Excitatory effect of noradrenaline on pacemaker cells in spinal cord primary cultures

Intracellular recordings were made from dissociated fetal mouse spinal cord neurons in primary culture. One particular type of neuron, with a large cell body (40-50 micron) and three to five thick neurites, exhibited rhythmic electrical activity of two different types, consisting of either spontaneous burst discharges or tonic action potential firing. Both types of activity appeared to be triggered by an endogenous membrane potential oscillation. Micropressure application of noradrenaline (10(-5) M in the delivery pipette) onto the surface of such cells evoked, in a dose-dependent manner, an increase in the input resistance with a depolarization of the membrane potential. The response to NA was potential-dependent. The maximum change in input resistance was observed at membrane potential values between -60 mV and -45 mV and the response was suppressed at membrane potentials lower than -80 mV. No modification of the response was observed in the presence of 50 mM of tetraethylammonium. The extrapolated reversal potential, close to -90 mV, was modified by increasing extracellular K+ concentration and unaltered by increasing the intracellular Cl- concentration. The decrease in K+ conductance induced by noradrenaline was Ca2+-dependent and reversibly suppressed by Ba2+ (6 mM) and Cd2+ (0.1 mM). This response to noradrenaline was suppressed in the presence of muscarine (10 microM) suggesting that noradrenaline decreases a K+ conductance related to M current. The noradrenaline evoked increase in input resistance was mediated by activation of an alpha 1 receptor site. Prazosin, an alpha 1 antagonist and phentolamine, an alpha 1 alpha 2 antagonist, reversibly suppressed the response in a competitive manner. Yohimbine, a competitive alpha 2 antagonist, also blocked the response, but in a noncompetitive manner. Clonidine, an alpha 2 agonist, isoprenaline, a beta agonist and L-alprenolol, a beta antagonist, had no effect.     

Neurite outgrowth-regulating properties of GABA and the effect of serum on mouse spinal cord neurons in culture

Time-lapse photography was used to examine the effects of γ-aminobutyric acid (GABA) on the outgrowth and motility of neurites in cultures from mouse spinal cord. GABA at concentrations of 100, 10 and 1 μ m caused significant inhibition of neurite outgrowth and the motility of growth cones was significantly reduced by treatment with 100 and 10 μ m GABA. This effect was mimicked by the GABA_B receptor agonist baclofen, whereas the GABA_A receptor agonist muscimol had no effect. The effect of GABA on outgrowth and motility seems to be dependent on the type of serum employed. The results reported here were obtained only when heat-inactivated serum was used and not when non heat-inactivated serum was added to the culture medium. They suggest that GABA has a role in the regulation of process outgrowth within the embryonic mouse spinal cord.     

Glutamate neurotoxicity in spinal cord cell culture.

The neurotoxicity of glutamate was investigated quantitatively in mixed neuronal and glial spinal cord cell cultures from fetal mice at 12-13 days of gestation. Five-minute exposure to 10-1000 microM glutamate produced widespread acute neuronal swelling, followed by neuronal degeneration over the next 24 h (EC50 for death about 100-200 microM); glia were not injured. Glutamate was neurotoxic in cultures as young as four days in vitro, although greater death was produced in older cultures. By 14-20 days in vitro, 80-90% of the neuronal population was destroyed by a 5-min exposure to 500 microM glutamate. Acute neuronal swelling following glutamate exposure was prevented by replacement of extracellular sodium with equimolar choline, with minimal reduction in late cell death. Removal of extracellular calcium enhanced acute neuronal swelling but attenuated late neuronal death. Both acute neuronal swelling and late degeneration were effectively blocked by the noncompetitive N-methyl-D-aspartate receptor antagonist dextrorphan and by the novel competitive antagonist CGP 37849. Ten micromolar 7-chlorokynurenate also inhibited glutamate neurotoxicity; protection was reversed by the addition of 1 mM glycine to the bathing medium. These observations suggest that glutamate is a potent and rapidly acting neurotoxin on cultured spinal cord neurons, and support involvement of excitotoxicity in acute spinal cord injury. Similar to telencephalic neurons, spinal neurons exposed briefly to glutamate degenerate in a manner dependent on extracellular Ca2+ and the activation of N-methyl-D-aspartate receptors.     

The effect of serotonin on GABA synthesis in cultured rat spinal dorsal horn neurons

The spinal dorsal horn (SDH) is the first step in the integration of primary nociceptive information, which is controlled by the descending serotonin (5-HT) system as well as the principal inhibitory neurotransmitter gamma-aminobutyric acid (GABA). However, the influence exerted by 5-HT on GABA synthesis remains poorly understood. The major pathway for GABA synthesis is the enzymatic decarboxylation of glutamate by glutamic acid decarboxylase (GAD) 65 and 67. In the present research, western blotting results show a time- and dose-dependent enhancement of GAD65 and GAD67 expression induced by 5-HT treatment and a concentration of 100nM 5-HT applied for 3 days is shown to be the optimal condition for maximal expression of GAD67 and a significant expression of GAD65. Under the stimulation of such 5-HT application the phosphorylation of Akt and p42/p44 mitogen-activated protein (MAP) kinase is activated and specifically blocked by inhibitors of phosphatidylinositol 3-kinase (PI3-K) (LY294002) or the p42/p44 MAP kinase (PD98059 and U0126) pathways. Moreover, LY294002, or PD98059, or U0126 partially inhibit 5-HT-stimulated increases in GAD67 or GAD65 expression. Further, 5-HT application has no effect on the number of GAD65/GAD67-immunopositive neuronal cells; but it can induce an increase in the total area, process length and number of primary neurites of GAD65/67-positive neurons, an increase that appears to involve LY294002 and PD98059. The results of this study provide an in vitro model of the regulation of 5-HT on synthesis of GABA in the SDH that is putatively thought to occur in vivo as a result of excitatory neural activity.     

Transplantation of dissociated foetal serotonin neurons into the transected spinal cord of adult rats

Twenty adult rats underwent a complete section of the spinal cord at the lower thoracic level. One week later, 15 of them received a cell suspension obtained from raphe nuclei of 14-day-old foetuses into the distal fragment of the spinal cord. They were sacrificed after survival periods of 10-60 days, and vibratome sections of the spinal cord were processed for immunocytochemical detection of serotonin (5-HT). The control, non-transplanted animals showed a total absence of 5-HT immunoreactivity below the section, whereas the transplanted rats showed many immunoreactive 5-HT perikarya in the graft region, some at a distance of up to 10 mm, and a progressive innervation of the whole grey matter extending at least over 20 mm from the graft site.     

Stimulatory effect of substance P on sympathetic ganglia and spinal cord in culture

Studies with organotypic culture of sympathetic ganglia and spinal cord revealed that substance P at concentrations of 10(-5) to 10(-12) M and 10(-5) to 10(-14) M exerts a marked growth-stimulating effect on sympathetic ganglia and spinal cord, respectively. In the presence of substance P, the intensity of sympathetic ganglion growth exceeds control values 3.0-4.8 times. The growth zone size of spinal cord explants increases under these conditions 2.0-5.2-fold. A feasible physiological significance of regulatory peptides in the growth and regeneration of nervous tissue as well as the role of noci-antinociceptive systems in histogenesis and regeneration are discussed.     

Insulin modulates neuronal plasma membrane development in human fetal spinal cord neurons in culture

The number of intramembrane protein particles (IMP) in the protoplasmic face of the perikaryal plasma membrane was evaluated in neurons from 9 week-old and 12 week-old human fetal spinal cord in culture. An increased number of IMP was observed in membranes from 12 week-old fetal neurons when compared to membranes from 9 week-old fetal neurons. The addition of insulin (100-2500 microU/ml) to the culture media resulted in a significantly increased number of IMP in neuronal membranes. Incubation with glucose (1.5-6 mg/ml) did not modify the number of IMP and glucose did not potentiate the effect of insulin when both glucose (3 mg/ml) and insulin (500 microU/ml) were added to the culture media. The results suggest that insulin may modulate the development of neuronal membranes and that this effect is not mediated by an increased glucose utilization.     

Differential distribution of excitatory amino acid receptors on embryonic rat spinal cord neurons in culture.

1. Excitatory amino acid (EAA) receptors mediate synaptic transmission in many areas of the vertebrate CNS. To map the distribution of the EAA receptors, three agonists selective for EAA receptor subtypes [kainate, quisqualate, and N-methyl-D-aspartate (NMDA)] were applied by pressure to the cell bodies and neurites of voltage-clamped, embryonic rat spinal cord neurons in culture. 2. Current loss along the neurite between the site of activation and the recording electrode at the soma was estimated independently of variations in receptor density by focal application of high [K+] solution. This estimate was used to compensate agonist-evoked responses for current loss due to leak. K(+)-evoked current amplitudes progressively decreased as applications were made at more distal positions along the neurite. 3. Response amplitudes to EAAs showed a monotonic decay, similar to that with high [K+] solution, in only a few of the cells tested with each agonist. The majority of neurons had areas of high agonist sensitivity along the neurites, implying a nonuniform density of receptors. Such regions sometimes occurred at the most proximal segment of the neurite. Most neurites had distal regions of agonist sensitivity that ranged from 40 to 300% higher than at the soma. 4. The relative distributions of the three EAA receptors were compared by applying two agonists to the same sites along neurites and observing variations in normalized response amplitudes. When comparing NMDA versus non-NMDA receptor distributions on the same neurite, approximately 40% of the cells had similar patterns to two agonists, and the remaining 60% had different patterns. When sensitivity to the two non-NMDA agonists kainate and quisqualate were compared, about one-half of the cells tested had different patterns of agonist sensitivity. 5. Areas of high sensitivity persisted after block of calcium channels by addition of La3+ to the bath solution and after prevention of evoked transmitter release by a low [Ca2+]/high [Mg2+] solution. 6. These results show that spinal cord neurons can have regions of high agonist sensitivity to NMDA, kainate, and quisqualate along their neurites and that the sensitivity to any one of the EAA receptor subtypes can be elevated independently of the others.     

Excitatory amino acids and potassium release in the frog spinal cord

Synaptic release of excitatory amino acids such as L-glutamate and/or L-aspartate and subsequent activation of specific receptors by these putative transmitters appears necessary for the release of K+ by afferent stimulation in the isolated frog spinal cord. This conclusion is based on the findings that (-)baclofen, which is thought to reduce the presynaptic release of putative excitatory amino acid transmitters, and some amino dicarboxylic amino acids (D, L-alpha-aminoadipic acid, 2-amino-4-phosphonobutyric acid, and D, L-alpha, epsilon-diaminopimelic acid), which are believed to interfere with the activation of receptors by these same excitatory amino acids, significantly attenuate the increment in extracellular K+ evoked by tetanic dorsal root stimulation.     

Evidence for bursting pacemaker neurones in cultured spinal cord cells

Intracellular recordings were made from dissociated mouse spinal cord cells in primary culture. One type of spinal cord neurone, with a large cell body (40-50 micron), 3-5 short neurites, and a mean resting potential of -65 mV, was found to fire rhythmic bursts of action potentials with a phase duration of approximately 1s when the membrane potential was depolarized to -55 mV. These bursts did not arise from spontaneous synaptic input, but appeared to result from endogenous ionic conductance properties of the membrane resembling those observed in molluscan bursting pacemaker neurones. Ionic conductances underlying this bursting activity were studied pharmacologically by local application of ionic conductance blockers. Pacemaker potentials depended on Na+ conductance, since tetrodotoxin and Na-free medium were the most potent agents for blocking spontaneous rhythmic activity. However, a Ca2+ conductance was involved in the depolarizing phase of membrane potential oscillations, since Ba2+ application increased oscillation amplitude. Action potentials observed during the bursts were Na+- and Ca2+-dependent. They did not differ significantly from those observed in other spinal cord neurones in culture. Application of tetraethylammonium, CoCl2, BaCl2 and 4-aminopyridine revealed at least three different potassium conductances which controlled this bursting pacemaker activity. A delayed potassium conductance controlled spike duration, a Ca-dependent potassium conductance controlled the duration of the burst and underlay the hyperpolarizing phase terminating the burst, and finally, a transient potassium conductance appeared to be involved in the control of phase duration. The demonstration that spinal cord neurones growing in monolayer culture display typical bursting pacemaker activity raises the possibility that bursting pacemaker neurones in the mammalian spinal cord may be involved in a phasic pattern generator that could control such activities as walking and the respiratory rhythm.     

Excitatory amino acids increase glycogen phosphorylase activity in the rat spinal cord

Glycogen phosphorylase is present in nervous tissue in an active and inactive form. Using a histochemical technique, an investigation into which putative neurotransmitters have the capacity to modify the activity of the enzyme, has been performed on the rat spinal cord. Intrathecal injections of L-glutamate and L-aspartate elevate glycogen phosphorylase activity in the dorsal horn, while substance P has no effect and only high doses of adenosine triphosphate (ATP) increase the enzyme activity. In addition the N-methyl-D-aspartate receptor antagonist, 5-amino-phosphonovaleric acid was found to block the elevation of glycogen phosphorylase activity in the dorsal horn produced by the peripheral activation of chemo-sensitive primary afferents. Excitatory amino-acid neurotransmitters can therefore, acting via second messengers and protein kinases, modify glycogen metabolism in the spinal cord.     

Excitatory neurotransmission activates voltage-dependent properties in neurons in spinal motor system of lamprey

1. Ventral horn neurons were studied under voltage clamp during episodes of sensory-evoked rhythmic coactivation in the in vitro lamprey spinal cord-tailfin preparation in the presence of strychnine. 2. Voltage clamp under a range of holding potentials during episodes of rhythmic coactivation revealed inward currents coincident with ventral root bursting in the same hemisegment and an apparent reversal potential of about -10 mV. 3. The current-voltage relationship of the peak inward current during each burst of this activity demonstrated a marked voltage dependency. 4. The voltage dependence of the inward current was eliminated by the specific NMDA-receptor blocker, APV, and by removal of Mg2+ from the bathing solution. 5. At depolarized potentials a long-lasting outward current could be observed, indicating an apparent voltage-dependent conductance for K+ and/or Cl-. This current was also blocked by APV and increased by the removal of Mg2+. 6. These results provide evidence that during rhythmic coactivation in strychnine, endogenous release of excitatory amino acid transmitter induces nonlinear conductance properties in ventral horn neurons by the activation of NMDA receptors. The results provide additional evidence that such activation contributes to the membrane potential oscillations that underlie rhythmic locomotory activity.     

Excitatory amino acid receptors involved in primary afferent-evoked polysynaptic EPSPs of substantia gelatinosa neurons in the adult rat spinal cord slice.

Intracellular recordings were made from substantia gelatinosa (SG) neurons in spinal cord slices to determine a subclass of excitatory amino acid receptors involved in polysynaptic excitatory postsynaptic potentials (EPSPs). In the majority of neurons, polysynaptic EPSPs evoked by A delta fiber were not affected by 2-amino-5-phosphonovaleric acid (APV), while all EPSPs including monosynaptic EPSPs were depressed by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). All spontaneous EPSPs were blocked by CNQX, while spontaneous EPSPs in a few SG neurons were attenuated by APV. These observations suggest that polysynaptic EPSPs evoked through A delta fibers are predominantly mediated by activation of the non-N-methyl-D-aspartate (non-NMDA) receptor subclass.     

The effect of stimulants on the electrical activity of solitary neurons of the spinal cord

Summary The effect exerted by strychine, securinine and corazole (pentamethylenetetrazole) on the electric activity of single motor and neurons of the spinal cord, was studied by intracellular recording of the potentials. The data obtained demonstrated that the stimulants studied had a different effect on inhibitory processes in the spinal cord. Due to this, with the action of some substances (corazole) there was not only a rise, but also a depression of the activity of the individual neurons. The excitatory action of the stimulants was mainly directed upon the neurons of the spinal cord.(Presented by Active Member AMN SSSR V. V. Zakusov) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 54, No. 10, pp. 70–76, October, 1962     

The effect of strychnine on the activity of individual intercalary neurons of the spinal cord

Summary Experiments were conducted on decerebrated curarized cats. With the aid of capillary microelectrodes discharges of individual units of the posterior horns of the VI–VII lumbar spinal cord segments were led off. Strychnine (0.1–0.2 mg/kg) depressed spontaneous rhythmic discharges slowing their rhythm and reducing their amplitude. It increased the frequency of group discharges, facilitated the appearance of responses to the afferent stimuli and intensified the impulse after effect. No change was seen in the inhibition of the interneurones against the background of strychnine action. A suggestion is made that strychnine tetanus caused by intensified group discharges and the appearance of new sources of group discharges, which become syndronized as a facilitated synaptic conductivity.(Presented by Active Member AMN SSSR V. V. Zakusov) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 53, No. 4, pp. 7–11, April, 1962     

Chlormethiazole acts on chloride channels in cultured spinal cord neurons

Cellular effects of chlormethiazole (CMZ), a sedative-hypnotic substance, were studied using intracellular recording techniques. As a model system primary cultures of mouse spinal cord neurons were used. CMZ (200 microM) increased the rheobase and decreased the cell input resistance. In addition, the spike after depolarization was decreased. The reversal potential for the CMZ-induced effect was dependent on the chloride ion gradient. It is concluded that CMZ opens calcium dependent chloride ion channels in cultured spinal cord neurons which will result in enhancement of inhibitory neurotransmission.