- Email: [email protected]
47. Toxin I has multiple effects on synaptic transmission in hippocampal pyramidal neurones
A20
Abstracts / Journal of Neuroscience Methods (1994) A 1-/128
buffer lacking calcium and containing 3 mM EDTA, pH 7.4) during the anoxic-aglycemic insult did not attenuate the protein synthesis inhibition. However, removal of extracellular calcium during the recovery period did lessen the degree of protein synthesis inhibition 2 h after the insult. These preliminary results suggest that extracellular calcium, while not responsible for the onset of the protein synthesis inhibition triggered by anoxia-aglycemia, is necessary for the maintenance of the protein synthesis inhibition following the insult.
thiourea alone did not preserve cell morphology. These data support the notion that ascorbate is an endogenous neuroprotective agent and suggest that loss of such antioxidants contributes to cell degeneration in vitro. (Supported by NIH grant NS-28480.)
46. RS-ZACOPRIDE FACILITATES LTP IN THE RAT HIPPOCAMPUS VIA INTERACTION WITH 5-HT 3 AND 5-HT 4 RECEPTORS
R. Santamaria, D. Caille 45. MAINTENANCE OF TISSUE ASCORBATE LEVELS PRESERVES CELL MORPHOLOGY IN HIP-
Synthelabo Recherche, Preclinical Research Department, Rueil-Malmaison, France
POCAMPAL SLICES
M.E. Rice, T. Lee, R. Kinkhabwala, M.A. P6rez-Pinz6n
Departments of Neurosurgery and Physiology and Biophysics, New York University Medical Center, New York, N Y 10016, USA Ascorbate and glutathione (GSH) are endogenous, water-soluble antioxidants and free-radical scavengers. Both are rapidly lost from mammalian brain slices incubated in ascorbate- and GSH-free media. This loss of endogenous antioxidants leaves tissue vulnerable to damage from reactive oxygen species in the hyperoxygenated in vitro environment generated by equilibration with 95% O z / 5 % CO 2. We determined the ascorbate and GSH content of Vibratome-cut slices (400 txm) of rat hippocampus and cortex after 6 h incubation at room temperature and correlated these levels with the histological quality of the slices. Ascorbate and GSH were determined using HPLC with electrochemical detection. Cell morphology in incubated slices was compared with that in intact tissue in cresyl violet-stained sections. Tissue ascorbate content in intact hippocampus was 2.81 _+ 0.05 tzmol • g-~ tissue wet weight and that of GSH 1.96 _+ 0.04 (n = 35). In cortex, ascorbate was 2.51 + 0.05 and GSH was 1.91 _+ 0.05 (n = 43). The ascorbate and GSH content of slices fell by 70-80% during incubation in vitro. Ascorbate loss could be prevented by including 400 IxM ascorbate (normal extracellular concentration) in the incubation media. By contrast, loss of GSH could not be prevented by incubation with GSH. The morphology of cells in hippocampal slices incubated under conditions that maintained ascorbate content were similar to those of intact tissue. Ascorbate protected pyramidal cells in CA1 and CA3 regions of the hippocampus from the degeneration that was seen in slices incubated in ascorbate-free media. The best results were obtained when 400 /zM thiourea was included with ascorbate to minimize ascorbate oxidation. Incubation with 400 /xM GSH or
RS-zacopride is a potent 5-HT 3 receptor antagonist and 5-HT 4 receptor agonist which improves learning and memory in animal models. In the present study, we have evaluated the effects of RS-zacopride on LTP of the CA1 area of hippocampal slices in the rat. RSzacopride was tested alone or in combination with tropisetron (ICS 205-930) either at low concentrations at which this compound shows only 5-HT 3 receptor antagonism or at higher concentrations at which it also displays 5-HT 4 receptor antagonism. Extracellular EPSPs were recorded from male Sprague-Dawley rat hippocampal slices maintained in an interface chamber and stimulated at 0.03 Hz. LTP, induced by a tetanic stimulation (100 Hz, 0.7 s × 2) was measured as the increase of the EPSP height 30 min after the induction. RS-zacopride (10 -6 M) substantially increased the LTP amplitude and the number of slices developping a LTP as compared to untreated slices. Tropisetron (10 -s M) enhanced the effect of RS-zacopride on the amplitude of LTP. In contrast, tropisetron (10 -7 M) markedly reduced the RSzacopride induced facilitation of LTP. These results suggest that the facilitation by RSzacopride of hippocampal LTP is effected via the combination of 5-HT 3 antagonist and 5-HT 4 agonist effects. Such dual interaction with 5 - H T 3 / 5 - H T 4 receptors regulating learning and memory processes may be of therapeutic utility.
47. TOXIN I HAS MULTIPLE EFFECTS ON SYNAPTIC TRANSMISSION IN HIPPOCAMPAL PYRAMIDAL NEURONES
A.P. Southan, D.G. Owen Wyeth Research (UK), Huntercombe Lane South, Taplow, Berks. SL6 OPH, UK Brief application of K + channel blockers to CA1 neurones of the in vitro rat hippocampal slice has been
Abstracts/Journal of Neuroscience Methods (1994) A1-A28 '
I
-
"
Control
t
A21
5OhM T o x i n
I
L3~OOpA 10ms
"
Fig. 1 (abstract 47).
reported to induce a long-lasting (TEA, MCDP) or short-term (4-AP, caesium) potentiation of excitatory synaptic responses. Subsequently we extended these observations to include some dendrotoxin homologues, which are potent and selective blockers of voltageactivated K + currents. The present study presents actions of Toxin I upon both excitatory and inhibitory synaptic transmission (Fig. 1). Extracellular field potential responses were recorded from the CA1 region of hippocampal slices using conventional methods and solutions. Potentials were evoked by constant stimulation of the Schaffer collaterals at 0.1 H z / 0 . 0 2 ms duration, at 30°C. In control experiments (n = 4 ) spike amplitude was 111.49+ 15.18% of its initial value following 70 min of stimulation. Following a 10 min exposure to Toxin I (10 nM, n = 3; 100 nM, n = 3; 500 nM, n = 2) and washout, a slowly developing increase in the amplitude of the population spike to 184.6 + 31.3,489.0 _+ 88.9 and 656% respectively, was observed at 60 min wash. Potentiation was also observed in the presence of 100 /zM APV (n = 3), suggesting that N M D A receptor activation is not required for this form of synaptic potentiation. Whole cell patch-clamp recordings were also made from CA1 neurones in 200 /zm slices using standard solutions and techniques. Neurones were voltage clamped at - 7 0 mV in the presence of 1 / z M TTX. In 5 experiments, Toxin I (50 nM) was found to markedly enhance the frequency and amplitude of spontaneous synaptic currents without affecting voltage-activated K ÷ currents recorded in the postsynaptic cell. The increase in spontaneous synaptic activity was observed within 2 min of adding Toxin I and persisted throughout exposure to the toxin. Bicuculline (50 /xM) abolished all large amplitude synaptic currents, consistent with the enhancement of spontaneous G A B A release by Toxin I. It is likely that the observed effects of Toxin I are due to blockade of pre-synaptic voltage-activated K + channels and not somatic K ÷ channels. The possible role of GABA-ergic transmission in the potentiation of field potentials and LTP induced by K ÷ channel blockers, is being studied further.
48. CORRELATION OF ENDOGENOUS NORADRENALINE RELEASE AND NEURONAL INHIBITION IN RAT LOCUS COERULEUS SLICES BY COMBINED VOLTAMMETRIC AND ELECTROPHYSIOLOGICAL RECORDING J.A. Stamford a, C.M. Jorm a, C. Davidson a, p. Palij a, J. Millar b
a Anaesthetics Unit, London Hospital Medical College, London E1 1BB, UK; b Physiology Department, Queen Mary and Westfield College, London E1 4NS, UK Locus coeruleus (LC) a 2 autoreceptors control cell firing and noradrenaline (NA) release. We were interested to examine the relation between the level of endogenously released NA and the firing of LC cells. In this study we combined single-unit recording and fast cyclic voltammetry (FCV) at single carbon-fibre microelectrodes (CFMs) in superfused slices of rat LC. The FCV polarograph monitored stimulated NA efflux every 500 ms and was used in a voltage follower mode between scans. The "spikes" were discriminated and counted using Neurolog modules. Twenty-two stable spontaneously active cells were recorded. Electrical stimulation caused an increase in firing rate in 16 units (73%) and inhibited activity or produced a biphasic response in 6 (27%). In the excited cells the magnitude of excitation did not correlate with NA efflux (not shown). Inhibited cells showed much clearer correlation between NA efflux and the duration of inhibition. The figure below shows a typical example (Fig. 1, page A22). Cell firing only recommenced as the NA level fell. The duration of inhibition was shortened by rauwolscine (1 /zM) showing that it was mediated via o~2 receptors. This study shows that FCV and single-unit recording may readily be combined at single CFMs in brain slices and that simultaneous recording of NA efflux and unit activity at the same site should facilitate comparison of drug effects at pre- and postsynaptic sites.
Abstracts / Journal of Neuroscience Methods (1994) A 1-/128
buffer lacking calcium and containing 3 mM EDTA, pH 7.4) during the anoxic-aglycemic insult did not attenuate the protein synthesis inhibition. However, removal of extracellular calcium during the recovery period did lessen the degree of protein synthesis inhibition 2 h after the insult. These preliminary results suggest that extracellular calcium, while not responsible for the onset of the protein synthesis inhibition triggered by anoxia-aglycemia, is necessary for the maintenance of the protein synthesis inhibition following the insult.
thiourea alone did not preserve cell morphology. These data support the notion that ascorbate is an endogenous neuroprotective agent and suggest that loss of such antioxidants contributes to cell degeneration in vitro. (Supported by NIH grant NS-28480.)
46. RS-ZACOPRIDE FACILITATES LTP IN THE RAT HIPPOCAMPUS VIA INTERACTION WITH 5-HT 3 AND 5-HT 4 RECEPTORS
R. Santamaria, D. Caille 45. MAINTENANCE OF TISSUE ASCORBATE LEVELS PRESERVES CELL MORPHOLOGY IN HIP-
Synthelabo Recherche, Preclinical Research Department, Rueil-Malmaison, France
POCAMPAL SLICES
M.E. Rice, T. Lee, R. Kinkhabwala, M.A. P6rez-Pinz6n
Departments of Neurosurgery and Physiology and Biophysics, New York University Medical Center, New York, N Y 10016, USA Ascorbate and glutathione (GSH) are endogenous, water-soluble antioxidants and free-radical scavengers. Both are rapidly lost from mammalian brain slices incubated in ascorbate- and GSH-free media. This loss of endogenous antioxidants leaves tissue vulnerable to damage from reactive oxygen species in the hyperoxygenated in vitro environment generated by equilibration with 95% O z / 5 % CO 2. We determined the ascorbate and GSH content of Vibratome-cut slices (400 txm) of rat hippocampus and cortex after 6 h incubation at room temperature and correlated these levels with the histological quality of the slices. Ascorbate and GSH were determined using HPLC with electrochemical detection. Cell morphology in incubated slices was compared with that in intact tissue in cresyl violet-stained sections. Tissue ascorbate content in intact hippocampus was 2.81 _+ 0.05 tzmol • g-~ tissue wet weight and that of GSH 1.96 _+ 0.04 (n = 35). In cortex, ascorbate was 2.51 + 0.05 and GSH was 1.91 _+ 0.05 (n = 43). The ascorbate and GSH content of slices fell by 70-80% during incubation in vitro. Ascorbate loss could be prevented by including 400 IxM ascorbate (normal extracellular concentration) in the incubation media. By contrast, loss of GSH could not be prevented by incubation with GSH. The morphology of cells in hippocampal slices incubated under conditions that maintained ascorbate content were similar to those of intact tissue. Ascorbate protected pyramidal cells in CA1 and CA3 regions of the hippocampus from the degeneration that was seen in slices incubated in ascorbate-free media. The best results were obtained when 400 /zM thiourea was included with ascorbate to minimize ascorbate oxidation. Incubation with 400 /xM GSH or
RS-zacopride is a potent 5-HT 3 receptor antagonist and 5-HT 4 receptor agonist which improves learning and memory in animal models. In the present study, we have evaluated the effects of RS-zacopride on LTP of the CA1 area of hippocampal slices in the rat. RSzacopride was tested alone or in combination with tropisetron (ICS 205-930) either at low concentrations at which this compound shows only 5-HT 3 receptor antagonism or at higher concentrations at which it also displays 5-HT 4 receptor antagonism. Extracellular EPSPs were recorded from male Sprague-Dawley rat hippocampal slices maintained in an interface chamber and stimulated at 0.03 Hz. LTP, induced by a tetanic stimulation (100 Hz, 0.7 s × 2) was measured as the increase of the EPSP height 30 min after the induction. RS-zacopride (10 -6 M) substantially increased the LTP amplitude and the number of slices developping a LTP as compared to untreated slices. Tropisetron (10 -s M) enhanced the effect of RS-zacopride on the amplitude of LTP. In contrast, tropisetron (10 -7 M) markedly reduced the RSzacopride induced facilitation of LTP. These results suggest that the facilitation by RSzacopride of hippocampal LTP is effected via the combination of 5-HT 3 antagonist and 5-HT 4 agonist effects. Such dual interaction with 5 - H T 3 / 5 - H T 4 receptors regulating learning and memory processes may be of therapeutic utility.
47. TOXIN I HAS MULTIPLE EFFECTS ON SYNAPTIC TRANSMISSION IN HIPPOCAMPAL PYRAMIDAL NEURONES
A.P. Southan, D.G. Owen Wyeth Research (UK), Huntercombe Lane South, Taplow, Berks. SL6 OPH, UK Brief application of K + channel blockers to CA1 neurones of the in vitro rat hippocampal slice has been
Abstracts/Journal of Neuroscience Methods (1994) A1-A28 '
I
-
"
Control
t
A21
5OhM T o x i n
I
L3~OOpA 10ms
"
Fig. 1 (abstract 47).
reported to induce a long-lasting (TEA, MCDP) or short-term (4-AP, caesium) potentiation of excitatory synaptic responses. Subsequently we extended these observations to include some dendrotoxin homologues, which are potent and selective blockers of voltageactivated K + currents. The present study presents actions of Toxin I upon both excitatory and inhibitory synaptic transmission (Fig. 1). Extracellular field potential responses were recorded from the CA1 region of hippocampal slices using conventional methods and solutions. Potentials were evoked by constant stimulation of the Schaffer collaterals at 0.1 H z / 0 . 0 2 ms duration, at 30°C. In control experiments (n = 4 ) spike amplitude was 111.49+ 15.18% of its initial value following 70 min of stimulation. Following a 10 min exposure to Toxin I (10 nM, n = 3; 100 nM, n = 3; 500 nM, n = 2) and washout, a slowly developing increase in the amplitude of the population spike to 184.6 + 31.3,489.0 _+ 88.9 and 656% respectively, was observed at 60 min wash. Potentiation was also observed in the presence of 100 /zM APV (n = 3), suggesting that N M D A receptor activation is not required for this form of synaptic potentiation. Whole cell patch-clamp recordings were also made from CA1 neurones in 200 /zm slices using standard solutions and techniques. Neurones were voltage clamped at - 7 0 mV in the presence of 1 / z M TTX. In 5 experiments, Toxin I (50 nM) was found to markedly enhance the frequency and amplitude of spontaneous synaptic currents without affecting voltage-activated K ÷ currents recorded in the postsynaptic cell. The increase in spontaneous synaptic activity was observed within 2 min of adding Toxin I and persisted throughout exposure to the toxin. Bicuculline (50 /xM) abolished all large amplitude synaptic currents, consistent with the enhancement of spontaneous G A B A release by Toxin I. It is likely that the observed effects of Toxin I are due to blockade of pre-synaptic voltage-activated K + channels and not somatic K ÷ channels. The possible role of GABA-ergic transmission in the potentiation of field potentials and LTP induced by K ÷ channel blockers, is being studied further.
48. CORRELATION OF ENDOGENOUS NORADRENALINE RELEASE AND NEURONAL INHIBITION IN RAT LOCUS COERULEUS SLICES BY COMBINED VOLTAMMETRIC AND ELECTROPHYSIOLOGICAL RECORDING J.A. Stamford a, C.M. Jorm a, C. Davidson a, p. Palij a, J. Millar b
a Anaesthetics Unit, London Hospital Medical College, London E1 1BB, UK; b Physiology Department, Queen Mary and Westfield College, London E1 4NS, UK Locus coeruleus (LC) a 2 autoreceptors control cell firing and noradrenaline (NA) release. We were interested to examine the relation between the level of endogenously released NA and the firing of LC cells. In this study we combined single-unit recording and fast cyclic voltammetry (FCV) at single carbon-fibre microelectrodes (CFMs) in superfused slices of rat LC. The FCV polarograph monitored stimulated NA efflux every 500 ms and was used in a voltage follower mode between scans. The "spikes" were discriminated and counted using Neurolog modules. Twenty-two stable spontaneously active cells were recorded. Electrical stimulation caused an increase in firing rate in 16 units (73%) and inhibited activity or produced a biphasic response in 6 (27%). In the excited cells the magnitude of excitation did not correlate with NA efflux (not shown). Inhibited cells showed much clearer correlation between NA efflux and the duration of inhibition. The figure below shows a typical example (Fig. 1, page A22). Cell firing only recommenced as the NA level fell. The duration of inhibition was shortened by rauwolscine (1 /zM) showing that it was mediated via o~2 receptors. This study shows that FCV and single-unit recording may readily be combined at single CFMs in brain slices and that simultaneous recording of NA efflux and unit activity at the same site should facilitate comparison of drug effects at pre- and postsynaptic sites.