- Email: [email protected]
Excitatory actions of homocysteic acid on hippocampal neurons
282
Brain Research, 238 (1982) 282-285 Elsevier Biomedical Press
Excitatory actions of homocysteic acid on hippocampal neurons
SATSUKI SAWADA, SHOBU TAKADA and CHOSABURO YAMAMOTO Department of Physiology, Faculty of Medicine, Kanazawa University, Kanazawa 920 (Japan} (Accepted December 31st, 1981) Key words: homocysteic acid - - hippocampus - - glutamate receptor - - brain slice
The actions of D- and D,L-homocysteate(DH and DLH) on CA3 neurons were studied in thin sections of the guinea pig hippocampus. DH and DLH administered to the stratum radiatum induced large depolarizations in CA3 neurons. The responses to DH and DLH were accompanied by increases in membrane conductance. The amplitudes of the responses increased and decreased, respectively, during tonic hyperpolarizingand depolarizing currents. In neurons injected with Cs+, these responses were reversed in polarity at membrane potentials of--13 to --19 mV. These results indicate that receptors for homocysteate in the hippoeampus have different properties from those found in the spinal cord and are quite similar to glutamate receptors.
Since glutamate (Glu) is a prominent candidate as an excitatory transmitter in the mammalian brain1, 5, it is of considerable importance to elucidate properties of receptors for Glu and other excitatory amino acids. In 1979, Engberg and his associates reported that the excitatory action of homocysteate on spinal motoneurons was different from that of Glu 2. Whereas membrane conductance increased during Glu-induced depolarization, it decreased with depolarization induced by homocysteate. While Glu-induced responses have been reported in other work to decrease in amplitude during tonic depolarizing currents and to reverse in polarity at a level of 20-30 mV below zero 9, homocysteate-induced responses became smaller with hyperpolarization of the impaled neuronsL Although reversal of the responses to homocysteate was not actually observed, the reversal potential estimated by extrapolation was about --100 mV. From these observations, Engberg et al, suggested that receptors for Glu and homocysteate were different from each other, and that activation of Glureceptors increased permeability for both Na ~ and K ÷, while that of homocysteatereceptors caused a decrease in K + permeability. Since this difference may have fundamental implications for the understanding of neurotransmitter receptors, we have pursued the issue by comparing excitatory actions of Glu and homocysteate on hippocampal neurons in thin slices maintained in an artificial medium. Our results are not in accord with those by Engberg et al. in spinal motoneurons. Transverse sections of the hippocampus of the guinea pig were prepared as described previously 7 and were incubated in the standard medium at 37 °C for more than 40 min. They were then transferred one by one into an observation vessel which 0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press
283 was continuously perfused with the standard medium at 36-37 °C. Amino acids were administered iontophoretically either with single- or double-barrel glass pipettes. These pipettes were filled with 0.5 or 1 M Glu, 0.25 or 0.5 M D-homocysteate (DH) or 0.2 M D,L-homocysteate (DLH). The pH of these amino acid solutions was adjusted to 8.0. Intracellular potentials were recorded from neurons in region CA3 with micropipettes filled with 4 M potassium acetate or 3 M cesium acetate. Cs + was injected into impaled neurons electrophoretically for 2-5 rain with 0.5 s pulses at 1 Hz. The Cs ÷injection markedly broadened action potentials and depolarized neurons by 20-30 mV. Rectangular, hyperpolarizing current pulses of 50 ms duration were used to measure membrane conductance. The amplitudes of the voltage transients produced by the injected current across the cell membrane were taken as inversely proportional to membrane conductance. Intracellular potentials were recorded with a pen-writer. The composition of the standard medium was (mM): NaC1 124, KCI 5, KH~PO4 1.24, MgSO4 1.3, CaC12 2.4, NaHCOa 26 and glucose 10. The medium was saturated with 95 ~ O~ and 5 ~ CO2. DLH, D H or Glu induced large depolarizations in CA3 neurons when ejected iontophoretically at appropriate sites in the stratum radiatum (apical dendrite region) 100-200/zm from the cell-body layer s. In all of the 7 neurons examined, membrane conductance increased consistently during depolarizations induced by DLH. As shown in Fig. 1, the conductance increase started with onset of DLH-induced depolarization and reached maximum at its peak. After ejecting currents were turned off, conductance returned to the control level with repolarization. The amount of conductance changes increased with intensity of ejecting currents. In the same cell, conductance changes were examined during depolarization induced by tonic outward currents (dotted lines). When depolarizations of almost the same amplitudes as those induced by D L H were generated by tonic currents, membrane conductance slightly decreased rather than increased. Depolarizations induced by D H were also accompanied by a considerable conductance increase (Fig. 2A). In the experiment depicted in Fig. 2B, D H and Glu was administered alternately to a neuron which had been injected with Cs +. The amplitudes of responses to D H as 2
extrQ
80
0.9 0.7 ! nA outward current
60
40 13L H
80
I
_.+ 20mY 24 sec
Fig. 1. Conductance changes are shown during tonic depolarizing currents and DLH ejections. Hyperpolarizing pulses of 0.35 nA for 50 ms were passed through the recording electrode at about 0.5 Hz to measure membrane conductance. Record 1 is an intracellular record. Effects of outward currents of indicated intensities (dotted lines) and of DLH (solid lines) were examined. In this and in the following dlustration, intensities of ejection currents are given in nanoamperes. Record 2 shows a recording just outside the cell.
284 A
DH 90
L 20mY 12sec
B 2O _m~
• DH x GIu
O £: ~p C:L
X
•~ 10 m
x
O
._c E
x
=
2 Em
El
0
i
•~o
%
~ : 6
~s v
x x
3 --
=
*-I 1omv E
--12
O
sec
-I0
Fig. 2. Effects of DH are presented. A: conductance changes are shown during and after administration of DH (solid line). B: Amplitudes of responses to Glu ( × ) and DH (O) at various membrane potentials are given. Recording was from a Cs-injected neuron. Resting potential was --34 mV after Cs-injection. Records 1-3 : specimen records. Records 1 and 2 were obtained at membrane potentials of--50 mV and + 13 mV, respectively. Record 3: obtained just outside the cell. Single and double bars indicate periods of Glu (80 nA) and DH (30 nA) ejections, respectively. well as Glu increased during a tonic hyperpolarizing current and decreased progressively with an increase in depolarizing current intensity. At a membrane potential of a b o u t - - 1 4 mV, the responses reversed in polarity and further increases in intensity of current resulted in augmentation o f reversed responses. In 7 neurons injected with Cs ~, responses to D L H as well as D H were similarly reversed at membrane potentials of - - 1 3 to - - 1 8 inV. In the neurons which were not injected with Cs +, the amplitudes of the responses to all of the three amino acids similarly increased with hyperpolarization and decreased with depolarization. However, no polarity reversal was observed in these neurons even with currents five times as intense as required to reverse the responses in Cs-injected neurons. M e m b r a n e conductance usually increases in depolarized neurons because of an increase in K ~ permeability (delayed rectification). As reported before, however, it decreases during moderate depolarization induced by outward currents in hippocampal neurons 3. This p h e n o m e n o n has been attributed to anomalous rectification. On the other hand, membrane conductance increases during depolarization induced by D L H or D H . This indicates that the observed conductance increase was not brought a b o u t by increased K + permeability accompanying depolarization, but that D L H or D H directly caused an increase in permeability for cations such as N a + and K ÷.
285 As reported by Langmoen and Hablits, responses to Glu increased in size during tonic hyperpolarizing currents and decreased during depolarizing currents 6. Without Cs-injection, no reversal of polarity occurred even at extreme depolarization. Identical phenomena were observed with D H and D L H . The lack of reversal without Csinjection may be explained by large delayed rectification which develops during excessive depolarization. Cs-injection was expected to minimize delayed rectification 4. In the neurons injected with Cs ÷, the responses to D L H and D H as well as Glu reversed in polarity at membrane potentials between --13 to - - 1 8 mV. Langmoen and Hablits previously reported a similar reversal of Glu-induced responses in Cs-injected hippocampal neurons 6. The reversal potential reported by them was about 0 mV. The difference in the values of reversal potential found in their experiments and ours may be explained in part by some degree of uncertainty inherent in measuring membrane potentials with single electrodes through which currents are being passed. Although voltages generated across the electrodes were compensated for by balancing a bridge circuit in our work, it was impossible to determine whether the bridge circuit was exactly balanced. Since in the present study, responses to D L H and D H were accompanied by increased membrane conductance and they reversed in polarity at extreme depolarization, it seems reasonable to think that at least in the hippocampus, D H and D L H , as Glu, produce depolarizations by increasing permeability for cations (probably N a ÷ and K÷). The discrepancy between observations of Engberg et al.~ and ours may be explained by functional differences in the hippocampus and spinal cord. It remains to be clarified how widely the homocysteate-specific receptors reported by Engberg et al. are distributed in the brain. We thank Dr. R, D. Freeman for reviewing this manuscript. This work was supported by a grant from the Ministry of Education of Japan. 1 Curtis, D. R. and Johnston, G. A. R., Amino acid transmitters in the mammalian central nervous system, Ergebn. Physiol., 69 (1974) 97-188. 2 Engberg, I., Flatman, J. A. and Lambert, J. D. C., The actions of excitatory amino acids on motoneurones in the feline spinal cords, J. Physiol. (Lond.), 288 (1979) 227-261. 3 Hotson, J. R., Prince, D. A. and Schwartzkroin, P. A., Anomalous inward rectification in hippocampal neurons, J. Neurophysiol., 42 (1979) 889-895. 4 Johnston, D. and Hablitz, J. J., Voltage clamp discloses slow inward current in hippocampal burst-firing neurones, Nature (Lond.), 286 (1980) 391-393. 5 Krnjevic, K., Chemical nature of synaptic transmission in vertebrates, Physiol. Rev., 54 (1974) 418-540. 6 Langmoen, I. A. and Hablitz, J. J., Reversal potential for glutamate responses in hippocampal pyramidal cells, Neurosci. Lett., 23 (1981) 61-65. 7 Yamamoto, C., Activation of hippocampal neurons by mossy fiber stimulation in thin brain section in vitro, Exp. Brain Res., 14 (1972) 423-435. 8 Yamamoto, C. and Sawada, S., Sensitivity of hippocampal neurons to glutamic acid and its analogues, Brain Research, in press. 9 Zieglgansberger, W. and Puil, E. A., Actions of glutamic acid on spinal neurones, Exp. Brain Res., 17 (1973) 35-49.
Brain Research, 238 (1982) 282-285 Elsevier Biomedical Press
Excitatory actions of homocysteic acid on hippocampal neurons
SATSUKI SAWADA, SHOBU TAKADA and CHOSABURO YAMAMOTO Department of Physiology, Faculty of Medicine, Kanazawa University, Kanazawa 920 (Japan} (Accepted December 31st, 1981) Key words: homocysteic acid - - hippocampus - - glutamate receptor - - brain slice
The actions of D- and D,L-homocysteate(DH and DLH) on CA3 neurons were studied in thin sections of the guinea pig hippocampus. DH and DLH administered to the stratum radiatum induced large depolarizations in CA3 neurons. The responses to DH and DLH were accompanied by increases in membrane conductance. The amplitudes of the responses increased and decreased, respectively, during tonic hyperpolarizingand depolarizing currents. In neurons injected with Cs+, these responses were reversed in polarity at membrane potentials of--13 to --19 mV. These results indicate that receptors for homocysteate in the hippoeampus have different properties from those found in the spinal cord and are quite similar to glutamate receptors.
Since glutamate (Glu) is a prominent candidate as an excitatory transmitter in the mammalian brain1, 5, it is of considerable importance to elucidate properties of receptors for Glu and other excitatory amino acids. In 1979, Engberg and his associates reported that the excitatory action of homocysteate on spinal motoneurons was different from that of Glu 2. Whereas membrane conductance increased during Glu-induced depolarization, it decreased with depolarization induced by homocysteate. While Glu-induced responses have been reported in other work to decrease in amplitude during tonic depolarizing currents and to reverse in polarity at a level of 20-30 mV below zero 9, homocysteate-induced responses became smaller with hyperpolarization of the impaled neuronsL Although reversal of the responses to homocysteate was not actually observed, the reversal potential estimated by extrapolation was about --100 mV. From these observations, Engberg et al, suggested that receptors for Glu and homocysteate were different from each other, and that activation of Glureceptors increased permeability for both Na ~ and K ÷, while that of homocysteatereceptors caused a decrease in K + permeability. Since this difference may have fundamental implications for the understanding of neurotransmitter receptors, we have pursued the issue by comparing excitatory actions of Glu and homocysteate on hippocampal neurons in thin slices maintained in an artificial medium. Our results are not in accord with those by Engberg et al. in spinal motoneurons. Transverse sections of the hippocampus of the guinea pig were prepared as described previously 7 and were incubated in the standard medium at 37 °C for more than 40 min. They were then transferred one by one into an observation vessel which 0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press
283 was continuously perfused with the standard medium at 36-37 °C. Amino acids were administered iontophoretically either with single- or double-barrel glass pipettes. These pipettes were filled with 0.5 or 1 M Glu, 0.25 or 0.5 M D-homocysteate (DH) or 0.2 M D,L-homocysteate (DLH). The pH of these amino acid solutions was adjusted to 8.0. Intracellular potentials were recorded from neurons in region CA3 with micropipettes filled with 4 M potassium acetate or 3 M cesium acetate. Cs + was injected into impaled neurons electrophoretically for 2-5 rain with 0.5 s pulses at 1 Hz. The Cs ÷injection markedly broadened action potentials and depolarized neurons by 20-30 mV. Rectangular, hyperpolarizing current pulses of 50 ms duration were used to measure membrane conductance. The amplitudes of the voltage transients produced by the injected current across the cell membrane were taken as inversely proportional to membrane conductance. Intracellular potentials were recorded with a pen-writer. The composition of the standard medium was (mM): NaC1 124, KCI 5, KH~PO4 1.24, MgSO4 1.3, CaC12 2.4, NaHCOa 26 and glucose 10. The medium was saturated with 95 ~ O~ and 5 ~ CO2. DLH, D H or Glu induced large depolarizations in CA3 neurons when ejected iontophoretically at appropriate sites in the stratum radiatum (apical dendrite region) 100-200/zm from the cell-body layer s. In all of the 7 neurons examined, membrane conductance increased consistently during depolarizations induced by DLH. As shown in Fig. 1, the conductance increase started with onset of DLH-induced depolarization and reached maximum at its peak. After ejecting currents were turned off, conductance returned to the control level with repolarization. The amount of conductance changes increased with intensity of ejecting currents. In the same cell, conductance changes were examined during depolarization induced by tonic outward currents (dotted lines). When depolarizations of almost the same amplitudes as those induced by D L H were generated by tonic currents, membrane conductance slightly decreased rather than increased. Depolarizations induced by D H were also accompanied by a considerable conductance increase (Fig. 2A). In the experiment depicted in Fig. 2B, D H and Glu was administered alternately to a neuron which had been injected with Cs +. The amplitudes of responses to D H as 2
extrQ
80
0.9 0.7 ! nA outward current
60
40 13L H
80
I
_.+ 20mY 24 sec
Fig. 1. Conductance changes are shown during tonic depolarizing currents and DLH ejections. Hyperpolarizing pulses of 0.35 nA for 50 ms were passed through the recording electrode at about 0.5 Hz to measure membrane conductance. Record 1 is an intracellular record. Effects of outward currents of indicated intensities (dotted lines) and of DLH (solid lines) were examined. In this and in the following dlustration, intensities of ejection currents are given in nanoamperes. Record 2 shows a recording just outside the cell.
284 A
DH 90
L 20mY 12sec
B 2O _m~
• DH x GIu
O £: ~p C:L
X
•~ 10 m
x
O
._c E
x
=
2 Em
El
0
i
•~o
%
~ : 6
~s v
x x
3 --
=
*-I 1omv E
--12
O
sec
-I0
Fig. 2. Effects of DH are presented. A: conductance changes are shown during and after administration of DH (solid line). B: Amplitudes of responses to Glu ( × ) and DH (O) at various membrane potentials are given. Recording was from a Cs-injected neuron. Resting potential was --34 mV after Cs-injection. Records 1-3 : specimen records. Records 1 and 2 were obtained at membrane potentials of--50 mV and + 13 mV, respectively. Record 3: obtained just outside the cell. Single and double bars indicate periods of Glu (80 nA) and DH (30 nA) ejections, respectively. well as Glu increased during a tonic hyperpolarizing current and decreased progressively with an increase in depolarizing current intensity. At a membrane potential of a b o u t - - 1 4 mV, the responses reversed in polarity and further increases in intensity of current resulted in augmentation o f reversed responses. In 7 neurons injected with Cs ~, responses to D L H as well as D H were similarly reversed at membrane potentials of - - 1 3 to - - 1 8 inV. In the neurons which were not injected with Cs +, the amplitudes of the responses to all of the three amino acids similarly increased with hyperpolarization and decreased with depolarization. However, no polarity reversal was observed in these neurons even with currents five times as intense as required to reverse the responses in Cs-injected neurons. M e m b r a n e conductance usually increases in depolarized neurons because of an increase in K ~ permeability (delayed rectification). As reported before, however, it decreases during moderate depolarization induced by outward currents in hippocampal neurons 3. This p h e n o m e n o n has been attributed to anomalous rectification. On the other hand, membrane conductance increases during depolarization induced by D L H or D H . This indicates that the observed conductance increase was not brought a b o u t by increased K + permeability accompanying depolarization, but that D L H or D H directly caused an increase in permeability for cations such as N a + and K ÷.
285 As reported by Langmoen and Hablits, responses to Glu increased in size during tonic hyperpolarizing currents and decreased during depolarizing currents 6. Without Cs-injection, no reversal of polarity occurred even at extreme depolarization. Identical phenomena were observed with D H and D L H . The lack of reversal without Csinjection may be explained by large delayed rectification which develops during excessive depolarization. Cs-injection was expected to minimize delayed rectification 4. In the neurons injected with Cs ÷, the responses to D L H and D H as well as Glu reversed in polarity at membrane potentials between --13 to - - 1 8 mV. Langmoen and Hablits previously reported a similar reversal of Glu-induced responses in Cs-injected hippocampal neurons 6. The reversal potential reported by them was about 0 mV. The difference in the values of reversal potential found in their experiments and ours may be explained in part by some degree of uncertainty inherent in measuring membrane potentials with single electrodes through which currents are being passed. Although voltages generated across the electrodes were compensated for by balancing a bridge circuit in our work, it was impossible to determine whether the bridge circuit was exactly balanced. Since in the present study, responses to D L H and D H were accompanied by increased membrane conductance and they reversed in polarity at extreme depolarization, it seems reasonable to think that at least in the hippocampus, D H and D L H , as Glu, produce depolarizations by increasing permeability for cations (probably N a ÷ and K÷). The discrepancy between observations of Engberg et al.~ and ours may be explained by functional differences in the hippocampus and spinal cord. It remains to be clarified how widely the homocysteate-specific receptors reported by Engberg et al. are distributed in the brain. We thank Dr. R, D. Freeman for reviewing this manuscript. This work was supported by a grant from the Ministry of Education of Japan. 1 Curtis, D. R. and Johnston, G. A. R., Amino acid transmitters in the mammalian central nervous system, Ergebn. Physiol., 69 (1974) 97-188. 2 Engberg, I., Flatman, J. A. and Lambert, J. D. C., The actions of excitatory amino acids on motoneurones in the feline spinal cords, J. Physiol. (Lond.), 288 (1979) 227-261. 3 Hotson, J. R., Prince, D. A. and Schwartzkroin, P. A., Anomalous inward rectification in hippocampal neurons, J. Neurophysiol., 42 (1979) 889-895. 4 Johnston, D. and Hablitz, J. J., Voltage clamp discloses slow inward current in hippocampal burst-firing neurones, Nature (Lond.), 286 (1980) 391-393. 5 Krnjevic, K., Chemical nature of synaptic transmission in vertebrates, Physiol. Rev., 54 (1974) 418-540. 6 Langmoen, I. A. and Hablitz, J. J., Reversal potential for glutamate responses in hippocampal pyramidal cells, Neurosci. Lett., 23 (1981) 61-65. 7 Yamamoto, C., Activation of hippocampal neurons by mossy fiber stimulation in thin brain section in vitro, Exp. Brain Res., 14 (1972) 423-435. 8 Yamamoto, C. and Sawada, S., Sensitivity of hippocampal neurons to glutamic acid and its analogues, Brain Research, in press. 9 Zieglgansberger, W. and Puil, E. A., Actions of glutamic acid on spinal neurones, Exp. Brain Res., 17 (1973) 35-49.