Exp Brain Res (1992) 92:85-93
9 Springer-Verlag 1992
Inhibitory effects of excitatory amino acids on pyramidal cells of the in vitro turtle medial cortex Raul E. Russo l'z and Julio C. Velluti 1
1 Divisidn Neurofisiologia, Instituto de Investigaciones Bioldgicas Clemente Estable, Avda. Italia 3318, 11600 Montevideo, Uruguay 2 Departamento de Fisiologia, Facultad de Medicina, Gral. Flores 2125, 11800 Montevideo, Uruguay Received January 14, 1992/Accepted May 29, 1992 Summary. The electroencephalogram of the in vitro brain
of the turtle Chrysemysd' orbigny shows spontaneous random large sharp waves (LSWs) which may be compared to interictal spikes. In order to evaluate the role of excitatory amino acids (EAAs) in particular through the N-methy,l-I>aspartate (NMDA) receptor in the generation of LSWs, the bath application of N M D A and its antagonists 3-(( _+)-2-carboxypiperazin-4y)-propyl-1phosphonic acid (CPP) and DI.-2-amino-5-phosphonovaleric acid (APV), was performed in the whole open hemisphere (WOH) in vitro. Field recordings in W O H showed that both CPP and APV unexpectedly increased LSW amplitude. Consistently, N M D A in the bath suppressed the LSWs. Iontophoretically applied glutamate, kainate and N M D A produced a hyperpolarization of intracellularly recorded medial cortex pyramidal cells both in W O H and in slices. The EAA-induced hyperpolarization was tetrodotoxin (TTX) and bicuculline sensitive and reversed close to - 70 mV. It would therefore seem to be due to the activation of gamma-aminobutyric acid (GABA) interneurons. The N M D A could also produce an excitation of pyramidal cells - always following a previous inhibitory phase. In some cases rhythmic bursting discharges or plateau potentials were observed. These N M D A effects were mainly elicited by a direct effect on pyramidal cells. A long-lasting hyperpolarizing response following the N M D A excitatory phase was also observed. This long-lasting response was an intrinsic property of pyramidal cells since it was TTX resistant. This study demonstrates that GABAergic interneurons from the turtle medial cortex can be activated by EAAs, a mechanism that can account for the effects of N M D A antagonists on LSWs. Key words: L-Glutamate - N-Methyl-o-aspartate - Kai-
nate GABAergic inhibition - In vitro turtle cortex Turtle
Correspondence to. J.C. Velluti, Divisidn Neurotisiologia, Instituto de Investigaciones Biol6gicas Clemente Estable, Avda. Italia 3318, 11600 Montevideo, Uruguay
Introduction
The turtle cerebral cortex represents a valuable experimental paradigm from a phylogenetic, anatomical and metabolic point of view. Phylogenetically, turtles are thought to have originated as an early branch arising from the Cotylosaurs, the basic stock reptiles from which all living mammals have evolved (Romer 1972). On the other hand, the relative anatomical simplicity of the reptilian cortex, with only two types of cells, arranged in a threelayered fashion (Ram6n y Cajal 1891; Desan 1984), facilitates the study and comprehension of this structure. Finally, the high resistance of aquatic turtles to hypoxia has made them particularly useful for in vitro intact preparations (Mori et al. 1980; Chan and Nicholson 1986; Kriegstein 1987; Chan et al. 1988; Larson-Prior et al. 1990). We have recently developed a whole hemisphere preparation that allows the simultaneous recording of the electroencephalogram (EEG) and transmembrane potentials (Velluti et al. 1991). Spontaneous large sharp waves (LSWs) were observed in the turtle EEG both in vivo (Walker and Berger 1973; Gaztelu et al. 1991) and in vitro (Velluti et al. 1991). During these LSWs (Velluti et al. 1991) the intracellular records frequently show a burst of action potentials (APs) riding on a slow depolarizing wave quite similar to the paroxysmal depolarization shift (PDS) described by Matsumoto and Ajmone-Marsan (1964). In this sense, turtle LSWs could be homologous to the interictal spikes extensively studied in mammalian brains (Prince 1978, 1985). It has been claimed that excitatory amino acids (EAAs) are the neurotransmitters used by the vast majority of excitatory synapses in the central nervous system (CNS) (Krnjevic 1974; Monhagan et al. 1989). Furthermore, it has become evident that a particular EAA receptor subclass - the N-methyl-I>aspartate (NMDA) receptor could be involved in epilepsy (Herron et at. 1985; MacDonald et al. 1988). Anticonvulsant properties of N M D A antagonists have been reported to act against audiogenic (Croucher et al. 1982) photogenic (Meldrum et al. 1983) and kindled (Peterson et al. 1983) seizures.
86 We have taken advantage of the in vitro turtle brain preparation (Velluti et al. 1991) to study the role of EAAs in the generation of turtle LSWs. Perfusion with selective N M D A antagonists p r o d u c e d an unexpected increase in the amplitude of spontaneous LSWs. Results from experiments using iontophoretic application of some EAAs provided an explanation for this paradoxical effect of N M D A receptor antagonists on a cellular level.
Materials and methods The experiments were performed in juvenile specimens (4-6 cm carapace length) of the turtle Chrysemysd' orbigny, under the guidelines established by the Ministerio de Ganaderia, Agricultura y Pesca, Divisi6n Fauna, Uruguay.
Tissue preparation The animals were anesthetized by cooling on crushed ice; they were then decapitated and the brain was removed. Two types of in vitro preparations were performed: (1) The whole open hemisphere (WOH) was used for recording the in vitro EEG and/or transmembrane potentials. Details of the techniques for obtaining the WOH are described elsewhere (Velluti et al. 1991). Briefly, one hemisphere was opened through its lateral surface, unfolded and placed in the recording chamber with its ependymal surface upward. (2) Coronal brain slices (500 #m thick) were utilized as an alternative to the WOH preparation. The slice preparation has the advantage of allowing the electrodes to be placed more easily within different cortical layers, and was therefore preferred for iontophoretic experiments. Both preparations were continuously superfused with Ringer solution bubbled with 5% CO 2 and 95% 02, at room temperature (20-22~ The composition (Mori et al. 1980) of standard Ringer was (in mM): NaC196.5; KCI 2.6; CaC12 4.0; MgCI 2 2.0; NaHCO3 31.5; glucose 10. Sometimes, when using WOH preparation the normal perfusate was modified by replacing NaHCO 3 with HEPES without bubbling gases in order to increase the frequency of spontaneous LSWs (Velluti et al. 1991). In all cases the pH of the media was maintained at 7.6.
Drugs The following drugs were added to the normal medium: NMDA (5-10 #M) and its antagonists 3-((_+)-2-carboxypiperazin-4y)propyl-l-phosphonic acid (CPP; 5-10 #M), and oL-2-amino-5phosphonovaleric acid (APV; 10-50 pM), tetrodotoxin (TTX; 1 #M), and bicuculline (50 #M). Single or multibarreled micropipettes (20 50 Mff~)were filled with NMDA (50 mM in 150 mM NaC1, pH 8), L-glutamate (Glu; 0.5 M, pH 8) or kainate (KA; 0.5 M, pH 8) for iontophoretic ejection. In a few cases the Glu concentration in the iontophoretic electrode was raised to 1.5 M. Ejection currents varied between 5 and 300 hA. Retaining current was used when necessary. Control experiments showed that these iontophoretic currents did not directly affect the neuronal excitability.
Electrophysiological recordings and stimulation Field activity was recorded with glass micropipettes (1 2 Mf~) filled with 1 M NaC1 by means of a high-sensitivity low-noise amplifier (0.3-60 Hz bandwidth). Conventional intracellular recordings were
made with glass micropipettes filled with 4 M potassium acetate (100-200 Mr2). In two experiments, electrodes filled with 3 M KC1 (100-150 Mfl) were also used. Recording and iontophoretic micropipettes were visually positioned in the medial cortex, either in WOH or slices, using a dissecting microscope (50 x ). Orthodromic activity was evoked by means of electrical stimuli (100 #s duration) through a bipolar nichrome electrode - insulated except at the tip - placed in the septal area. The signals were simultaneously monitored on a oscilloscope and stored in a digital recorder. The data were later amplified, filtered and fed into a personal computer. The transmembrane potential was filtered at 6 kHz and sampled at 15 kHz, while EEG was filtered at 60 Hz and sampled at 200 Hz. Hard copies of digitized data were obtained with a laser printer.
Results The in vitro E E G consisted of irregular b a c k g r o u n d activity ( 5 - 1 0 #V) interrupted by spontaneous LSWs, with an amplitude between 20 and 400 #V, occurring r a n d o m l y at frequencies varying between 0.5 and 11 min - 1 (Fig. 1A, D, control). S p o n t a n e o u s LSWs were not observed in 21% of W O H preparations. O n l y those preparations showing L S W frequencies higher than 1.5 min - i were used in the present study. A detailed description of the in vitro E E G concerning b a c k g r o u n d activity, L S W s and their cellular basis has been given (Velluti et al. 1991).
Effects of NMDA antagonists on LS Ws In order to evaluate the participation of EAAs (in particular t h r o u g h the N M D A receptor) in the genesis of L S W s we added C P P ( 5 - 1 0 #M, n = 4) or A P V (10-50 #M, n = 6) to the n o r m a l medium. The N M D A antagonists produced an increase of a b o u t 80% in L S W amplitude (Fig. 1A, B). In some cases (4 out of 10) an increase in L S W frequency was also observed. The N M D A antagonists also modified the evoked activity p r o d u c e d by septal stimulation. In control conditions the only response elicited by septal stimulation was a triphasic field potential (Fig. 1C, control). After treatment with A P V or C P P , an afterdischarge appeared while the evoked potential remained almost unchanged except for a small (16%) amplitude increase (Fig. 1C, APV). These evoked afterdischarges were similar to the spontaneous L S W s and will thus be referred to as evoked LSWs. They occurred with a delay of a b o u t 1 s, which decreased to 0.2 s with stronger stimuli. Waveform, amplitude and duration of the evoked L S W s could vary from one stimulus to the other (Fig. 1C, APV). N M D A antagonist treatment resulted in evoked L S W s that persisted even after prolonged washing (more than 1 h). In line with these results, the addition of N M D A (5-10 pM, n = 3 ) to the n o r m a l medium abolished the L S W s within a few minutes (Fig. 1D). This effect was reversed after 20 min washing with n o r m a l perfusate. An increase in b a c k g r o u n d activity was seen together with the suppression of L S W s in the N M D A - c o n t a i n i n g medium. Higher concentrations of N M D A (up to 100 #M) had the same effect but were more difficult to reverse.
87 A
Control
C
Control
A
C I
APV
Irl
ML
d.. A P V
' I
-
Glu
CL SCLJ
w
D
B gV
300.
j Control .
.... L.,..,J,I I .... r '
200,
',]
...... ,,I.. "~ . . . . . . T-
NMDA 100
NMDA
1
Wash " ~'I ~,tt[...l .... .,,li,O0.V APV
Fig. 1A-D. Effects of APV and NMDA on turtle EEG. A Control conditions showing spontaneous LSWs. APV (25 #M) increases LSW amplitude. B Bar plot of LSW amplitude (mean-t-SD) from 18 min data of same experiment. Note that APV produces an increase in LSW amplitude that is reversed after washing (P
9 Springer-Verlag 1992
Inhibitory effects of excitatory amino acids on pyramidal cells of the in vitro turtle medial cortex Raul E. Russo l'z and Julio C. Velluti 1
1 Divisidn Neurofisiologia, Instituto de Investigaciones Bioldgicas Clemente Estable, Avda. Italia 3318, 11600 Montevideo, Uruguay 2 Departamento de Fisiologia, Facultad de Medicina, Gral. Flores 2125, 11800 Montevideo, Uruguay Received January 14, 1992/Accepted May 29, 1992 Summary. The electroencephalogram of the in vitro brain
of the turtle Chrysemysd' orbigny shows spontaneous random large sharp waves (LSWs) which may be compared to interictal spikes. In order to evaluate the role of excitatory amino acids (EAAs) in particular through the N-methy,l-I>aspartate (NMDA) receptor in the generation of LSWs, the bath application of N M D A and its antagonists 3-(( _+)-2-carboxypiperazin-4y)-propyl-1phosphonic acid (CPP) and DI.-2-amino-5-phosphonovaleric acid (APV), was performed in the whole open hemisphere (WOH) in vitro. Field recordings in W O H showed that both CPP and APV unexpectedly increased LSW amplitude. Consistently, N M D A in the bath suppressed the LSWs. Iontophoretically applied glutamate, kainate and N M D A produced a hyperpolarization of intracellularly recorded medial cortex pyramidal cells both in W O H and in slices. The EAA-induced hyperpolarization was tetrodotoxin (TTX) and bicuculline sensitive and reversed close to - 70 mV. It would therefore seem to be due to the activation of gamma-aminobutyric acid (GABA) interneurons. The N M D A could also produce an excitation of pyramidal cells - always following a previous inhibitory phase. In some cases rhythmic bursting discharges or plateau potentials were observed. These N M D A effects were mainly elicited by a direct effect on pyramidal cells. A long-lasting hyperpolarizing response following the N M D A excitatory phase was also observed. This long-lasting response was an intrinsic property of pyramidal cells since it was TTX resistant. This study demonstrates that GABAergic interneurons from the turtle medial cortex can be activated by EAAs, a mechanism that can account for the effects of N M D A antagonists on LSWs. Key words: L-Glutamate - N-Methyl-o-aspartate - Kai-
nate GABAergic inhibition - In vitro turtle cortex Turtle
Correspondence to. J.C. Velluti, Divisidn Neurotisiologia, Instituto de Investigaciones Biol6gicas Clemente Estable, Avda. Italia 3318, 11600 Montevideo, Uruguay
Introduction
The turtle cerebral cortex represents a valuable experimental paradigm from a phylogenetic, anatomical and metabolic point of view. Phylogenetically, turtles are thought to have originated as an early branch arising from the Cotylosaurs, the basic stock reptiles from which all living mammals have evolved (Romer 1972). On the other hand, the relative anatomical simplicity of the reptilian cortex, with only two types of cells, arranged in a threelayered fashion (Ram6n y Cajal 1891; Desan 1984), facilitates the study and comprehension of this structure. Finally, the high resistance of aquatic turtles to hypoxia has made them particularly useful for in vitro intact preparations (Mori et al. 1980; Chan and Nicholson 1986; Kriegstein 1987; Chan et al. 1988; Larson-Prior et al. 1990). We have recently developed a whole hemisphere preparation that allows the simultaneous recording of the electroencephalogram (EEG) and transmembrane potentials (Velluti et al. 1991). Spontaneous large sharp waves (LSWs) were observed in the turtle EEG both in vivo (Walker and Berger 1973; Gaztelu et al. 1991) and in vitro (Velluti et al. 1991). During these LSWs (Velluti et al. 1991) the intracellular records frequently show a burst of action potentials (APs) riding on a slow depolarizing wave quite similar to the paroxysmal depolarization shift (PDS) described by Matsumoto and Ajmone-Marsan (1964). In this sense, turtle LSWs could be homologous to the interictal spikes extensively studied in mammalian brains (Prince 1978, 1985). It has been claimed that excitatory amino acids (EAAs) are the neurotransmitters used by the vast majority of excitatory synapses in the central nervous system (CNS) (Krnjevic 1974; Monhagan et al. 1989). Furthermore, it has become evident that a particular EAA receptor subclass - the N-methyl-I>aspartate (NMDA) receptor could be involved in epilepsy (Herron et at. 1985; MacDonald et al. 1988). Anticonvulsant properties of N M D A antagonists have been reported to act against audiogenic (Croucher et al. 1982) photogenic (Meldrum et al. 1983) and kindled (Peterson et al. 1983) seizures.
86 We have taken advantage of the in vitro turtle brain preparation (Velluti et al. 1991) to study the role of EAAs in the generation of turtle LSWs. Perfusion with selective N M D A antagonists p r o d u c e d an unexpected increase in the amplitude of spontaneous LSWs. Results from experiments using iontophoretic application of some EAAs provided an explanation for this paradoxical effect of N M D A receptor antagonists on a cellular level.
Materials and methods The experiments were performed in juvenile specimens (4-6 cm carapace length) of the turtle Chrysemysd' orbigny, under the guidelines established by the Ministerio de Ganaderia, Agricultura y Pesca, Divisi6n Fauna, Uruguay.
Tissue preparation The animals were anesthetized by cooling on crushed ice; they were then decapitated and the brain was removed. Two types of in vitro preparations were performed: (1) The whole open hemisphere (WOH) was used for recording the in vitro EEG and/or transmembrane potentials. Details of the techniques for obtaining the WOH are described elsewhere (Velluti et al. 1991). Briefly, one hemisphere was opened through its lateral surface, unfolded and placed in the recording chamber with its ependymal surface upward. (2) Coronal brain slices (500 #m thick) were utilized as an alternative to the WOH preparation. The slice preparation has the advantage of allowing the electrodes to be placed more easily within different cortical layers, and was therefore preferred for iontophoretic experiments. Both preparations were continuously superfused with Ringer solution bubbled with 5% CO 2 and 95% 02, at room temperature (20-22~ The composition (Mori et al. 1980) of standard Ringer was (in mM): NaC196.5; KCI 2.6; CaC12 4.0; MgCI 2 2.0; NaHCO3 31.5; glucose 10. Sometimes, when using WOH preparation the normal perfusate was modified by replacing NaHCO 3 with HEPES without bubbling gases in order to increase the frequency of spontaneous LSWs (Velluti et al. 1991). In all cases the pH of the media was maintained at 7.6.
Drugs The following drugs were added to the normal medium: NMDA (5-10 #M) and its antagonists 3-((_+)-2-carboxypiperazin-4y)propyl-l-phosphonic acid (CPP; 5-10 #M), and oL-2-amino-5phosphonovaleric acid (APV; 10-50 pM), tetrodotoxin (TTX; 1 #M), and bicuculline (50 #M). Single or multibarreled micropipettes (20 50 Mff~)were filled with NMDA (50 mM in 150 mM NaC1, pH 8), L-glutamate (Glu; 0.5 M, pH 8) or kainate (KA; 0.5 M, pH 8) for iontophoretic ejection. In a few cases the Glu concentration in the iontophoretic electrode was raised to 1.5 M. Ejection currents varied between 5 and 300 hA. Retaining current was used when necessary. Control experiments showed that these iontophoretic currents did not directly affect the neuronal excitability.
Electrophysiological recordings and stimulation Field activity was recorded with glass micropipettes (1 2 Mf~) filled with 1 M NaC1 by means of a high-sensitivity low-noise amplifier (0.3-60 Hz bandwidth). Conventional intracellular recordings were
made with glass micropipettes filled with 4 M potassium acetate (100-200 Mr2). In two experiments, electrodes filled with 3 M KC1 (100-150 Mfl) were also used. Recording and iontophoretic micropipettes were visually positioned in the medial cortex, either in WOH or slices, using a dissecting microscope (50 x ). Orthodromic activity was evoked by means of electrical stimuli (100 #s duration) through a bipolar nichrome electrode - insulated except at the tip - placed in the septal area. The signals were simultaneously monitored on a oscilloscope and stored in a digital recorder. The data were later amplified, filtered and fed into a personal computer. The transmembrane potential was filtered at 6 kHz and sampled at 15 kHz, while EEG was filtered at 60 Hz and sampled at 200 Hz. Hard copies of digitized data were obtained with a laser printer.
Results The in vitro E E G consisted of irregular b a c k g r o u n d activity ( 5 - 1 0 #V) interrupted by spontaneous LSWs, with an amplitude between 20 and 400 #V, occurring r a n d o m l y at frequencies varying between 0.5 and 11 min - 1 (Fig. 1A, D, control). S p o n t a n e o u s LSWs were not observed in 21% of W O H preparations. O n l y those preparations showing L S W frequencies higher than 1.5 min - i were used in the present study. A detailed description of the in vitro E E G concerning b a c k g r o u n d activity, L S W s and their cellular basis has been given (Velluti et al. 1991).
Effects of NMDA antagonists on LS Ws In order to evaluate the participation of EAAs (in particular t h r o u g h the N M D A receptor) in the genesis of L S W s we added C P P ( 5 - 1 0 #M, n = 4) or A P V (10-50 #M, n = 6) to the n o r m a l medium. The N M D A antagonists produced an increase of a b o u t 80% in L S W amplitude (Fig. 1A, B). In some cases (4 out of 10) an increase in L S W frequency was also observed. The N M D A antagonists also modified the evoked activity p r o d u c e d by septal stimulation. In control conditions the only response elicited by septal stimulation was a triphasic field potential (Fig. 1C, control). After treatment with A P V or C P P , an afterdischarge appeared while the evoked potential remained almost unchanged except for a small (16%) amplitude increase (Fig. 1C, APV). These evoked afterdischarges were similar to the spontaneous L S W s and will thus be referred to as evoked LSWs. They occurred with a delay of a b o u t 1 s, which decreased to 0.2 s with stronger stimuli. Waveform, amplitude and duration of the evoked L S W s could vary from one stimulus to the other (Fig. 1C, APV). N M D A antagonist treatment resulted in evoked L S W s that persisted even after prolonged washing (more than 1 h). In line with these results, the addition of N M D A (5-10 pM, n = 3 ) to the n o r m a l medium abolished the L S W s within a few minutes (Fig. 1D). This effect was reversed after 20 min washing with n o r m a l perfusate. An increase in b a c k g r o u n d activity was seen together with the suppression of L S W s in the N M D A - c o n t a i n i n g medium. Higher concentrations of N M D A (up to 100 #M) had the same effect but were more difficult to reverse.
87 A
Control
C
Control
A
C I
APV
Irl
ML
d.. A P V
' I
-
Glu
CL SCLJ
w
D
B gV
300.
j Control .
.... L.,..,J,I I .... r '
200,
',]
...... ,,I.. "~ . . . . . . T-
NMDA 100
NMDA
1
Wash " ~'I ~,tt[...l .... .,,li,O0.V APV
Fig. 1A-D. Effects of APV and NMDA on turtle EEG. A Control conditions showing spontaneous LSWs. APV (25 #M) increases LSW amplitude. B Bar plot of LSW amplitude (mean-t-SD) from 18 min data of same experiment. Note that APV produces an increase in LSW amplitude that is reversed after washing (P
