic neurons in slice preparations.     

Acidic and basic fibroblast growth factors delay the maturation of neural crest-derived neurons

Neural crest (NC) cultures were prepared from lumbosacral segments of 12 day rat embryos and maintained in a defined medium. Post-mitotic, flat, neurofilament+ neurons with broad neuritic processes ('nascent neurons') appeared within 24 h. Timing of the next stage in neuronal differentiation, the formation of bipolar, phase-bright cells that bound tetanus toxin with long, slender neurites ('bipolar neurons'), was markedly influenced by acidic or basic fibroblast growth factor (FGF). The transition from nascent to bipolar neuron occurred several days prematurely in medium without added FGF, but took place with a time-course like that in vivo when 10 ng/ml of acidic or basic FGF was added.     

Differential regulation of SHC proteins by nerve growth factor in sensory neurons and PC12 cells

We have characterized some of the nerve growth factor (NGF) stimulated receptor tyrosine kinase (TrkA) signalling cascades in adult rat primary dorsal root ganglia (DRG) neuronal cultures and compared the pathways with those found in PC12 cells. TrkA receptors were phosphorylated on tyrosine residues in response to NGF in DRG neuronal cultures. We also saw phosphorylation of phospholipase Cgamma1 (PLCgamma1). We used recombinant glutathione-S-transferase (GST)-PLCgamma1 SH2 domain fusion proteins to study the site of interaction of TrkA receptors with PLCgamma1. TrkA receptors derived from DRG neuronal cultures bound preferentially to the amino terminal Src homology-2 (SH2) domain of PLCgamma1, but there was enhanced binding with tandemly expressed amino- and carboxy-terminal SH2 domains. The most significant difference in NGF signalling between PC12 cells and DRG was with the Shc family of adapter proteins. Both ShcA and ShcC were expressed in DRG neurons but only ShcA was detected in PC12 cells. Different isoforms of ShcA were phosphorylated in response to NGF in DRG and PC12 cells. NGF phosphorylated only one whereas epidermal growth factor phosphorylated both isoforms of ShcC in DRG cultures. Activation of the downstream mitogen-activated protein (MAP) kinase, p42Erk2 was significantly greater than p44Erk1 in DRG whereas both isoforms were activated in PC12 cells. Blocking the MAP kinase cascade using a MEK1/2 inhibitor, PD98059, abrogated NGF dependent capsaicin sensitivity, a nociceptive property specific to sensory neurons.     

Monaural sound localization: acute versus chronic unilateral impairment

We report that choline acetyltransferase (ChAT) activity and neuronal survival were enhanced in rat septal neurons cocultured with hippocampal neurons. The enhancement of ChAT activity also occurred as a result of the addition of hippocampal conditioned medium (HpCM). When septal neurons from embryonic day 17 (E17) rats were cocultured with hippocampal neurons, ChAT activity was increased 2-fold compared with homogeneous culture of septal neurons. By contrast, no increase in ChAT activity was observed in coculture of septal and neocortical neurons. Treatment with HpCM obtained from cultured E19 rat hippocampal neurons enhanced the ChAT activity of E17 rat septal neurons. The enhancement of ChAT activity caused by coculture with hippocampal neurons and that caused by the addition of HpCM were not blocked by the addition of anti-nerve growth factor (NGF) antibody, suggesting that NGF, which is known to increase the ChAT activity of septal neurons both in vivo and in vitro, did not participate in the increase of ChAT activity. These findings indicate that possible target-derived neurotrophic factor(s), other than NGF, from hippocampal neurons enhance(s) the ChAT activity of septal neurons.     

Neuronal sprouting in mouse sensory ganglia infected with herpes simplex virus type 2 (HSV-2): induction of growth-associated protein (GAP-43) and ultrastructural evidence

Herpes simplex virus (HSV) is neurotropic and when inoculated on the mouse footpad is retrogradely transported to the associated dorsal root ganglia (DRG), where infection is established. Previous observations suggest that, after HSV infection, sensory ganglion neurons may mount a sprouting response. In our HSV-infected DRG model, we investigate this issue by (1) examining expression of growth-associated protein (GAP-43), a molecule known to be induced by growing axons, and (2) determining ultrastructurally whether HSV-infected dorsal roots contain neurites. In a time course study, we show that GAP-43 is induced both in HSV-infected DRG and their central processes. The increase in GAP-43 is first seen 2 weeks following unilateral footpad inoculation in both cell bodies and dorsal roots, and is sustained at 1 month post inoculation in roots but not in perikarya. Large bundles of unmyelinated small caliber axons, lacking Schwann cell ensheathment, are observed by electron microscopy in dorsal roots 2 weeks and 1 month following inoculation. These profiles resemble developing or regenerating neurites and are rarely seen in roots of mock-infected or uninfected controls. The increased GAP-43 immunoreactivity and ultrastructural changes shown here, in conjunction with previously documented selective neuropeptide and enzyme alterations, confirm that a sprouting response is mounted in sensory ganglia following acute HSV infection.     

Astrocytic gap junctional communication decreases neuronal vulnerability to oxidative stress-induced disruption of Ca2+ homeostasis and cell death

We investigated the effect of uncoupling astrocytic gap junctions on neuronal vulnerability to oxidative injury in embryonic rat hippocampal cell cultures. Mixed cultures (neurons growing on an astrocyte monolayer) treated with 18-alpha-glycyrrhetinic acid (GA), an uncoupler of gap junctions, showed markedly enhanced generation of intracellular peroxides (2,7-dichlorofluorescein fluorescence), impairment of mitochondrial function [(dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction], and cell death (lactate dehydrogenase release) following exposure to oxidative insults (FeSO4 and 4-hydroxynonenal). GA alone had little or no effect on basal levels of peroxides, mitochondrial function, or neuronal survival. Intercellular dye transfer analyses revealed extensive astrocyte-astrocyte coupling but no astrocyte-neuron or neuron-neuron coupling in the mixed cultures. Studies of pure astrocyte cultures and microscope analyses of neurons in mixed cultures showed that the increased oxidative stress and cell death in GA-treated cultures occurred only in neurons and not in astrocytes. Antioxidants (propyl gallate and glutathione) blocked the death of neurons exposed to FeSO4/GA. Elevations of neuronal intracellular calcium levels ([Ca2+]i) induced by FeSO4 were enhanced in neurons in mixed cultures exposed to GA. Removal of extracellular Ca2+ and the L-type Ca2+ channel blocker nimodipine prevented impairment of mitochondrial function and cell death induced by FeSO4 and GA, whereas glutamate receptor antagonists were ineffective. Finally, GA exacerbated kainate- and FeSO4-induced injury to pyramidal neurons in organotypic hippocampal slice cultures. The data suggest that interastrocytic gap junctional communication decreases neuronal vulnerability to oxidative injury by a mechanism involving stabilization of cellular calcium homeostasis and dissipation of oxidative stress.     

Acidic and basic fibroblast growth factors down-regulate collagen gene expression in keloid fibroblasts

The effects of acidic and basic fibroblast growth factors (FGFs) on collagen expression by keloid fibroblasts were examined in the absence and presence of heparin. Collagen biosynthesis and gene expression of type I collagen were down-regulated by the FGFs in the presence of heparin. Acidic FGF, in a concentration range of 0.4 to 50 ng/ml, had little or no effect on collagen synthesis after a 4-day incubation. However, in the presence of heparin (100 micrograms/ml) acidic FGF, in concentrations ranging from 2 to 50 ng/ml, decreased [3H]hydroxyproline synthesis by 44 to 68%, compared with untreated control cultures. Total [3H]hydroxyproline synthesis was similar between control and heparin-treated cultures. Basic FGF (2.0 to 50 ng/ml) was effective in suppressing [3H]hydroxyproline synthesis by 50 to 90% after a 4-day incubation without heparin in keloid and normal fibroblast cultures. The steady-state levels of type I collagen messenger RNA were significantly decreased by acidic FGF in the presence of heparin, as well as by basic FGF without heparin. The data suggest that the FGFs are effective in down-regulating excess collagen production by keloid fibroblasts and that this inhibitory effect is apparently associated with pretranslational events. Moreover, acidic FGF is apparently dependent on heparin, whereas basic FGF is not, for potentiation of the down-regulatory effects of the FGFs.     

Novaco Anger Scale: reliability and validity within an adult criminal sample

Serum is used widely for culturing neurons and glial cells, and is thought to provide essential, albeit undefined, factors such as hormones, growth factors, and trace elements that promote the growth of cells in vitro. Moreover, serum can have profound effects on cell proliferation, differentiation, and cell morphology, and may even influence cell fate decisions. Despite the overall growth-promoting influence of serum on cell culture, frequent media changes have been shown to be detrimental to neuronal cultures, significantly reducing the yield of viable neurons. The reason for this loss of neurons by frequent media changes has been puzzling. We demonstrate that bovine and horse sera, the most popular serum complements for CNS cell culture, are a significant source for glutamate, supplying glutamate at concentrations sufficient to kill primary cultured hippocampal neurons. By using the bioluminescence detection method, we determined the glutamate concentration [Glu] in several batches of fetal bovine (calf) sera (FBS) to be close to 1 mM, and that of horse sera to be approximately 0.3 mM. Thus 10% serum supplement to culture media results in [Glu] of 30-100 microM due to serum alone. We subsequently produced glutamate depleted media (GDM) by using primary cultures of hippocampal astrocytes to absorb glutamate from media containing 10% FBS. Within 3 h, astrocytes reduced the [Glu] in the medium from approximately 90 microM to less than 1 microM. Sister cultures of hippocampal neuron that underwent frequent media changes with GDM or GDM + partial untreated media demonstrated that GDM significantly increase neuronal survival (10-fold at 21 DIV). Subsequent exposure to glutamate provided by either untreated serum or by equivalent doses of exogenous glutamate added to GDM led to dose-dependent neuronal cell death. The relative sensitivity of hippocampal neurons to glutamate increased with increasing culture age from initial ED50 values of > 100 microM (< 6 DIV) to approximately 6 microM in cultures maintained for 3 weeks or longer. The relative sensitivity to exogenous glutamate was at least 2-fold higher in neurons cultured in GDM than in sister cultures maintained in media containing untreated serum. The death of neurons exposed to untreated media was blocked by the NMDA receptor antagonist MK-801. These experiments suggest that the vulnerability of neurons to media changes can be solely explained by excitotoxicity resulting from serum-borne glutamate. Moreover, we propose that use of GDM may be advantageous for culturing hippocampal neurons and may eliminate the possible selection for glutamate resistant neurons. The use of GDM could be particularly important for studies of excitotoxicity; our study predicts that the ED50 for neuronal culture with regular serum will be artificially high and may not adequately reflect the in vivo state.     

Physiological control on the expression and secretion of Candida rugosa lipase

The presence and distribution of functional, high-affinity receptors for fibroblast growth factors (FGFs) in the neonatal organ of Corti were probed using the intracellular toxin saporin conjugated to basic FGF (FGF-2). FGFs that bind to high-affinity FGF receptors are internalized as part of the normal process of receptor inactivation. The receptor can thus be used for the targeted delivery of molecules conjugated to FGF into the cytoplasm. Incubation of postnatal day 5 (P5) rat organ of Corti cultures with FGF-saporin caused a dose dependent destruction of outer hair cells, Deiters cells and outer pillar cells. Inner hair cells and other cells were unaffected. Organ of Corti cultures at P0 and P10 showed much less damage than at P5. The results suggest that outer hair cells and adjacent supporting cells in the organ of Corti transiently express high-affinity FGF receptors, and that these receptors can mediate the intracellular delivery of bioactive molecules.     

Novel synthesis and release of GABA in cerebellar granule cell cultures after infection with defective herpes simplex virus vectors expressing glutamic acid decarboxylase

The inhibitory amino acid neurotransmitter gamma-aminobutyric acid (GABA) is synthesized from glutamate in a single step by the enzyme glutamatic acid decarboxylase (GAD). We sought to determine whether viral vectors containing GAD cDNA could be used to enhance synthesis and stimulation-evoked release of GABA in cultures of CNS neurons. For this purpose, we generated double-cassette defective herpes simplex virus (HSV) vectors that expressed one of the two GAD isoforms (GAD65 or GAD67), and Escherichia coli LacZ. Infection of cerebellar granule cell (CGC) cultures with vectors containing GAD cDNA resulted in a significant increase in isoform-specific expression of GAD, synthesis of GABA, and stimulation-evoked GABA release. GAD65 and GAD67 vector-infected neurons exhibited a comparable profile of GABA levels, synthesis and release, as well as GAD protein distribution. In CGCs cultured for 6 days in vitro (DIV), GABA synthesized after vector-derived GAD expression was released by treatment with glutamate or veratridine, but only in a Ca2+-independent fashion. In more mature (10 DIV) cultures, both Ca2+-dependent, K+ depolarization-induced, as well as Ca2+-independent, veratridine-induced, GABA release was significantly enhanced by GAD vector infection. Treatment of CGCs with kainic acid, which destroys most of the GABAergic neurons (<1% remaining), did not prevent vector-derived expression of GAD nor synthesis of GABA. This suggests that defective HSV vector-derived GAD expression can be used to increase GABA synthesis and release in CNS tissue, even in the relative absence of GABAergic neurons. The use of such GAD vectors in the CNS has potential therapeutic value in neurologic disorders such as epilepsy, chronic pain, Parkinson's and Huntington's disease.     

Loss of rhythmically bursting neurons in rat medial septum following selective lesion of septohippocampal cholinergic system

The medial septum contains cholinergic and GABAergic neurons that project to the hippocampal formation. A significant proportion of the septohippocampal neurons (SHN) exhibit a rhythmically bursting (RB) activity that is involved in the generation of the hippocampal theta rhythm. The neurochemical nature of septal RB neurons is not firmly established. To address this question, the septal unit activity has been recorded in rats after selective destruction of the cholinergic septal neurons by the immunotoxin 192 IgG-saporin. Experiments have been performed in urethan-anesthetized and unanesthetized rats, 14-21 days after lesion. Acetylcholinesterase (AChE) histochemistry revealed a near-complete loss of cholinergic septal neurons and of cholinergic fibers in the hippocampus. The recorded neurons were located in the medial septum-diagonal band of Broca area. A number of these neurons were identified as projecting to the hippocampus (SHN) by their antidromic response to the electrical stimulation of the fimbria-fornix. In urethan-anesthetized lesioned rats, the percentage of RB neurons decreased significantly as compared with controls (17 vs. 41% for SHNs and 5 vs. 19% for unidentified septal neurons). The axonal conduction velocity and the burst frequency of the SHNs that retained a RB activity were higher in lesioned as compared with control rats. The number of spikes per burst was lower and the burst duration was shorter in lesioned rats as compared with controls. The urethan-resistant hippocampal theta was altered both in terms of frequency and amplitude. In unanesthetized lesioned rats, no RB septal neurons were found during arousal, as compared with 25% in controls. Their number was also markedly reduced during paradoxical sleep (9.7 vs. 38.5%). Histochemistry in 192 IgG-saporin-treated rats showed that RB neurons were found in areas devoid of AChE-positive neurons but containing parvalbumine-positive (presumably GABAergic) neurons. These data show that RB activity is considerably reduced after selective lesion of the cholinergic medial septal neurons. They suggested that the large majority of the RB septal neurons are cholinergic and that the few neurons that display RB activity in lesioned rats are GABAergic.     

Peripheral and central target requirements for survival of embryonic rat dorsal root ganglion neurons in slice cultures

Developmental cell death in the nervous system usually is controlled by the availability of target-derived trophic factors. It is well established that dorsal root ganglia (DRG) neurons require the presence of their peripheral target for survival, but because of their central projections, it is possible that the spinal cord also may be required. Before examining this possibility in rat embryos, we first used terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) to determine that thoracic DRG cell death occurred from embryonic day 15 (E15) to E18. To determine the target requirements of DRG neurons, we used organotypic slice cultures of E15 thoracic trunk segments. After peripheral target removal, essentially all DRG neurons disappeared within 5 d. In contrast, after removal of the spinal cord, approximately half of the DRG neurons survived for at least 8 d. Hence, some E15 DRG neurons could survive without the spinal cord. However, those DRG neurons that died after spinal cord ablation apparently required trophic factors from both central and peripheral targets, because the presence of only one of these tissues was not adequate by itself to support this cell group. Addition of neurotrophin-3 (NT-3) to the culture medium rescued some DRG neurons after CNS removal, suggesting a possible role for NT-3 in vivo. In other experiments, cultures were established from older (E16) embryos, and essentially all neurons survived after spinal cord ablation, even without added factors. These and other experiments indicated that approximately 65% of DRG neurons are transiently dependent on the CNS early in development.     

Antagonism of neuronal kainate receptors by lanthanum and gadolinium

The effects of lanthanum and gadolinium on currents evoked by excitatory amino acids were studied in cultured rat hippocampal and cortical neurons, in freshly dissociated dorsal root ganglion neurons, and in human embryonic kidney 293 cells expressing the GluR6 kainate receptor subunit. In all of these cells, currents mediated by kainate-preferring receptors were antagonized by low micromolar concentrations of the trivalent ions. At negative holding potentials, the IC50 values for inhibition in DRG cells were 2.8 microM for La and 2.3 microM for Gd. Kainate receptor-mediated currents in hippocampal neurons and in 293 cells expressing GluR6 were blocked by La with IC50 values of 2.1 and 4.4 microM, respectively. Steady-state inhibition by the lanthanides showed very slight dependence on membrane potential, however, we were not able to resolve any systematic variation with membrane potential in the kinetics of block onset or recovery. Inhibition was not use-dependent and was not overcome by increasing the concentration of agonist. These results indicate that lanthanides probably do not bind deep within the ion pore or directly compete for the agonist binding site. In contrast to neuronal AMPA receptors, which require more than 100 microM lanthanides for half-maximal blockade, the inhibition of neuronal and recombinant kainate receptors by these ions displays significantly higher potency.     

The expression of P2X3 purinoreceptors in sensory neurons: effects of axotomy and glial-derived neurotrophic factor

We have studied the distribution and regulation of the P2X3 receptor (a ligand-gated ion channel activated by ATP) in adult dorsal root ganglion (DRG) neurons using a polyclonal antibody. P2X3 receptor immunoreactivity was normally present in about 35% of L4/5 DRG neurons, virtually all small in diameter. In the dorsal horn, P2X3 receptor expression was restricted to the terminals of sensory neurons terminating in lamina IIinner. P2X3 receptors were expressed in approximately equal numbers of sensory neurons projecting to skin and viscera but in very few of those innervating skeletal muscle. P2X3 receptors were found mostly in sensory neurons that bind the lectin IB4. After sciatic nerve axotomy, P2X3 receptor expression dropped by more than 50% in L4/5 DRG. Glial cell line-derived neurotrophic factor (GDNF), delivered intrathecally, completely reversed axotomy-induced down-regulation of the P2X3 receptor. We conclude that P2X3 receptors are normally expressed in nociceptive primary sensory neurons, predominantly the nonpeptidergic nociceptors. P2X3 receptors are down-regulated following peripheral nerve injury and their expression can be regulated by GDNF.