Rapid pH and PO2 changes in the tissue recording chamber during stoppage of a gas-equilibrated perfusate: effects on calcium currents in ventral horn neurons

In vitro studies often use bicarbonate-buffered saline solutions to mimic the normal extracellular environment of tissues. These solutions are typically equilibrated with gaseous O2 and CO2, the latter interacting with bicarbonate ions to maintain a physiological pH. In vitro tissue chambers, like those used for electrophysiology, are usually continually perfused with the gassed buffer, but stopping the perfusion to add expensive chemicals or acquire imaging data is a common practice. The present study demonstrates that this procedure leads to rapid (< 30 s) increases in pH and decreases in PO2 of the detained solution in the tissue chamber. During the first 200 s, pH increased by 0.4 units and resulted in a 25% PO2 reduction of the detained solution. The rates of these changes were dependent on the volume of solution in the chamber. In experiments using acute transverse slices from the lumbar spinal cord of neonatal (postnatal day 0-10) mice, perfusion stoppage of the same duration was accompanied by a 34.7% enhancement of the peak voltage-gated calcium current recorded from ventral horn neurons. In these cells both low voltage-activated and high voltage-activated currents were affected. These currents were unaffected by decreasing PO2 when a CO2-independent buffer was used, suggesting that changes in pH were responsible for the observed effects. It is concluded that the procedure of stopping a bicarbonate/CO2-buffered perfusate results in rapid changes in pH and PO2 of the solution detained in the tissue chamber, and that these changes have the potential to covertly influence experimental results.     

Characterization of calcium currents in functionally mature mouse spinal motoneurons

Motoneurons integrate synaptic input and produce output in the form of trains of action potentials such that appropriate muscle contraction occurs. Motoneuronal calcium currents play an important role in the production of this repetitive firing. Because these currents change in the postnatal period, it is necessary to study them in animals in which the motor system is 'functionally mature', that is, animals that are able to weight-bear and walk. In this study, calcium currents were recorded using whole-cell patch-clamp techniques from large (> 20 microm) ventral horn cells in lumbar spinal cord slices prepared from mature mice. Ninety percent (nine out of 10) of the recorded cells processed for choline acetyltransferase were found to be cholinergic, confirming their identity as motoneurons. A small number of motoneurons were found to have currents with low-voltage-activated (T-type) characteristics. Pharmacological dissection of the high-voltage-activated current demonstrated omega-agatoxin-TK- (P/Q-type), omega-conotoxin GVIA- (N-type), and dihydropyridine- and FPL-64176-sensitive (L-type) components. A cadmium-sensitive component of the current that was insensitive to these chemicals (R-type) was also seen in these cells. These results indicate that the calcium current in lumbar spinal motoneurons from functionally mature mice is mediated by a number of different channel subtypes. The characterization of these calcium channels in mature mammalian motoneurons will allow for the future study of their modulation and their roles during behaviours such as locomotion.     

nov基因在不同种属动物脊髓中的表达

用地高辛标记的cRNA探针原位杂交方法研究了鲢、青蛙、蛇、鸡、牛、犬和猫脊髓中肾母细胞瘤过度表达基因（nov）mRNA阳性神经元的种系发育。结果显示，低等脊椎动物鲢、青蛙和蛇脊髓中仅有少量novmRNA阳性神经元，分布于灰质腹角。鸡脊髓中阳性神经元除主要分布于脊髓腹角外，中央灰质也有少量分布。哺乳动物牛、大和猫脊髓灰质中novmRNA阳性神经元分布广泛，背腹角、中央灰质及中央核区都检测到很强的杂交信号。以上结果表明，nov基因在从低等脊椎动物到高等脊椎动物的进化过程中非常保守，这种保守性提示nov基因在脊髓神经元发育、分化及正常生理功能中可能具有重要作用。     

Substance P neurons sprout in the cervical spinal cord of the wobbler mouse: a model for motoneuron disease

The mutant mouse, wobbler, possesses a recessively inherited degeneration of motoneurons and other ventral horn cells in the cervical spinal cord, and therefore it has been proposed as an animal model of human motoneuron disease. Affected mice have been identified by behavioral tests that also determined the extent of the motor deficit. The results from these tests were combined and used to define distinct stages of the disease process that could then be correlated histochemically with the amount of acetylcholinesterase (AChE) staining in the cervical spinal cord. AChE is used as a marker for cholinergic neurons and is known to hydrolyze the neuropeptide modulator substance P (SP). SP, a peptide neuromodulator of primary afferent transmission in the dorsal horn, excites motoneurons in the ventral horn, and may possess secondary functions in neuronal maintenance. Therefore, the levels of immunoreactive (IR) SP and AChE were examined in an attempt to determine the possible interaction between these factors in motoneuron degeneration. By enzyme histochemistry, the cervical spinal cord, taken from wobbler mice at behaviorally identified stages of the motor deficit, exhibited decreased levels of AChE throughout the ventral horn. The decrease detected in the AChe staining intensity was linear and correlated with the decrease in the number of AChE-positive cells in the ventral cervical spinal cord, as the motor deficit progressed. Presumably, the continual decrease in AChE staining represents the degeneration of cholinergic perikarya and neuronal processes in the ventral horn as the motoneuron disease proceeds. At two well-established stages of the motor deficit, the amount of immunoreactive SP increased in the ventral horn compared with the control mice. The elevated levels of immunoreactive SP suggest sprouting may have occurred preceding, or in response to, the motoneuron degeneration. Several additional hypotheses are discussed in respect to phenomena that might contribute to the increase of immunoreactive SP in the degenerating ventral horn of the wobbler mouse.     

HCN1 ion channel immunoreactivity in spinal cord and medulla oblongata

Hyperpolarization-activated cyclic nucleotide-gated (HCN) non-selective cation channels in neurons carry currents proposed to perform diverse functions, including the hyperpolarization activated Ih current. The 4 HCN subunits have unique but overlapping patterns of expression in the CNS. Here, we examined the distribution of HCN1 channel subunits in the brainstem and spinal cord using immunohistochemistry. At all levels of the spinal cord dorsal horn, HCN1 immunoreactivity (HCN1-IR) was predominantly absent from laminae I and II, while a dense band of punctate labeling was visible in lamina III. Labeled neurons were identified in close vicinity to the central canal, in the lateral spinal nucleus, in the ventral horn and occasionally in lamina II and III. Those in the ventral horn were identified as alpha motor neurons using retrograde tracing and/or double or triple immunostaining with neuronal markers neurofilament 200 (NF200) and choline acetyltransferase. HCN1-IR neurons in the brainstem included neurons in sensory pathways such as the dorsal column nuclei, the area postrema, the spinal trigeminal nucleus as well as identified motor neurons in motor nuclei. In the nucleus ambiguus, a mixed visceral/motor nucleus, HCN1-IR was present only in NF200-IR cells, suggesting that it is expressed in motor but not autonomic preganglionic neurons. HCN1-IR motor neurons in the nucleus ambiguus also expressed the neurokinin 1 receptor and were labeled retrogradely from the larnyx. At the light microscopic level, the NTS and inferior olive contained punctate labeling, which ultrastructural examination revealed to be present in predominantly synaptic terminals or dendrites respectively. These data therefore described the first localization of the HCN1 subunit in the spinal cord and extend previous reports from the brainstem.     

Interneuronal synapses formed by motor neurons appear to be glutamatergic

Acetylcholine release at motor neuron synapses has been long established; however, recent discoveries indicate that synaptic transmission by motor neurons is more complex than previously thought. Using whole-cell patch clamp, we show that spontaneous excitatory postsynaptic currents of rat motor neurons in primary ventral horn cultures are entirely glutamatergic, although the cells respond to exogenous acetylcholine. Motor neurons in cultures express the vesicular glutamate transporter VGlut2, and culturing motor neurons for weeks with glutamate receptors blocked upregulates glutamate signaling without increasing cholinergic signaling. In spinal cord slices, motor neurons showed no decrease in spontaneous excitatory synaptic potentials after blocking acetylcholine receptors. Our results suggest that motor neuron synapses formed on other neurons are largely glutamatergic in culture and the spinal cord.     

Calcium-dependent inactivation of the monosynaptic NMDA EPSCs in rat hippocampal neurons in culture.

The effects of increased dendritic calcium concentration ([Ca2+]i) induced by single action potentials on monosynaptic glutamatergic excitatory postsynaptic currents (EPSCs) were studied in cultured rat hippocampal neurons. To investigate the respective roles of pre- and postsynaptic elements in the depolarization-induced NMDAR inactivation, we have performed simultaneous paired whole-cell recordings from monosynaptically connected pre- and postsynaptic hippocampal neurons. We report that the single firing of the postsynaptic neuron did not result in inactivation of the NMDAR-EPSC, whereas a burst of depolarizing steps transiently depressed the NMDAR-EPSCs in both pyramidal cells and interneurons. This effect was mediated by postsynaptic voltage-gated Ca2+ influx, as it was prevented by: (i) buffering postsynaptic [Ca2+]i with 30 mM BAPTA; (ii) removing extracellular Ca2+; or (iii) applying Cd2+o (100 microM), a voltage-gated calcium channel blocker. It does not involve presynaptic mechanisms as it selectively affected NMDA but not alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor-mediated EPSCs. These results suggest that inactivation of NMDAR-channels by voltage-gated Ca influx is a general property of hippocampal neurons, which may play an important role in reducing postsynaptic NMDAR Ca2+ influx that leads to plasticity or excitotoxicity during sustained neuronal activity.     

Contributions of T-Type Voltage-Gated Calcium Channels to Postsynaptic Calcium Signaling within Purkinje Neurons

Low threshold voltage-gated T-type calcium channels have long been implicated in the electrical excitability and calcium signaling of cerebellar Purkinje neurons although the molecular composition, localization, and modulation of T-type channels within Purkinje cells have only recently been addressed. The specific functional roles that T-type channels play in local synaptic integration within Purkinje spines are also currently being unraveled. Overall, Purkinje neurons represent a powerful model system to explore the potential roles of postsynaptic T-type channels throughout the nervous system. In this review, we present an overview of T-type calcium channel biophysical, pharmacological, and physiological characteristics that provides a foundation for understanding T-type channels within Purkinje neurons. We also describe the biophysical properties of T-type channels in context of other voltage-gated calcium channel currents found within Purkinje cells. The data thus far suggest that one specific T-type isoform, Ca_v3.1, is highly expressed within Purkinje spines and both physically and functionally couples to mGluR1 and other effectors within putative signaling microdomains. Finally, we discuss how the selective potentiation of Ca_v3.1 channels via activation of mGluR1 by parallel fiber inputs affects local synaptic integration and how this interaction may relate to the overall excitability of Purkinje neuron dendrites.     

Transient expression of GABA immunoreactivity in the developing rat spinal cord.

The development of GABAergic neurons in the spinal cord of the rat has been investigated by immunocytochemical staining of frozen sections with anti-gamma-aminobutyric acid (GABA) antiserum. In the cervical cord, GABA-immunoreactive fibers first appeared at embryonic day (E) 13 in the presumptive white matter within the ventral commissure, ventral funiculus, and dorsal root entrance zone, and in the ventral roots. There were no GABA-immunoreactive cell bodies detected at this age. By E14, motoneurons, the earliest generated spinal cells, were the first cell population to become GABA-immunoreactive at the cell body level. Thereafter, GABA-immunoreactive neurons increased progressively in number and extended from ventral to dorsal regions. GABA-immunoreactive relay neurons within lamina I of the dorsal horn were initially detected at E17. Interneurons in the substantia gelatinosa, the latest generated cells in the spinal cord, were also the last to express the GABA immunoreactivity at E18. Immunoreactive neurons peaked in intensity and extent at E18 and 19. GABA immunoreactivity was only detectable in neurons within the intermediate and marginal zones 1-3 days after they withdrew from the cell cycle. This contrasts to glutamate decarboxylase immunoreactivity, which is detected in precursor cells in the ventricular zone prior to, or during, withdrawal from the cell cycle. Toward the end of gestation, GABA immunoreactivity declined in intensity and extent. This regression began in the ventral horn of the cervical region and ended in the dorsal horn of the lumbosacral region. During the first week after birth, immunoreactivity in motoneurons and in many other neurons within the ventral horn, intermediate gray, and deeper layers of the dorsal horn disappeared, and only in those neurons predominantly within the superficial layers of the dorsal horn did it persist into adulthood. Thus, the expression and regression of GABA immunoreactivity in the spinal cord followed ventral-to-dorsal, rostral-to-caudal, and medial-to-lateral gradients. These observations indicate that the majority of embryonic spinal neurons pass through a stage of transient expression of GABA immunoreactivity. The functional significance of this transient expression is unknown, but it coincides with the period of intense neurite growth of motoneurons, sensory neurons, and interneurons, and of neuromuscular junction formation, suggesting that the transient presence of GABA may play an important role in the differentiation of sensorimotor neuronal circuits.     

Amyotrophic lateral sclerosis: glutamate dehydrogenase and transmitter amino acids in the spinal cord.

Measurements were taken of the activity of glutamate dehydrogenase (GDH) and the levels of transmitter amino acids in anatomically dissected regions of cervical and lumbar spinal cord in eight patients dying with amyotrophic lateral sclerosis (ALS) and in 11 neurologically normal controls. GDH activity was considerably increased in lateral and ventral white matter and in the dorsal horn of the ALS cervical spinal cord, but normal in the ventral horn and the dorsal columns. Similar, although less pronounced, GDH changes were found in the lumbar enlargement. The mean concentrations of aspartate and glutamate were reduced in all regions of ALS spinal cord investigated. Taurine concentrations were significantly increased in several subdivisions of cervical spinal cord, but normal in lumbar regions. Glycine levels were significantly reduced in lumbar ventral and dorsal horns. There was no striking change in spinal cord GABA levels in our ALS patients. It is suggested that the reduced levels of glutamate and aspartate as well as the elevated GDH activity in the spinal cord of ALS patients may reflect an overactivity of the neurons releasing these potentially excitotoxic amino acids and thus may be causally related to the spinal neuro-degenerative changes characteristic of ALS.     

Transient ischemia-induced changes of excitatory amino acid carrier 1 in the ventral horn of the lumbar spinal cord in rabbits

OBJECTIVES: To examine temporal changes of EAAC1 immunoreactivity and its protein level in the spinal ventral horn after transient ischemia in the rabbit to investigate the correlation between neuronal cell death and EAAC1 in the ventral horn of spinal cord. METHODS: White rabbits weighing 2.5-3.0 kg were anesthetized with a mixture of 2.5% isoflurane in 30% oxygen and 70% nitrous oxide, and the abdominal aortic artery below the left renal artery was occluded for 15 minutes. At designated times after reperfusion, the immunohistochemical and Western blot analysis for EAAC1 was conducted using tissues of the seventh lumbar spinal segment. RESULTS: EAAC1 immunoreactivity was detected in the neurons of the normal spinal cord. EAAC1 immunoreactivity and protein level reduced significantly 30 minutes after ischemia/reperfusion, but EAAC1 immunoreactivity and protein level again increased by 80% versus sham 3 hours after ischemia. At this time point, neurological defect in hindlimb was also detected. Thereafter, EAAC1 immunoreactivity and protein levels remained to be attenuated in the ventral horn of spinal cord until 48 hours after ischemia. CONCLUSION: The significant change in EAAC1 expression and motor defects at early time after transient spinal cord ischemia relates to the acute events following ischemia/reperfusion. These results indicate that EAAC1 has an important role in the modulation of glutamate homeostasis in ischemic neurons in the spinal ventral horn.     

Cholinergic, noradrenergic, and serotonergic inhibition of fast synaptic transmission in spinal lumbar dorsal horn of rat

It is known that spinal nociceptive sensory transmission receives descending inhibitory and facilitatory modulation from supraspinal structures. Glutamate is the major fast excitatory transmitter between primary afferent fibers and spinal dorsal horn neurons. In whole-cell patch clamp recordings from dorsal horn neurons in spinal slices, we investigated synaptic mechanisms for inhibitory modulation at the lumbar level of the spinal cord. Application of the cholinergic receptor agonist carbachol produced a dose-dependent inhibition of glutamate-mediated excitatory postsynaptic currents (EPSCs) (IC(50) 13 microM). Postsynaptic injection of two different types of G-protein inhibitors, guanosine 5'-O-2-thiophosphate or guanosine 5'-O-3-thiotriphosphate, blocked the inhibition produced by carbachol. Clonidine, a selective alpha-adrenergic receptor agonist, also produced a dose-dependent inhibition of EPSCs (IC(50) 7 microM) that was reduced by postsynaptic inhibition of G-proteins. The inhibitory effect of serotonin was likewise mediated by postsynaptic G-proteins. Our results suggest that activation of postsynaptic neurotransmitter receptors plays a critical role in inhibition of glutamate mediated sensory responses by acetylcholine, norepinephrine, and serotonin. Our results support the hypothesis that descending sensory modulation may be mediated by multiple neurotransmitter receptors in the spinal cord.     

Changes in the electrical activity of spinal cord neurons of adrenalectomized rats under the effect of adrenal cortex hormones

Effect of dexamethasone and desoxycorticosterone on the electrical activity of neurons in dorsal and ventral horn of spinal cord evoked by sciatic nerve stimulation were studied in adrenalectomized rats as well as effect of the same hormones on the background activity of single cells in the dorsal horn. The results demonstrated that both hormones (dexamethasone and desoxycorticosterone) provided enhancement of the amplitude of the field potentials recorded from the dorsal half of the spinal cord and facilitation of the background neuronal discharges of the single cells under investigation. It was stated that gluco- and mineralocorticoid hormones exerted different effects on the activity of ventral horn neurons of the spinal cord: dexamethasone++ potentiated and desoxycorticosterone depressed the amplitudes of the field potentials recorded from the region of motoneurons. The presented data have shown the modulatory effects of neurosteroids on the electrical activity of the spinal cord neurons.     

Cholecystokinin octapeptide reverses the κ-opioid-receptor-mediated depression of calcium current in rat dorsal root ganglion neurons

Although the cholecystokinin (CCK-8) is reported to antagonize the κ-opioid-receptor-mediated analgesic effect in spinal cord, its mechanism and sites of action remain obscure. In the present study, the whole-cell patch-clamp recording technique was employed to examine the effect of κ-opioid agonist U50488H on voltage-gated calcium channels and the interaction between the CCK-8 and U50488H in acutely isolated rat dorsal root ganglion neurons. The results indicate that the calcium currents elicited in dorsal root antiopioid peptide CCK-8. The effect of the CCK-8 can be abolished by the CCK-B receptor antagonist, L365, 260. While CCK-8 showed a potent opioid-reversal effect, it by itself exerted a slight inhibitory effects on calcium current. This novel observation in the dorsal root ganglion neurons indicates that CCK-8 can antagonize the κ-opioid-receptor-mediated depressant effect on voltage-gated calcium current, and this antagonizing effect appears to be mediated via CCK-B receptor.     

Expression of metabotropic glutamate receptor mRNAs in the human spinal cord: implications for selective vulnerability of spinal motor neurons in amyotrophic lateral sclerosis

To elucidate the relevance of metabotropic glutamate receptors (mGluRs) to the selective vulnerability of motor neurons in the spinal cord in patients with amyotrophic lateral sclerosis (ALS), we investigated the distribution of mRNAs coding mGluR1-5 in the normal human spinal cord. The mRNAs for mGluR1, 4 and 5 were observed in the spinal gray matter, whereas mGluR2 mRNA was absent in the spinal cord and mGluR3 mRNA was displayed only on glial cells in the white matter. Signals for mGluR1 and mGluR5 were enriched in the dorsal horn, while mGluR4 mRNA was abundant in the ventral horn. Since agonists to group I mGluRs (mGluR 1 and 5) have been demonstrated to have neuroprotective effects on spinal motor neurons, less expression of mRNAs coding mGluR1 and mGluR5 in the ventral horn than in the dorsal horn may be implicated in the selective susceptibility of spinal motor neurons in ALS.     

The role of dihydropyridine-sensitive voltage-gated calcium channels in potassium-mediated neuronal survival

The survival of isolated neurons from chick embryo ciliary, sympathetic, and dorsal root ganglia is greatly enhanced by concentrations of extracellular potassium that significantly depolarize the neurons (ED50 = 20-25 mM). The survival-promoting effect of elevated potassium on each of these 3 types of neurons appears to be the result of the opening of voltage-gated calcium channels. The dihydropyridine, Bay K 8644, which increases calcium influx through L-type voltage-gated calcium channels in neurons, strongly potentiated the survival-promoting action of elevated potassium (ED50 = 10.8 +/- 7.0 nM). In contrast, chemically closely related dihydropyridines, PN200-110 (ED50 = 0.33 +/- 0.15 nM) and nitrendipine (ED50 = 1.3 +/- 0.3 nM), which block calcium influx through the same voltage-gated channels, completely inhibited potassium-mediated neuronal survival. Chemically different agents that also block calcium influx through voltage-gated channels also inhibited potassium-mediated neuronal survival: the phenylalkylamine verapamil (ED50 = 0.78 +/- 0.38 microM), the benzothiazepine diltiazem (ED50 = 1.7 microM), and the inorganic ion cadmium (ED50 = 5.8 microM). These calcium-channel blockers are not simply toxic to neurons, since they did not inhibit neuronal survival mediated by the neurotrophic proteins, nerve growth factor, basic fibroblast growth factor, or ciliary neurotrophic factor, also suggesting that voltage-gated calcium channels are not involved in the action of these factors. These results suggest that neuronal survival in elevated potassium in ciliary, sympathetic, and dorsal root ganglion neurons is the result of calcium influx through dihydropyridine-sensitive, L-type voltage-gated calcium channels. These findings are discussed in relation to the neuronal toxicity of excitatory amino acids which is also thought to occur through increased calcium influx.     

Spinal cord afferent systems containing the nerve terminal protein NT75

In the adult spinal cord, immunocytochemical staining for NT75 is concentrated in nerve terminals in the superficial laminae of the dorsal horn. Deeper laminae of the dorsal horn contain moderate immunocytochemical labeling, but the ventral horn is only sparsely stained. The origin of spinal nerve terminals containing NT75 was investigated with lesion techniques, colchicine treatment, and retrograde tracing in combination with immunocytochemical staining. Primary afferent neurons express NT75 immunoreactivity and account for most of the dense staining in the superficial dorsal horn and part of the labeling in the deeper laminae. It was found that corticospinal and virtually all brainstem neurons with descending projections to the spinal cord also express NT75 immunoreactivity, including those terminating in the ventral horn. Colchicine treatment of the spinal cord also resulted in NT75 staining in most, if not all, spinal neurons. It appears that neurons in all three major sources of spinal afferents (primary sensory, descending, and intrinsic systems) can express NT75 immunoreactivity, but that some neurons normally contain higher levels of the protein in their nerve terminals. Previous analysis of developing spinal cord has shown widespread, dense NT75 labeling throughout the spinal gray in the early postnatal period, which later becomes restricted to the adult pattern. These studies support the hypothesis that many spinal pathways express high levels of NT75 immunoreactivity during development, but that only certain pathways maintain high levels in the adult. © 1993 Wiley-Liss, Inc.     

Development changes in the distribution of gamma-aminobutyric acid-immunoreactive neurons in the embryonic chick lumbosacral spinal cord

The development of γ-aminobutyric acid (GABA)-immunoreactive neurons was investigated in the embryonic and posthatch chick lumbosacral spinal cord by using pre- and postembedding immunostaining with an anti-GABA antiserum. The first GABA-immunoreactive cells were detected in the ventral one-half of the spinal cord dorsal to the lateral motor exception of the lateral motor column, appeared throughout the entire extent of the ventral one-half of the spinal gray matter by E6. Thereafter, GABA-immunoreactive neurons extended from ventral to dorsal regions. Stained perikarya first appeared at E8 and then progressively accumulated in the dorsal horn, while immunoreactive neurons gradually declined in the ventral horn. The general pattern of GABA immunoreactivity characteristic of mature animals had been achieved by E12 and was only slightly altered afterwards. In the dorsal horn, most of the stained neurons were observed in laminae I–III, both at the upper (LS 1–3) and at the lower (LS 5–7) segments of the lumbosacral spinal cord. In the ventral horn, the upper and lower lumbosacral segments showed marked differences in the distribution of stained perikarya. GABAergic neurons were scattered in a relatively large region dorsomedial to the lateral motor column at the level of the upper lumbosacral segments, whereas they were confined to the dorsalmost region of lamina VII at the lower segments. The early expression of GABA immunoreactivity may indicate a trophic and synaptogenetic role for GABA in early phases of spinal cord development. The localization of GABAergic neurons in the ventral horn and their distribution along the rostrocaudal axis of the lumbosacral spinal cord coincide well with previous physiological findings, suggesting that some of these GABAergic neurons may be involved in neural circuits underlying alternating rhythmic motor activity of the embryonic chick spinal cord. © 1994 Wiley-Liss, Inc.     

Quantitative assay of capsaicin-sensitive thiamine monophosphatase and beta-glycerophosphatase activity in rodent spinal cord

The axon terminals of some capsaicin-sensitive sensory neurons in the spinal cord of the rat contain high amounts of acid phosphatase (EC 3.1.3.1) activity. We quantitated this activity in control and capsaicin-treated rats and mice in a biochemical assay using beta-glycerophosphate (beta-GP) and thiamine monophosphate (TMP), which have both been used in previous histological investigations, as substrates and measured the amount of phosphate liberated from particular fractions. The ventral spinal cord of rats yielded 209 +/- 9 (mean +/- S.E.M.) nmol phosphate/mg protein/h from beta-GP and 18 +/- 5 nmol from TMP; the values for the upper dorsal horn are 544 +/- 42 and 198 +/- 12 for beta-GP and TMP respectively. Values for mouse spinal cord tissue are quite similar; the spinal cord of guinea pigs contains lower amounts of beta-GPase and very little TMPase activity per mg protein. There was a fairly broad pH optimum between 5.4 and 6.3. After capsaicin (50 mg/kg s.c.) pretreatment, beta-GPase activity in the upper dorsal horn was decreased by 29% in rats and by 17% in mice; TMPase activity was reduced by 48% and 37% respectively. Values in the ventral spinal cord were unchanged. It is proposed that biochemical measurement of TMPase activity might be useful in quantitative investigations of acid phosphatase activity (e.g. "FRAP") in capsaicin-sensitive sensory neurons.     

Amyotrophic lateral sclerosis patient antibodies label Ca2+ channel α1 subunit

Sporadic amyotrophic lateral sclerosis is an idiopathic human degenerative disease of spinal cord and brain motor neurons. Prior studies demonstrated that most patients with amyotrophic lateral sclerosis posses immunoglobulins that bind to purified L-type voltage-gated calcium channels, that titers of anti–voltage-gated calcium channel antibodies correlate with disease progression rates, and that amyotrophic lateral sclerosis patient-derived antibodies (ALS IgG) produce electrophysiological changes in the function of voltage-gated calcium channels. Using Western transfer immunoblots and enzyme-linked immunosorbent assays, the calcium ionophore–forming α₁ subunig of the voltage-gated calcium channel is now identified as the major voltage-gated calcium channel antigen to which ALS IgG binds. Additionally, the binding of an L-type voltage-gated calcium channel α₁ subunit–directed monoclonal antibody, which itself mimics the effects of ALS IgG on skeletal muscle voltage-gated calcium channel currents, is selectively prevented by preaddition of ALS IgG. Voltage-gated calcium channel–binding IgG from patients with Lambert-Eaton myasthenic syndrome appears to be differentiated from ALS IgG by the reactivity of the former to both α₁ and β subunits of the calcium channel. These assays provide further evidence linking amyotrophic lateral sclerosis to an autoimmune process, and suggest one means to differentiate immunoglobulins from patients with amyotrophic lateral sclerosis from those of patients with another autoimmune disease expressing calcium channel antibodies.