Superior Cervical Ganglion Cells Research Articles

Extracellular metabolism of nucleotides in neuroblastoma x glioma NG108-15 cells determined by capillary electrophoresis.

1. The metabolism of extracellular nucleotides in NG108-15 cells, a neuroblastoma x glioma hybrid cell line, was studied by means of capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC). 2. In NG108-15 cells ATP, ADP, AMP, UTP, UDP, and UMP were hydrolyzed to the nucleosides adenosine and uridine indicating the presence of ecto-nucleotidases and ectophosphatases. The hydrolysis of the purine nucleotides ATP and ADP was significantly faster than the hydrolysis of the pyrimidine nucleotides UTP and UDP. 3. ATP and UTP breakdown appeared to be mainly due to an ecto-nucleotide-diphosphohydrolase. ADP, but not UDP, was initially also phosphorylated to some extent to the corresponding triphosphate, indicating the presence of an adenylate kinase on NG108-15 cells. The alkaline phosphatase (ALP) inhibitor levamisole did not only inhibit the hydrolysis of AMP to adenosine and of UMP to uridine, but also the degradation of ADP and to a larger extent that of UDP. ATP and UTP degradation was only slightly inhibited by levamisole. 4. These results underscore the important role of ecto-alkaline phosphatase in the metabolism of adenine as well as uracil nucleotides in NG108-15 cells Dipyridamole, a potent inhibitor of nucleotide breakdown in superior cervical ganglion cells, had no effect on nucleotide degradation in NG108-15 cells. 5. Dipyridamole, which is a therapeutically used nucleoside reuptake inhibitor in humans, reduced the extracellular adenosine accumulation possibly by allosteric enhancement of adenosine reuptake into the cells.

Delayed Rectifier K+Currents,IK, Are Encoded by Kv2 α-Subunits and Regulate Tonic Firing in Mammalian Sympathetic Neurons

Previous studies have revealed the presence of four kinetically distinct voltage-gated K+ currents, I(Af), I(As), I(K), and I(SS), in rat superior cervical ganglion (SCG) neurons and demonstrated that I(K) and I(SS) are expressed in all cells, whereas I(Af) and I(As) are differentially distributed. Previous studies have also revealed the presence of distinct components of I(Af) encoded by alpha-subunits of the Kv1 and Kv4 subfamilies. In the experiments described here, pore mutants of Kv2.1 (Kv2.1W365C/Y380T) and Kv2.2 (Kv2.2W373C/Y388T) that function as Kv2 subfamily-specific dominant negatives (Kv2.1DN and Kv2.2DN) were generated to probe the functional role(s) of Kv2 alpha-subunits. Expression of Kv2.1DN or Kv2.2DN in human embryonic kidney-293 cells selectively attenuates Kv2.1- or Kv2.2-encoded K+ currents, respectively. Using the Biolistics Gene Gun, cDNA constructs encoding either Kv2.1DN or Kv2.2DN [and enhanced green fluorescent protein (EGFP)] were introduced into SCG neurons. Whole-cell recordings from EGFP-positive Kv2.1DN or Kv2.2DN-expressing cells revealed selective decreases in I(K). Coexpression of Kv2.1DN and Kv2.2DN eliminates I(K) in most (75%) SCG cells and, in the remaining (25%) cells, I(K) density is reduced. Together with biochemical data revealing that Kv2.1 and Kv2.2 alpha-subunits do not associate in rat SCGs, these results suggest that Kv2.1 and Kv2.2 form distinct populations of I(K) channels, and that Kv2 alpha-subunits underlie (most of) I(K) in SCG neurons. Similar to wild-type cells, phasic, adapting, and tonic firing patterns are evident in SCG cells expressing Kv2.1DN or Kv2.2DN, although action potential durations in tonic cells are prolonged. Expression of Kv2.2DN also results in membrane depolarization, suggesting that Kv2.1- and Kv2.2-encoded I(K) channels play distinct roles in regulating the excitability of SCG neurons.

Ionic basis of the resting membrane potential in cultured rat sympathetic neurons.

The conductances which determine the resting membrane potential of rat superior cervical ganglia (SCG) neurons were investigated using perforated voltage- and current-clamp whole-cell techniques. The resting potential of SCG cells varied from -47 to -80 mV (-58.3 +/- 0.8 mV, n = 55). Blockade of M and h currents induced a depolarisation (7.4 +/- 0.7 mV, n = 22) and a hyperpolarisation (7.2 +/- 0.7 mV, n = 20) respectively; however, no correlation between the amplitude of these currents and the resting potential was found. The inhibition of the Na/K pump also induced membrane depolarisation (3.2 +/- 0.2 mV, n = 8). Inhibition of voltage-gated currents unmasked a voltage-independent resting conductance reversing at -50 mV. The reversal potential of the voltage-independent conductance, which included the electrogenic contribution of the Na/K pump, was strongly correlated with the resting potential (R = 0.87, p < 0.0001, n = 30). Ionic substitution experiments confirmed the existence of a voltage-independent conductance (leakage) with four components, a main potassium conductance, two minor sodium and chloride conductances and a small contribution of the Na/K pump. It is concluded that the resting potential of SCG cells strongly depends on the reversal potential of the voltage-independent conductance, with voltage-activated M and h currents playing a prominent stabilising role.

Involvement of TLCK-sensitive serine protease in colchicine-induced cell death of sympathetic neurons in culture.

Superior cervical ganglion (SCG) cells from neonatal rats underwent apoptosis upon treatment with colchicine, a microtubule-disrupting agent. Western blotting and activity measurements showed that caspase-3 was indeed activated, but its peptide inhibitor (Ac-DEVD-CHO) neither suppressed nuclear fragmentation nor rescued the neurons from cell death. z-VAD-fmk, the general inhibitor of caspases, prevented nuclear fragmentation and delayed the cell death. Moreover, N-alpha-tosyl-L-lysine chloromethyl ketone (TLCK), but not N-alpha-tosyl-L-phenylalanine chloromethyl ketone (TPCK), prevented nuclear fragmentation and provided neuronal protection as well. The combination of both z-VAD-fmk and TLCK provided a long-term neuronal protection (>4 days), whereas neither one alone could do so, suggesting that there are both caspase-dependent and -independent pathways. TLCK-sensitive serine protease is also likely to act upstream of caspase-3 in a caspase-dependent pathway. Electron microscopic observations demonstrated that z-VAD-fmk suppressed nuclear fragmentation and improved mitochondrial swelling, but failed to prevent vesicular formation, which resulted in a slowly-occurring necrosis. More importantly, TLCK effectively blocked this abundant vesicular formation along with suppressing chromatin condensation. Thus, the combination of both conferred a nearly normal morphology, which is consistent with the results of cell survival experiments. These findings clearly indicate that TLCK-sensitive serine protease plays multiple roles in caspase-dependent and -independent pathways of colchicine-induced cell death, and suggest a novel mechanism underlying a necrotic pathway involving ER swelling and vesicular formation.

Molecular Heterogeneity of the Voltage-Gated Fast Transient Outward K+ Current, IAf, in Mammalian Neurons

Recently, we identified four kinetically distinct voltage-gated K(+) currents, I(Af), I(As), I(K), and I(SS), in rat superior cervical ganglion (SCG) neurons and demonstrated that I(Af) and I(As) are differentially expressed in type I (I(Af), I(K), I(SS)), type II (I(Af), I(As), I(K), I(SS)), and type III (I(K), I(SS)) SCG cells. In addition, we reported that I(Af) is eliminated in most ( approximately 70%) SCG cells expressing Kv4.2W362F, a Kv4 subfamily-specific dominant negative. The molecular correlate(s) of the residual I(Af), as well as that of I(As), I(K), and I(SS), however, are unknown. The experiments here were undertaken to explore the role of Kv1 alpha-subunits in the generation of voltage-gated K(+) currents in SCG neurons. Using the Biolistics Gene Gun, cDNA constructs encoding a Kv1 subfamily-specific dominant negative, Kv1.5W461F, and enhanced green fluorescent protein (EGFP) were introduced into SCG neurons. Whole-cell recordings from EGFP-positive Kv1.5W461F-expressing cells revealed a selective decrease in the percentage of type I cells and an increase in type III cells, indicating that I(Af) is gated by Kv1 alpha-subunits in a subset of type I SCG neurons. I(Af) is eliminated in all SCG cells expressing both Kv1.5W461F and Kv4.2W362F. I(Af) tau(decay) values in Kv1.5W461F-expressing and Kv4.2W362F-expressing type I cells are significantly different, revealing that Kv1 and Kv4 alpha-subunits encode kinetically distinct I(Af) channels. Expression of Kv1.5W461F increases excitability by decreasing action potential current thresholds and converts phasic cells to adapting or tonic firing. Interestingly, the molecular heterogeneity of I(Af) channels has functional significance because Kv1- and Kv4-encoded I(Af) play distinct roles in the regulation of neuronal excitability.

Effects of ATP on intracellular calcium dynamics of neurons and satellite cells in rat superior cervical ganglia.

Adenosine 5'-triphosphate (ATP) which is released from neuronal and non-neuronal tissues interacts with cell surface receptors to produce a broad range of physiological responses. The present study addressed the issue of whether the cells of the superior cervical ganglia (SCG) respond to ATP. To this end, the dynamics of the intracellular calcium ion concentration ([Ca2+]i) of neurons and satellite cells in intact SCG was analyzed by laser scanning confocal microscopy. ATP produced an increase of [Ca2+]i in both neurons and satellite cells; initially, ATP elicited [Ca2+]i increase in satellite cells and, subsequently, a [Ca2+]i change in neurons was observed. P1 purinoceptor agonists had no effect on this process, but P2 purinoceptor agonists induced [Ca2+]i increase and suramin totally inhibited ATP-induced [Ca2+]i dynamics in both neurons and satellite cells. In satellite cells, Ca2+ channel blockers and the removal of extracellular Ca2+, but not thapsigargin pretreatment, abolished ATP-induced [Ca2+]i dynamics. In contrast, thapsigargin pretreatment abolished ATP-induced [Ca2+]i dynamics in neurons. Reactive blue-2 inhibited the ATP-induced reaction on neurons alone. Uridine 5'-triphosphate caused a [Ca2+]i increase in neurons and alpha,beta-methylene ATP caused a [Ca2+]i increase in satellite cells. We concluded that neurons respond to extracellular ATP mainly via P2Y purinoceptors and that satellite cells respond via P2X purinoceptors.

SERCA function declines with age in adrenergic nerves from the superior cervical ganglion.

1. Intracellular calcium is a universal second messenger integrating numerous cellular pathways. An age-related breakdown in the mechanisms controlling [Ca2+]i homeostasis could contribute to neuronal degeneration. One component of neuronal calcium regulation believed to decline with age is the function of sarco/endoplasmic reticulum calcium ATPase (SERCA) pumps. 2. Therefore we investigated the impact of age on the capacity of SERCA pumps to control high (68 mM) [K+]-evoked [Ca2+]i-transients in acutely dissociated superior cervical ganglion (SCG) cells from 6- and 20-month-old Fisher-344 rats. Calcium transients were measured by fura-2 microfluorometry in the presence of vanadate (0.1 microM) to selectively block plasma membrane calcium ATPase (PMCA) pumps, dinitrophenol (100 microM) to block mitochondrial calcium uptake and extracellular sodium replaced with tetraethylammonium to block Na+/Ca2+-exchanger, thus forcing the neuronal cells to rely on SERCA uptake to control [Ca2+]i homeostasis. 3. In the presence of these calcium buffering blockers, the rate of recovery of [Ca2+]i was significantly slower and time to recover to approximately 90% of resting [Ca2+]i was significantly greater in SCG cells from old (20 months) compared with young (6 months) animals. 4. This age-related change in the recovery phase of [K+]-evoked [Ca2+]i-transients could not be explained by differences in the sensitivity of SCG cells to the calcium buffering blockers, as no age-related difference in basal [Ca2+]i was observed. 5. These studies illustrate that when rat SCG cells are forced to rely on SERCAs to buffer [K+]-evoked [Ca2+]i-transients, an age-related decline in SERCA function is revealed. Such age-related declines in calcium regulation coupled with neuronal sensitivity to calcium overload underscore the importance of understanding the components of [Ca2+]i homeostasis and the functional compensation that may occur with advancing age.

Expression of arachidonate 12-lipoxygenase in rat tissues: a possible role in glucagon secretion.

There are three isoforms of arachidonate 12-lipoxygenase in mammals: platelet, leukocyte, and epidermal types. We found in this study that the leukocyte-type enzyme was present in rat pineal gland, lung, spleen, aorta, adrenal gland, spinal cord, and pancreas, as assessed by RT-PCR. Immunohistochemical analysis showed that the enzyme was localized in macrophages in lung and spleen, alpha-cells of pancreatic islet, zona glomerulosa cells of adrenal cortex, and neuronal cells of spinal cord and superior cervical ganglion. The presence of the 12-lipoxygenase in pancreatic alpha-cells was confirmed by glucagon staining in a consecutive section. We overexpressed the leukocyte-type 12-lipoxygenase cDNA in a glucagon-secreting alphaTC clone 6 cell line that had been established from a transgenic mouse. Glucagon secretion was stimulated by approximately twofold in the 12-lipoxygenase-expressing cells compared to the mock-transfected and original cells. The results suggest that the 12-lipoxygenase of the leukocyte type augments glucagon secretion from pancreatic islets.

Muscarinic Inhibition of Calcium Current and M Current in Gαq-Deficient Mice

Activation of M(1) muscarinic acetylcholine receptors (M(1) mAChR) inhibits M-type potassium currents (I(K(M))) and N-type calcium currents (I(Ca)) in mammalian sympathetic ganglia. Previous antisense experiments suggested that, in rat superior cervical ganglion (SCG) neurons, both effects were partly mediated by the G-protein Galpha(q) (Delmas et al., 1998a; Haley et al., 1998a), but did not eliminate a contribution by other pertussis toxin (PTX)-insensitive G-proteins. We have tested this further using mice deficient in the Galpha(q) gene. PTX-insensitive M(1) mAChR inhibition of I(Ca) was strongly reduced in Galpha(q) -/- mouse SCG neurons and was fully restored by acute overexpression of Galpha(q). In contrast, M(1) mAChR inhibition of I(K(M)) persisted in Galpha(q)-/- mouse SCG cells. However, unlike rat SCG neurons, muscarinic inhibition of I(K(M)) was partly PTX-sensitive. Residual (PTX-insensitive) I(K(M)) inhibition was slightly reduced in Galpha(q) -/- neurons, and the remaining response was then suppressed by anti-Galpha(q/11) antibodies. Bradykinin (BK) also inhibits I(K(M)) in rat SCG neurons via a PTX-insensitive G-protein (G(q) and/or G(11); Jones et al., 1995). In mouse SCG neurons, I(K(M)) inhibition by BK was fully PTX-resistant. It was unchanged in Galpha(q) -/- mice but was abolished by anti-Galpha(q/11) antibody. We conclude that, in mouse SCG neurons (1) M(1) mAChR inhibition of I(Ca) is mediated principally by G(q), (2) M(1) mAChR inhibition of I(K(M)) is mediated partly by G(q), more substantially by G(11), and partly by a PTX-sensitive G-protein(s), and (3) BK-induced inhibition of I(K(M)) is mediated wholly by G(11).

Sodium overload through voltage-dependent Na + channels induces necrosis and apoptosis of rat superior cervical ganglion cells in vitro

Using the failure to exclude trypan blue as a criterion for cell death, we found that veratridine, the voltage-dependent Na + channel activator, exerted its toxicity to cultured sympathetic neurons in a dose-dependent manner (half-maximal toxicity occurred at 2 μM). The co-presence of tetrodotoxin completely reversed the toxicity only at concentrations of veratridine <20μM. Veratridine neurotoxicity was due to the influx of Na +; a medium low in Na + (36 mM) completely abolished its neurotoxicity, whereas a Ca 2+-free medium did not attenuate its neurotoxicity. Furthermore, the buffering action of 1,2-Bis-(2-aminophenoxy)ethane-N,N,N′,N′,-tetraacetate (BAPTA) on veratridine-induced increase in intracellular Ca 2+ levels neither blocked veratridine neurotoxicity in normal medium, nor attenuated the low Na + effect. Elevated K + effectively blocked veratridine neurotoxicity in a Ca 2+-dependent manner. Cytoplasmic pH measurements using a fluorescent pH indicator demonstrated that cellular acidification (from pH 7.0 to pH 6.5) occurred upon treatment with veratridine. Both veratridine-induced acidification and cell death were ameliorated by 5-(N-ethyl-N-isopropyl)amiloride, the specific inhibitor of the Na +/H + exchanger (IC 50 = 0.5 μM). Finally, necrosis occurred predominantly in veratridine neurotoxicity, but both staining with bis-benzimide and TUNEL analysis showed nuclear features of apoptosis in sympathetic neurons undergoing cell death.

Adrenergic nerves compensate for a decline in calcium buffering during ageing

1. The ubiquitous involvement of intracellular calcium ([Ca2+]i) in multiple neuronal pathways has led investigators to suggest that dysfunction of calcium homeostasis may be the primary mediator of age-related neuronal degeneration. Recently, it was shown that sympathetic neurones from superior cervical ganglion (SCG) of aged rats demonstrate decreased sarco-/endoplasmic reticulum Ca2+-ATPase (SERCA) function and that aged neurones are more dependent upon mitochondria to control K+-evoked [Ca2+]i transients. 2. Therefore, in the present study we investigated age-related changes in ATP-dependent calcium pumps of plasma membrane Ca2+-ATPase (PMCA) and SERCA in acutely dissociated SCG cells from Fischer-344 rats aged 6 and 20 months. To distinguish between PMCA and SERCA pump activity, we applied the Ca2+-ATPase blocker vanadate and measured rates of recovery of K+-evoked [Ca2+]i transients by fura-2 microfluorometry. 3. Young SCG cells showed a biphasic response to vanadate over the vanadate concentration range (0.01-100 microM); however, old SCG cells showed only a single response over the same concentration range. Additionally, old SCG cells showed a greater sensitivity to Ca2+-ATPase blockade by vanadate. 4. The contribution of mitochondrial calcium uptake to regulate [Ca2+]i was also investigated. To measure the impact of mitochondrial calcium uptake, PMCAs and SERCAs were blocked with vanadate (100 microM) and extracellular sodium was replaced with tetraethylammonium (TEA) to block Na+/Ca2+-exchange. Treated SCG cells showed a decline of 50% in rate of recovery of [Ca2+]i in both 6- and 20-month-old cells; however, this effect did not vary with age. 5. These data suggest that there is an age-related decline in function of SERCAs, with an increased reliance on PMCAs to control high K+-evoked [Ca2+]i transients. In addition, there appears to be no age-related change in the capacity of the mitochondria to restore [Ca2+]i transients to basal levels.

Alteration of veratridine neurotoxicity in sympathetic neurons during development in vitro.

Neurotoxic effects of veratridine, the activator of voltage-dependent Na+ channels, were examined at various stages of in vitro development of superior cervical ganglion cells dissociated from newborn rats. Veratridine neurotoxicity did not occur in 1DIV (days in vitro) neurons, but occurred in 7DIV neurons, both of which depend on NGF for survival, but elevated K+ supports only the latter. TUNEL and electron microscopic analyses revealed that 7DIV neurons underwent both apoptotic and necrotic cell death. Veratridine was also toxic to 21DIV neurons which are independent of NGF for survival. Nuclear features of apoptosis, however, were greatly reduced in these neurons undergoing cell death, suggesting that nuclear vulnerability is also subject to developmental regulation in vitro.

Interleukin-12 promotes neurite outgrowth in mouse sympathetic superior cervical ganglion neurons

To determine the role of cytokines in the nervous system, we examined the effect of interleukin-12(IL-12) on the nerve regeneration of mouse superior cervical ganglion cells (SCG). IL-12 enhanced the neurite outgrowth in a concentration-dependent manner. Immunocytochemical studies demonstrated the expression of IL-12 receptors in neuronal bodies and neurites. The mRNA expression of IL-12 receptors in SCG cells was confirmed by reverse transcription-polymerase chain reaction. Our data demonstrated the presence of IL-12 receptors in sympathetic neurons and suggest that IL-12 plays an important role in neuronal regeneration.

Nitric oxide synthase in trigeminal ganglion cells projecting to the cochlea of rat and guinea pig.

Nitric oxide (NO) influences electrophysiological and morphological parameters of the mammalian cochlea. Recently, the isoform of the NO-producing enzyme neuronal NO synthase (nNOS) has been demonstrated in spiral ganglion cells and olivocochlear neurons. The cochlea is also innervated by fibers stemming from the trigeminal ganglion (TG) and superior cervical ganglion (SCG). Whether these ganglion cells contain nNOS is not known yet. We therefore identified TG and SCG cells upon injection of Fluoro-Gold (FG) into the cochlea and retrograde neuronal transport of FG in rat and guinea pig. These ganglion cells were investigated for neuronal NOS immunohistochemically. Perikarya labeled by FG were found in the ipsilateral TG and SCG. In both species investigated, a considerable number of FG-labeled TG cells were also nNOS-immunoreactive whereas SCG cells were not. These data, demonstrating the existence of nNOS-containing TG cells that project to the cochlea, provide evidence that these neurons are further sources of nitric oxide in the cochlea.

The role of ryanodine receptors in the cyclic ADP ribose modulation of the M-like current in rodent m1 muscarinic receptor-transformed NG108-15 cells.

1. The role of cyclic ADP ribose and ryanodine receptors in the inhibition of the M-like current (IK(M,ng)) by acetylcholine was investigated in m1 muscarinic receptor-transformed mouse neuroblastoma-rat glioma hybrid (NG108-15) cells using patch-clamp techniques and calcium microfluorimetry. 2. Acetylcholine (1-100 microM) decreased IK(M,ng) by up to 55 %. Application, via the patch pipette, of the cyclic ADP ribose antagonists 8-amino-cyclic ADP ribose (10-100 microM) and 8-bromo-cyclic ADP ribose (100-1000 microM) reduced this inhibition of IK(M,ng) in a concentration-dependent manner. The half-maximal inhibition concentrations for 8-amino- cyclic ADP ribose and 8-bromo-cyclic ADP ribose were around 40 microM and 1 mM, respectively. 3. Neither of the cyclic ADP ribose antagonists altered the amplitude of IK(M,ng) per se, or the incidence of the concurrent Ca2+-activated K+ current (IIK(Ca)) activation, also mediated by acetylcholine. 4. The ryanodine receptor modulators ryanodine (1-10 microM) and Ruthenium Red (10 microM) did not alter IK(M,ng) amplitude or IK(M,ng) inhibition mediated by acetylcholine. There was a statistically significant increase in the proportion of cells showing outward currents in the presence of Ruthenium Red. 5. Intracellular calcium levels measured with fura-2 microfluorimetry were increased with low concentrations of ryanodine (1 microM), more consistently with caffeine (10 mM), and in almost every case with both bradykinin (300 nM) and acetylcholine (100 microM). Caffeine-, but not bradykinin-evoked responses were abolished by preincubation with ryanodine (10 microM). 6. The fast 'rundown rate' of the M-current recorded in rat superior cervical ganglion cells under whole-cell conditions precluded an investigation of the effects of intracellular dialysis of cyclic ADP ribose. However, when cyclic ADP ribose (5 microM) was applied directly to the cytoplasmic face of inside-out membrane patches excised from rat superior cervical ganglion cells containing M-channels, it had no effect on the main parameters of single channel activity (conductance, mean open time or frequency of opening). 7. These results indicate that cyclic ADP ribose acts on a specific intracellular site to mediate IK(M,ng) inhibition. However, unlike previously established effects of cyclic ADP ribose, the ryanodine receptor is not required, suggesting that another molecular target may be involved. Studies at the single channel level indicate that cyclic ADP ribose may not act directly on the M-channels in inside-out patches.

Effects of glucagon on axoplasmic transport in mouse superior cervical ganglion cells.

The effects of glucagon on the axoplasmic transport of cultured mouse superior cervical ganglion cells were analyzed with the video-enhanced DIC microscope system. Glucagon increased the rate of fast axoplasmic transport by 30% in both anterograde and retrograde directions. The average velocity was increased from 1.36 +/- 0.48 microns/s to 1.74 +/- 0.43 microns/s (anterograde, n = 60) and from 1.37 +/- 0.48 microns/s to 1.62 +/- 0.39 microns/s (retrograde, n = 60). The stimulatory effect of glucagon on the axoplasmic transport was reversed in a glucose-free medium, whereas blocking the citrate cycle by pretreating neuronal cells with malonate did not alter the effect of glucagon. Together with our previous findings, our data suggest that neurotransmitters and hormones play a major role in the regulation of fast axoplasmic transport.

Growth cones exhibit enhanced cell–cell adhesion after neurotransmitter release

Evoked release of acetylcholine and subsequent cell–cell adhesive contacts between growth cones and acetylcholine sensing neurons were observed using cultured neurons dissociated from the diagonal band of Broca of the rat. Stimulation to the soma of the diagonal band of Broca neurons evoked release of acetylcholine from the growth cones. The release of acetylcholine was monitored using whole-cell patch-clamp recording from acetylcholine receptor-rich superior cervical ganglion neuron positioned on the growth cone as a sensor of acetylcholine release. By measuring changes in fluorescence from the growth cone using Ca 2+-sensitive dye while voltage-clamping the superior cervical ganglion neuron, transient intracellular Ca 2+ concentration increase and acetylcholine release from growth cone were recorded simultaneously. Video-enhanced differential interference contrast imaging of the growth cones demonstrated tether formation between the growth cone and superior cervical ganglion cell soma when the superior cervical ganglion cell soma was moved away from the growth cone after acetylcholine release, suggesting formation of adhesive contacts between the growth cone and the sensor neuron. Adhesive contacts between growth cones and sensor neurons were also detected when a high K + solution or α-latrotoxin was applied to the growth cone. Adhesions were also observed between growth cones and latex beads, when growth cones were exposed to high K + solution. The properties of the adhesive contacts at the growth cone were studied by optically manipulating a latex bead attached to the growth cone surface. These results suggest that growth cones exhibit cell–cell adhesion after neurotransmitter release.

Analysis of a novel mechanism of neuronal toxicity produced by an apolipoprotein E-derived peptide.

The apolipoprotein E (apoE)-derived peptide (141-155)2 has a neurotoxic effect, implying that apoE itself could be a source of toxicity in Alzheimer's disease brain. We characterized the toxicity of this peptide on superior cervical ganglion (SCG) neurons and compared the death with the apoptotic death that occurs after nerve growth factor (NGF) deprivation in these cells. A dose of 10 microM apoE (141-155)2 resulted in the death of approximately 50% of the neurons within 24 h. Nuclear condensation and DNA fragmentation preceded the death. However, most inhibitors of NGF deprivation-induced death, including the caspase inhibitor Boc-aspartyl(O-methyl)fluoromethyl ketone and genetic deletion of bax-/-, had no effect on the toxicity. Inclusion of depolarizing levels of potassium did block the toxicity. Receptor-associated peptide (RAP), an antagonist for apoE receptors, did not protect cells in either SCG or hippocampal cultures. In addition, RAP had no effect on internalization of the apoE peptide. These data support the observation that apoE (141-155)2 is neurotoxic but suggest that the neurotoxicity is distinct from classical apoptosis or necrosis. Furthermore, these results indicate that the toxic effect may occur independently of members of the low-density lipoprotein receptor gene family.

Transcriptional and Posttranscriptional Control of Tyrosine Hydroxylase Gene Expression During Persistent Stimulation of Pituitary Adenylate Cyclase‐Activating Polypeptide Receptors on PC12 Cells: Regulation by Protein Kinase A‐Dependent and Protein Kinase A‐Independent Pathways

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates catecholamine release and biosynthesis in sympathetic postganglionic cells. Moreover, PACAP receptor activation in cultured adrenal chromaffin and superior cervical ganglion cells has been reported to increase the expression of the gene coding for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis. However, the relative contribution of transcriptional and posttranscriptional mechanisms to the effects of PACAP on TH gene expression has not been evaluated. Therefore, in this study we compared the temporal effects of PACAP on TH gene transcription with the duration of its effects on TH mRNA levels. We had previously shown that vasoactive intestinal polypeptide, peptide histidine isoleucine, and secretin, peptides closely related to PACAP, induce TH gene expression through a cyclic AMP (cAMP)-dependent pathway. Therefore, using a mutant PC12 cell line deficient in cAMP-dependent protein kinase II (PKA), we also evaluated the role of the cAMP pathway in the effect of PACAP on TH gene expression. Continuous treatment of wild-type PC12 cells with PACAP (1 nM) increased TH mRNA levels maximally by 12 h and maintained TH mRNA at near maximal levels for at least 2 days. In contrast, the rate of TH gene transcription, as measured by a nuclear run-on assay, was maximal by 1 h and returned to basal levels by 3 h. The fact that a new steady-state level of TH mRNA was achieved and maintained for days in the absence of a sustained increase in TH gene transcription supports the involvement of posttranscriptional mechanisms. Removal of PACAP after 12 h, a time at which TH gene transcription was at basal levels, resulted in a subsequent return of TH mRNA to unstimulated levels within 36 h. Thus, continuous PACAP stimulation is required to maintain sustained increases in TH mRNA levels in the absence of a sustained elevation of transcription. To examine the role of the cAMP pathway in these effects, we compared the effects of PACAP in wild-type PC12 cells and in a mutant PC12 cell line (A126-1B2) that is deficient in PKA. PACAP failed to stimulate either TH mRNA levels or TH gene transcription in the mutant cells. In contrast to the effects of PACAP, dexamethasone increased TH mRNA levels by the same magnitude in both cell lines. It is noteworthy that stimulation of the PKA-deficient mutant cells with a combination of PACAP and dexamethasone (1 microM) produced a synergistic increase in TH mRNA levels, which was nearly twice that induced by dexamethasone stimulation alone. This synergistic effect was not transcriptionally mediated. The effect of the combined treatment on TH gene transcription was identical to the effect of dexamethasone alone. Taken together, these data indicate that PACAP regulates TH gene expression through a transcriptional mechanism requiring an intact cAMP pathway and through posttranscriptional mechanisms under the control of a cAMP-independent pathway(s).

CNS neuroblasts migrate in three-dimensional gels with extracellular matrix containing laminin

The effect of dibutyryl cyclic AMP (dbcAMP) on axoplasmic transport of cultured hippocampal neuron cells from postnatal 1-day mice was analyzed with a computer-assisted video-enhanced differential interference contrast microscope system. Dibutyryl cyclic AMP increased the axoplasmic transport in both anterograde and retrograde directions. The number of particles flowing in the neurites was increased by 0.5 mM dbcAMP. The peak reached about 160% of the initial value. The instantaneous velocity of axoplasmic transport was also increased by 0.5 mM dbcAMP. The average velocity of anterograde and retrograde direction changed respectively from 1.95±1.01 μm/s (n=55) to 2.66±1.26 μm/s (n=58) and from 1.94±0.85 (n=57) to 2.39±0.93 (n=57). Rates were 136.1 and 123.1%, respectively. Previously, we have found that acetylcholine suppressed and adrenaline increased the axoplasmic transport in superior cervical ganglion cells. These effects are related to the amount of endogeneous cAMP. The results of the present report suggest that endogeneous cAMP is also related to hippocampal axoplasmic transport.