Brain Research, 160 (1979) 95-104 © Elsevier/North-Holland BiomedicalPress
95
CAT SPINAL MOTONEURONES EXHIBIT TOPOGRAPHIC SENSITIVITY TO G L U T A M A T E AND GLYCINE*
W. ZIEGLG,~NSBERGER and J. CHAMPAGNAT Max-Planck-lnstitut fiir Psychiatrie, Department of Neuropharmacology, 8 Munich 40 (G.F.R.) and Centre National de la Recherche Scientifique, Laboratoire de Physiologic nerveuse, 91190 Gif-surYvette (France)
(Accepted April 20th, 1978)
SUMMARY Spinal neurones from the 6th and 7th lumbar segments of cat were recolded intracellularly. L-Glutamate (GLU) and glycine (GLY) were preferentially applied to 'somatic' or 'dendritic' regions. To accomplish this, single micropipette electrodes for intracellular recording were assembled alongside drug-delivering micropipettes at varying distances from the recording tip. Microiontophoretic application of G L U to predominantly 'dendritic' sites led to a depolarizing response after a significantly shorter delay than applications to more 'somatic' sites. 'Dendritic' applications did not cause measurable increases in membrane conductance at the recording site. 'Somatic' application of high amounts of G L U caused a depolarization associated with a progressively increasing membrane conductance. Thus, GLU may increase membrane conductance at 'dendritic' sites, but this effect is hardly detectable by conventional resistance measurements at the soma membrane. Microiontophoretic application of GLY at 'dendritic' or 'somatic' sites produced hyperpolarizing responses with marked increases of membrane conductance. The onset latency of the response was significantly longer for 'dendritic' applications than for 'somatic' applications. Action potentials, EPSPs and IPSPs were all attenuated by shunting during the GLY (or GLU) increases of membrane conductance. The equilibrium potentials measured for the hyperpolarizing action of GLY (range: --75 to --82 mV) and for the orthodromically evoked IPSPs (range: --74 to --78 mV) were similar and were closer to the resting potential than antidromically evoked IPSPs (range: --80 to --84 mV). The specific distribution of excitatory glutaminergic and inhibitory glycinergic * A preliminaryreport was given at the Spring Meeting of the German PhysiologicalSociety, March 1977.
96 receptive sites on cat spinal motoneurones further supports a physiologic role for these amino acids as synaptic transmitters.
INTRODUCTION Both L-glutamate (GLU) and glycine (GLY) fulfill criteria establishing them as likely candidates for excitatory and inhibitory neurotransmitters in a variety of areas in the mammalian central nervous system (see refs. 9, 11 and 14). However, a major problem confronts the analysis of the ionic mechanism underlying the actions of putative transmitters in the mammalian CNS: neurones with complex shape are difficult to influence uniformly either by microiontophoretic application of a test compound or by intracellular current or ion injection. The aim of the present study was to determine whether there are predominant sites of receptivity to iontophoretically applied GLU and GLY on spinal motoneurones of the cat. A combination of intracellular recording and the microiontophoretic application to different regions of a neurone was employed to provide information about the predominant location of amino acid receptive sites on these neurones. METHODS The experiments were performed in 28 adult cats of either sex (body weight: 2.5-3.5 kg). Surgery was performed during pentobarbital anaesthesia (35 mg/kg, i.p.). The anaesthesia was continued by repeated intravenous injections of pentobarbital (5 mg/kg/h). After surgery the animals were immobilized with gallamine triethiodide and artificially respired. A bilateral pneumothorax was made routinely. Blood pressure was monitored continually and experiments were discontinued when blood pressure dropped below 80 mm Hg. The rectal temperature was maintained between 37.8 and 38.2 °C by means of a thermostatically controlled electric blanket and heat radiators. During the experiments plasma-expanders (2 ml/h) and small amounts of dextrose were given intravenously. The spinal cord was exposed by laminectomy at the lumbar-sacral level (L3-$2) and covered with a pool of paraffin oil kept at body temperature. The dura mater was reflected and the pia mater incised at the recording site to facilitate insertion of the electrode assembly. Strands dissected from the dorsal roots of the segments L5 and L6 were stimulated by means of bipolar electrodes. The ventral roots were stimulated in situ. The electrode assembly consisted of a single and two double- (or triple-) barrelled micropipettes fixed together at variable distances (see insert, Fig. 1) with fastsetting epoxy glue. Only the upper parts of the electrode shafts were fixed together, thus leaving 3 mm of the tip free. Electrode intertip distances ranged from 80 to 150 #m for the inner ('somatic') application and from 150 to 350 #m for the remote ('dendritic') application. The recording barrel was filled with a 10:1 mixture of potassium citrate (1.6 M) and KCI (1.0 M). The tip diameter of the recording barrel
97 was less than 1 #m; DC resistance ranged from 10 to 15 MfL Intracellular recordings and conductance measurements were performed using conventional techniques. Recordings were stored on tape for later analysis. The solutions used for microelectrophoretic application were as follows: Monosodium-L-glutamate (0.5 M, pH 8.0), glycine (0.5 M, pH 5.0) and NaC1 (1.0 M, pH 5.0). Current neutralization was routinely employed to eliminate current artifacts, and was assumed to be complete when the baseline showed no detectable deflection when the ejection currents were switched on. Currents (GLU was applied as an anion, GLY as a cation) were provided from constant current sources and could be preset in magnitude and duration of flow. The voltage necessary to drive the current was monitored continuously. Since oscillations of this voltage indicate that the ohmic resistance of the application electrode is unstable and that is unlikely to release consistent amounts of material, only applications in which the voltage remained stable were included in this study. RESULTS The present results were obtained from 82 spinal motoneurones recorded intracellularly from the 6th and 7th lumbar segments. They all showed stable membrane potentials greater than --65 mV and action potentials larger than 80 mV during the test period. Subsequently the drug applications from electrodes arranged at intertip distances less than 150 ~m will be referred to as 'somatic', whereas those from electrodes more remotely spaced will be termed 'dendritic' (see insert, Fig. 1).
(1) Actions of L-glutamate (GLU) When GLU was applied microiontophoretically to more 'dendritic' sites, the majority of cells started to be depolarized after a clearly shorter latency than after the 'somatic' applications. The graph in Fig. 1 (B) summarizes the mean latency of onset of the depolarizing effect obtained from 77 motoneurones employing electrodes staggered at distances of 80-260 #m. The phoretic currents (range: 10-100 nA) were adjusted so as to induce a depolarizing response of 3 mV or more in amplitude. In most neurones the applications were repeated 2-6 times and the latencies did not vary by more than 10-15 ~ in a given neurone. Part A of Fig. 1 shows a test with two drug delivery electrodes arranged at 80/zm and 225/zm distances from the recording tip respectively. The onset of depolarization following the 'somatic' application of GLU (80 nA) was clearly delayed when compared with that of the 'dendritic' application. Employing equal ejection currents the responses to 'somatic' application were usually delayed but larger in amplitude (see Fig. 1A) than those induced at 'dendritic' sites. The depolarizations recorded following 'dendritic' applications were usually not accompanied by a measurable increase in conductance at the recording site, presumably the motoneuron soma. In 8 out of 38 cells the membrane conductance even decreased slightly (5-15~) during the GLU-induced depolarizations (range: 5-30 mV). When the depolarization reached the firing level most cells started to fire
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DiStar,~(jum) Fig. 1. Different latencies of the depolarizingeffectof GLU applied to spinal motoneurones at varying distances from the soma. A: superimposed pen-writer recordings of the membrane potential following applications from electrode assemblies (see insert) arranged at 80 #m and 225/tm, intertip distance respectively. GLU (80 nA) was applied for 10 see (bar, time calibration) from either electrode. The depolarizing effectfollowingthe 'somatic' application was clearlydelayed(dotted line). The membrane potential shifts to values below control, following GLU (see ref. 22). B" results obtained from 77 motoneurones. The mean value of the latencies is given when more than one test was performed. The phoretic currents were adjusted to evoke depolarizingresponses of more than 3 mV in amplitude. repetitively. The overshoot of ortho- or antidromically evoked spikes was not detectably altered in amplitude. An increase in dosage up to currents of 200-500 nA led to marked depolarizations after delays of 5-15 sec, associated with progressive conductance increases. During these responses all spontaneous or evoked postsynaptic transients were strongly diminished. Such excessive conductance changes were only observed with intertip distances less than 120 #m. Fig. 2 compares current/voltage plots obtained with either 'dendritic' or 'somatic' application of GLU. When G L U (60 nA) was applied by the remote application electrode (intertip distance: 250/~m), the depolarization started after a delay less than 500 msec. No conductance change was observed during the maximal depolarization which was reached within approximately 4 sec. 'Somatic' application (intertip distance: 100 #m) of G L U (100 nA) caused an increase in the conductance of the postsynaptic membrane which plateaued after 10 sec. The intersection point for the current/voltage lines obtained during the maximal drug effect and the control was between zero and - - 1 0 mV. Comparable measurements were performed in 15 neurones. An approximation of the equilibrium potential for G L U was found between --28 and + 10 mV for 'somatic' applications employing currents from 80 to 500 nA from electrodes with intertip distances ranging from 85 to 150/zm.
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Fig. 2. Current/voltage plots obtained for GLU effectselicited at different distances from the recording site. During the application of GLU (100 hA) from the electrode closest to the recording site (100 #m, 'somatic'), the slope (triangles) measured at maximal depolarization (10 see after onset of the application) was found to be decreased. It intersected the control slope (dots) at membrane potential values between zero and --10 mV. Following application of GLU (60 hA) from the electrode arranged at a distance of 250 #m, no detectable change in membrane resistance was associated with the depolarizing effect which plateaued 4 see after onset of application (open squares). The slopes drawn were estimated. The symbols represent mean values from 5 consecutive measurements. Duration of intracellularly applied current pulses for resistance measurement was 20 msec. All measurements were performed during maximal depolarization. The electrode resistance remained constant during the measurements.
(2} Actions of glycine (GL Y) Tests comparable to those described for G L U were performed to investigate the site of action of microiontophoretically applied G L Y (5-100 nA) on spinal neurones. Fig. 3A illustrates the hyperpolarizing action of G L Y (50 nA) when it is applied either close to the soma (70/~m) or preferentially to the dendrites (200/~m) of a lumbar motoneurone. In contrast to G L U applications, G L Y induced a response with shorter latencies when it was applied closer to the soma. Fig. 3B summarizes results (mean latencies) obtained from 38 motoneurones. The phoretic currents were adjusted as to evoke hyperpolarizing responses of more than 3 mV in amplitude. The hyperpolarizations were invariably associated with a strong conductance increase of the postsynaptie membrane (see current/voltage plot in Fig. 3C). The action potentials evoked antidromicaUy (Fig. 3C, a ) a n d orthodromically as well as EPSPs and IPSPs were maximally attenuated by shunting. Comparable tests were performed in 14 neurones. The equilibrium potential for the hyperpolarizing action of G L Y for both 'somatic' and 'dendritic' application was found in the range of----75 to - - 8 2 mV. In these tests the equilibrium potential for the recurrent IPSP was found at slightly more hyperpolarized levels (range: - - 8 0 to - - 8 4 mV) than the equilibrium potential for the IPSPs evoked by dorsal rootlet stimulation (range: - - 7 4 to - - 7 8 mV). In a number of cells (12 out o f 26) a tendency was observed for repolarization to occur despite continuous
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-tO0 Nembrane potential(mY) Fig. 3. Different latencies of the hyperpolarizing effect of GLY applied to spinal motoneurones from varying distances. A: superimposed pen-writer recordings of the membrane potential following applications (bar, time calibration) of GLY (50 nA/10 sec) at different distances from the recording site (70 #m and 200/~m, resp.). The application from the electrode closer to the soma (solid line) caused an almost immediate onset of the hyperpolarization. B: results obtained from 38 motoneurones. The phoretic currents were adjusted to evoked hyperpolarizing responses of more than 3 mV in amplitude. The intertip distances of the electrodes varied from 60 to 300/~m. Mean values for the latency of the hyperpolarizing responses are given when more than one measurement was performed. C: the current/voltage plots shows a marked increase in the conductance of the postsynaptic membrane. The arrow indicates the intersection point (EGzy) of the slopes during GLY administration with the control (triangles). The open circles represent the 'somatic' and the dots represent the 'dendritic' application of GLY. Measurements were performed during maximal hyperpolarization (mean values out of 4 measurements). There is no obvious difference in the current/voltage characterestics following these different sites of application. The antidromically evoked action potential (C, a) is maximally attenuated during such GLY applications due to shunting of the neuronal membrane. Calibration: 1 msec; 20 mV. application of G L Y (Fig. 3A); this effect was e n h a n c e d by repeated applications of the a m i n o acid at short intervals ( < 30 see).
(3) Current 'artefacts' The shift in the baseline of the D C recording of the m e m b r a n e potential ('coupling') induced by the phoretic current is, without current neutralization,
101 proportional to the magnitude of the current and equal when switching on and off. Following G L U (applied as an anion, tip negative), however, the coupling artefact at the termination of the application increased in size (Fig. 4). The initial depolarizing effect was superceded by a repolarization despite ongoing application of GLU. This effect was proportional to the amount of G L U released. In 5 neurones the firing initially induced by G L U disappeared, whereas with current neutralization the neuronal discharge persisted throughout the application. The onset of the depolarizing response was generally slower in the neutralized mode of application. Application of negative current of the same magnitude out from the NaCl-filled barrel showed the same coupling artefact for 'on' and 'off' and had no effect on the membrane potential of the neurone. DISCUSSION Applying G L U to more 'dendritic' regions of spinal motoneurones resulted in a measurable depolarization which occurred earlier than when comparable amounts of the amino acid were administered to more 'somatic' regions. These findings suggest a very specific distribution of the receptive sites for G L U on these neurones, although a different number of receptors or a less sensitive population of receptors on the 'somatic' sites can not be ruled out by these experiments. N o information can be provided as to whether dendritic sites more remote than 350~/~m are also sensitive to
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Fig. 4. Effect of current neutralization upon the time course of GLU-induced depolarization. Superimposed pen-writer recordings of the membrane potential following GLU (100 nA/20 sec) applied with (solid line) and without (dotted line) current neutralization (Cal: 10 mV). The insert illustrates the mechanism. Note: the anionic current during GLU application can enter the cell more easily when GLU has elicited an increase in conductance causing a repolarization in spite of continued drug application (Ag). The slower time course of the depolarizing response with current neutralization is due to the absence of an electric field. The larger response and the longer lasting effect at drug offsetarise most likely from the greater extracellular drug concentration reached without neutralizing current, which draws drug ions into the neutralizing barrel.
102 GLU, because the response to the amounts of G L U applied became too small to allow a clear measurement of the onset of the depolarizing action. Employing identical phoretic currents, the responses evoked by 'dendritic' application were usually smaller. This is most likely due to the fact that a smaller area of the neuronal membrane is influenced by such an application. Although the functional role of dendritically located synapses is well established 1,3,10,12,13,16,17,19, the peripheral location of synapses 6 introduces factors which seriously complicate the current/voltage analyses of ionic mechanisms involved in synaptic transmission 4. Dependent on the site of application, various virtual equilibrium potentials for the drug effect were measured. As described previously 2 rather small amounts of G L U applied 'dendritically' were able to induce a marked depolarization usually not accompanied by a measurable change of the membrane conductance at the (somatic) recording site. In some cases the depolarizations, induced by GLU, led to an increase in membrane resistance, most probably attributable to anomalous rectification18, although no detailed analysis was made of this membrane behavior. Increased doses of G L U usually caused a proportional increase in conductance as previously described z2. In the present study current neutralization through an adjacent NaCl-filled barrel was employed in most neurones studied to avoid artefacts originating from extracellular polarization. Without current neutralization the initially depolarizing action of G L U fades despite ongoing administration. It may be assumed that, due to a locally increased membrane conductance elicited by GLU, the phoretic current (anionic, tip negative) presumably enters the cell more readily and causes this repolarization, superceding the initial depolarizing effect of the amino acid. Such an artifactual displacement of the membrane potential by the application current can only be avoided by current neutralization. Passing positive or negative current through an NaCl-filled barrel before or after a drug test is not an appropriate control because there is no drug-associated conductance increase shunting the phoretic current into the cell. This phenomenon can also be seen when G L U is applied 'dendritically', indicating that the permeability of the postsynaptic membrane at the application site is increased, but these conductance increases are usually not detected by the electrode which is most likely lodged in the soma of the cell. In contrast to GLU, GLY application led to a hyperpolarization associated with an increase in conductanceT,8,15,21 with a clearly shorter delay when it was administered closer to the soma rather than to the dendritic areas. These findings indicate a predominantly 'somatic' location of glycinergic receptive sites. From the comparison of the equilibrium potential of the action of GLY with the reversal potential of directly evoked and recurrent IPSPs it became obvious that the reversal potential of the orthodromically evoked IPSP is close to that measured for electrophoretically applied GLY. The reversal point of the recurrent IPSPs occurred at slightly more hyperpolarized values. Assuming identical inhibitory transmitters it might be speculated that only the receptors mediating direct inhibition are located on the soma, therefore being more readily reached by intracellular current injection. The above results suggest that in cat spinal motoneurones G L U receptive sites
103 are preferentially localized o n p r o x i m a l dendrites in a m a n n e r similar to that reported in P u r k i n j e cells 5 a n d h i p p o c a m p a l cells 20. G L Y receptive sites, o n the other hand, appear to have a more somatic localization. Such a preferential localization of receptive sites complicates the analysis o f the ionic m e c h a n i s m u n d e r l y i n g the actions of electrophoretically applied putative t r a n s m i t t e r s in the m a m m a l i a n CNS. Nevertheless, the data strongly indicate a site-specific role for these two a m i n o acids as n e u r o t r a n s m i t t e r s at these particular synapses. ACKNOWLEDGEMENTS The a u t h o r s are i n d e b t e d to Drs. A. Herz a n d H. D. Lux for valuable c o m m e n t s a n d criticism d u r i n g the p r e p a r a t i o n of this manuscript.
REFERENCES 1 Barrett, J. N. and Crill, W. E., Influence of dendritic location and membrane properties on the effectiveness of synapses on cat motoneurons, J. Physiol. (Lond.), 239 (1974) 325-346. 2 Bernardi, G., Zieglg/insberger, W., Herz, A. and Puil, E. A., Intracellular studies on the action of L-glutamic acid on spinal neurones of the cat, Brain Research, 39 (1972) 523-525. 3 Burke, R. E. and Ten Bruggencate, G., Electrotonic characteristics of alpha motoneurone of varying size, J. Physiol. (Lond.), 212 (1971) 1-20. 4 Calvin, W. H., Dendritic synapses and reversal potentials: theoretical implications of the view from the soma, Exp. Neurol., 24 (1969) 248-264. 5 Chujo, T., Yamada, Y. and Yamamoto, C., Sensitivity of Purkinje cell dendrites to glutamic acid, Exp. Brain Res., 23 (1975) 293-300. 6 Conradi, S., On motoneuron synaptology in adult cats, Acta physiol, scand., Suppl. 332 (1969) 5-85. 7 Curtis, D. R., H6sli, L. and Johnston, G. A. R., Inhibition of spinal neurones by glycine, Nature (Lond.), 215 (1967) 1502-1503. 8 Curtis, D. R., Htisli, L., Johnston, G. A. R. and Johnston, I. H., The hyperpolarisation of spinal motoneurones by glycine and related amino acids, Exp. Brain Res., 5 (1968) 235-258. 9 Curtis, D. R. and Johnston, G. A. R., Amino acid transmitters in the mammalian central nervous system, Ergebn. PhysioL, PhysioL Rev., 69 (1974) 98-188. 10 Edwards, F. R., Redman, S. J. and Walmsley, B., The effect of polarizing currents on unitary Ia excitatory post-synaptic potentials evoked in spinal motoneurones, J. PhysioL (Lond.), 259 (1976) 705-723. 11 Freeman, A. R., Polyfunctional role of glutamic acid in excitatory synaptic transmission, Progr. Neurobiol., 6 (1976). 12 Iansek, R. and Redman, S. J., The amplitude, time course and charge of unitary excitatory post synaptic potentials evoked in spinal motoneurone dendrites, J. PhysioL (Lond.), 234 (1973) 665--668. 13 Jack, J. J. B. and Redman, S. J., An electrical description of the motoneurone and its application to the analysis of synaptic potentials, J. PhysioL (Lond.), 215 (1971) 321-352. 14 Krnjevi6, K., Chemical nature of synaptic neurotransmission in vertebrates, Physiol. Rev., 54 (1974) 418-540. 15 Krnjevi6, K., Puil, E. and Werman, R., GABA and glycine actions on spinal motoneurones, Canad. J. PhysioL PharmacoL, 55 (1977) 658-669. 16 Lux, H. D., Schubert, P. and Kreutzberg, G. W., Direct matching of morphological and electrophysiological data in cat spinal motoneurones. In P. Andersen and J. K. S. Janse (Eds.), Excitatory Synaptic Mechanism, Universitetsforlaget, Oslo, 1970, pp. 189-198. 17 Lux, H. D. and Schubert, P., Some aspects of the electroanatomy of dendrites. In G. W. Kreutzberg (Ed.), Advance in Neurology, Vol. 12, Raven Press, New York, 1975, pp. 29-44. 18 Nelson, P. G. and Frank, K., Anomalous rectification in cat spinal motoneurones and the effect of polarizing currents on the excitatory postsynaptic potential, J. NeurophysioL, 130 (1967) 10971113.
104 19 Rail, W., Burke, R. E., Smith, T. G., Nelson, P. G. and Frank, K., Dendritic location of synapses and possible mechanisms for the monosynaptic EPSP in motoneurons, J. Neurophysiol., 30 (1967) 1169-1193. 20 Schwartzkroin, P. A. and Andersen, P., Glutamic acid sensitivity of dendrites in hippocampal slices in vitro. In G. W. Kreutzberg (Ed.), Advances in Neurology, Fol. 12, Raven Press, New York, 1975, pp. 45-51. 21 Werman, R., Davidoff, R. A. and Aprison, M. H., Inhibition of motoneurones by iontophoresis of glycine, Nature (Lond.), 214 (1967) 681-683. 22 Zieglg~insberger,W. and Puil, E. A., Actions of glutamic acid on spinal neurons, Exp. Brain Res., 17 (1973) 35-49.
95
CAT SPINAL MOTONEURONES EXHIBIT TOPOGRAPHIC SENSITIVITY TO G L U T A M A T E AND GLYCINE*
W. ZIEGLG,~NSBERGER and J. CHAMPAGNAT Max-Planck-lnstitut fiir Psychiatrie, Department of Neuropharmacology, 8 Munich 40 (G.F.R.) and Centre National de la Recherche Scientifique, Laboratoire de Physiologic nerveuse, 91190 Gif-surYvette (France)
(Accepted April 20th, 1978)
SUMMARY Spinal neurones from the 6th and 7th lumbar segments of cat were recolded intracellularly. L-Glutamate (GLU) and glycine (GLY) were preferentially applied to 'somatic' or 'dendritic' regions. To accomplish this, single micropipette electrodes for intracellular recording were assembled alongside drug-delivering micropipettes at varying distances from the recording tip. Microiontophoretic application of G L U to predominantly 'dendritic' sites led to a depolarizing response after a significantly shorter delay than applications to more 'somatic' sites. 'Dendritic' applications did not cause measurable increases in membrane conductance at the recording site. 'Somatic' application of high amounts of G L U caused a depolarization associated with a progressively increasing membrane conductance. Thus, GLU may increase membrane conductance at 'dendritic' sites, but this effect is hardly detectable by conventional resistance measurements at the soma membrane. Microiontophoretic application of GLY at 'dendritic' or 'somatic' sites produced hyperpolarizing responses with marked increases of membrane conductance. The onset latency of the response was significantly longer for 'dendritic' applications than for 'somatic' applications. Action potentials, EPSPs and IPSPs were all attenuated by shunting during the GLY (or GLU) increases of membrane conductance. The equilibrium potentials measured for the hyperpolarizing action of GLY (range: --75 to --82 mV) and for the orthodromically evoked IPSPs (range: --74 to --78 mV) were similar and were closer to the resting potential than antidromically evoked IPSPs (range: --80 to --84 mV). The specific distribution of excitatory glutaminergic and inhibitory glycinergic * A preliminaryreport was given at the Spring Meeting of the German PhysiologicalSociety, March 1977.
96 receptive sites on cat spinal motoneurones further supports a physiologic role for these amino acids as synaptic transmitters.
INTRODUCTION Both L-glutamate (GLU) and glycine (GLY) fulfill criteria establishing them as likely candidates for excitatory and inhibitory neurotransmitters in a variety of areas in the mammalian central nervous system (see refs. 9, 11 and 14). However, a major problem confronts the analysis of the ionic mechanism underlying the actions of putative transmitters in the mammalian CNS: neurones with complex shape are difficult to influence uniformly either by microiontophoretic application of a test compound or by intracellular current or ion injection. The aim of the present study was to determine whether there are predominant sites of receptivity to iontophoretically applied GLU and GLY on spinal motoneurones of the cat. A combination of intracellular recording and the microiontophoretic application to different regions of a neurone was employed to provide information about the predominant location of amino acid receptive sites on these neurones. METHODS The experiments were performed in 28 adult cats of either sex (body weight: 2.5-3.5 kg). Surgery was performed during pentobarbital anaesthesia (35 mg/kg, i.p.). The anaesthesia was continued by repeated intravenous injections of pentobarbital (5 mg/kg/h). After surgery the animals were immobilized with gallamine triethiodide and artificially respired. A bilateral pneumothorax was made routinely. Blood pressure was monitored continually and experiments were discontinued when blood pressure dropped below 80 mm Hg. The rectal temperature was maintained between 37.8 and 38.2 °C by means of a thermostatically controlled electric blanket and heat radiators. During the experiments plasma-expanders (2 ml/h) and small amounts of dextrose were given intravenously. The spinal cord was exposed by laminectomy at the lumbar-sacral level (L3-$2) and covered with a pool of paraffin oil kept at body temperature. The dura mater was reflected and the pia mater incised at the recording site to facilitate insertion of the electrode assembly. Strands dissected from the dorsal roots of the segments L5 and L6 were stimulated by means of bipolar electrodes. The ventral roots were stimulated in situ. The electrode assembly consisted of a single and two double- (or triple-) barrelled micropipettes fixed together at variable distances (see insert, Fig. 1) with fastsetting epoxy glue. Only the upper parts of the electrode shafts were fixed together, thus leaving 3 mm of the tip free. Electrode intertip distances ranged from 80 to 150 #m for the inner ('somatic') application and from 150 to 350 #m for the remote ('dendritic') application. The recording barrel was filled with a 10:1 mixture of potassium citrate (1.6 M) and KCI (1.0 M). The tip diameter of the recording barrel
97 was less than 1 #m; DC resistance ranged from 10 to 15 MfL Intracellular recordings and conductance measurements were performed using conventional techniques. Recordings were stored on tape for later analysis. The solutions used for microelectrophoretic application were as follows: Monosodium-L-glutamate (0.5 M, pH 8.0), glycine (0.5 M, pH 5.0) and NaC1 (1.0 M, pH 5.0). Current neutralization was routinely employed to eliminate current artifacts, and was assumed to be complete when the baseline showed no detectable deflection when the ejection currents were switched on. Currents (GLU was applied as an anion, GLY as a cation) were provided from constant current sources and could be preset in magnitude and duration of flow. The voltage necessary to drive the current was monitored continuously. Since oscillations of this voltage indicate that the ohmic resistance of the application electrode is unstable and that is unlikely to release consistent amounts of material, only applications in which the voltage remained stable were included in this study. RESULTS The present results were obtained from 82 spinal motoneurones recorded intracellularly from the 6th and 7th lumbar segments. They all showed stable membrane potentials greater than --65 mV and action potentials larger than 80 mV during the test period. Subsequently the drug applications from electrodes arranged at intertip distances less than 150 ~m will be referred to as 'somatic', whereas those from electrodes more remotely spaced will be termed 'dendritic' (see insert, Fig. 1).
(1) Actions of L-glutamate (GLU) When GLU was applied microiontophoretically to more 'dendritic' sites, the majority of cells started to be depolarized after a clearly shorter latency than after the 'somatic' applications. The graph in Fig. 1 (B) summarizes the mean latency of onset of the depolarizing effect obtained from 77 motoneurones employing electrodes staggered at distances of 80-260 #m. The phoretic currents (range: 10-100 nA) were adjusted so as to induce a depolarizing response of 3 mV or more in amplitude. In most neurones the applications were repeated 2-6 times and the latencies did not vary by more than 10-15 ~ in a given neurone. Part A of Fig. 1 shows a test with two drug delivery electrodes arranged at 80/zm and 225/zm distances from the recording tip respectively. The onset of depolarization following the 'somatic' application of GLU (80 nA) was clearly delayed when compared with that of the 'dendritic' application. Employing equal ejection currents the responses to 'somatic' application were usually delayed but larger in amplitude (see Fig. 1A) than those induced at 'dendritic' sites. The depolarizations recorded following 'dendritic' applications were usually not accompanied by a measurable increase in conductance at the recording site, presumably the motoneuron soma. In 8 out of 38 cells the membrane conductance even decreased slightly (5-15~) during the GLU-induced depolarizations (range: 5-30 mV). When the depolarization reached the firing level most cells started to fire
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DiStar,~(jum) Fig. 1. Different latencies of the depolarizingeffectof GLU applied to spinal motoneurones at varying distances from the soma. A: superimposed pen-writer recordings of the membrane potential following applications from electrode assemblies (see insert) arranged at 80 #m and 225/tm, intertip distance respectively. GLU (80 nA) was applied for 10 see (bar, time calibration) from either electrode. The depolarizing effectfollowingthe 'somatic' application was clearlydelayed(dotted line). The membrane potential shifts to values below control, following GLU (see ref. 22). B" results obtained from 77 motoneurones. The mean value of the latencies is given when more than one test was performed. The phoretic currents were adjusted to evoke depolarizingresponses of more than 3 mV in amplitude. repetitively. The overshoot of ortho- or antidromically evoked spikes was not detectably altered in amplitude. An increase in dosage up to currents of 200-500 nA led to marked depolarizations after delays of 5-15 sec, associated with progressive conductance increases. During these responses all spontaneous or evoked postsynaptic transients were strongly diminished. Such excessive conductance changes were only observed with intertip distances less than 120 #m. Fig. 2 compares current/voltage plots obtained with either 'dendritic' or 'somatic' application of GLU. When G L U (60 nA) was applied by the remote application electrode (intertip distance: 250/~m), the depolarization started after a delay less than 500 msec. No conductance change was observed during the maximal depolarization which was reached within approximately 4 sec. 'Somatic' application (intertip distance: 100 #m) of G L U (100 nA) caused an increase in the conductance of the postsynaptic membrane which plateaued after 10 sec. The intersection point for the current/voltage lines obtained during the maximal drug effect and the control was between zero and - - 1 0 mV. Comparable measurements were performed in 15 neurones. An approximation of the equilibrium potential for G L U was found between --28 and + 10 mV for 'somatic' applications employing currents from 80 to 500 nA from electrodes with intertip distances ranging from 85 to 150/zm.
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Fig. 2. Current/voltage plots obtained for GLU effectselicited at different distances from the recording site. During the application of GLU (100 hA) from the electrode closest to the recording site (100 #m, 'somatic'), the slope (triangles) measured at maximal depolarization (10 see after onset of the application) was found to be decreased. It intersected the control slope (dots) at membrane potential values between zero and --10 mV. Following application of GLU (60 hA) from the electrode arranged at a distance of 250 #m, no detectable change in membrane resistance was associated with the depolarizing effect which plateaued 4 see after onset of application (open squares). The slopes drawn were estimated. The symbols represent mean values from 5 consecutive measurements. Duration of intracellularly applied current pulses for resistance measurement was 20 msec. All measurements were performed during maximal depolarization. The electrode resistance remained constant during the measurements.
(2} Actions of glycine (GL Y) Tests comparable to those described for G L U were performed to investigate the site of action of microiontophoretically applied G L Y (5-100 nA) on spinal neurones. Fig. 3A illustrates the hyperpolarizing action of G L Y (50 nA) when it is applied either close to the soma (70/~m) or preferentially to the dendrites (200/~m) of a lumbar motoneurone. In contrast to G L U applications, G L Y induced a response with shorter latencies when it was applied closer to the soma. Fig. 3B summarizes results (mean latencies) obtained from 38 motoneurones. The phoretic currents were adjusted as to evoke hyperpolarizing responses of more than 3 mV in amplitude. The hyperpolarizations were invariably associated with a strong conductance increase of the postsynaptie membrane (see current/voltage plot in Fig. 3C). The action potentials evoked antidromicaUy (Fig. 3C, a ) a n d orthodromically as well as EPSPs and IPSPs were maximally attenuated by shunting. Comparable tests were performed in 14 neurones. The equilibrium potential for the hyperpolarizing action of G L Y for both 'somatic' and 'dendritic' application was found in the range of----75 to - - 8 2 mV. In these tests the equilibrium potential for the recurrent IPSP was found at slightly more hyperpolarized levels (range: - - 8 0 to - - 8 4 mV) than the equilibrium potential for the IPSPs evoked by dorsal rootlet stimulation (range: - - 7 4 to - - 7 8 mV). In a number of cells (12 out o f 26) a tendency was observed for repolarization to occur despite continuous
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-tO0 Nembrane potential(mY) Fig. 3. Different latencies of the hyperpolarizing effect of GLY applied to spinal motoneurones from varying distances. A: superimposed pen-writer recordings of the membrane potential following applications (bar, time calibration) of GLY (50 nA/10 sec) at different distances from the recording site (70 #m and 200/~m, resp.). The application from the electrode closer to the soma (solid line) caused an almost immediate onset of the hyperpolarization. B: results obtained from 38 motoneurones. The phoretic currents were adjusted to evoked hyperpolarizing responses of more than 3 mV in amplitude. The intertip distances of the electrodes varied from 60 to 300/~m. Mean values for the latency of the hyperpolarizing responses are given when more than one measurement was performed. C: the current/voltage plots shows a marked increase in the conductance of the postsynaptic membrane. The arrow indicates the intersection point (EGzy) of the slopes during GLY administration with the control (triangles). The open circles represent the 'somatic' and the dots represent the 'dendritic' application of GLY. Measurements were performed during maximal hyperpolarization (mean values out of 4 measurements). There is no obvious difference in the current/voltage characterestics following these different sites of application. The antidromically evoked action potential (C, a) is maximally attenuated during such GLY applications due to shunting of the neuronal membrane. Calibration: 1 msec; 20 mV. application of G L Y (Fig. 3A); this effect was e n h a n c e d by repeated applications of the a m i n o acid at short intervals ( < 30 see).
(3) Current 'artefacts' The shift in the baseline of the D C recording of the m e m b r a n e potential ('coupling') induced by the phoretic current is, without current neutralization,
101 proportional to the magnitude of the current and equal when switching on and off. Following G L U (applied as an anion, tip negative), however, the coupling artefact at the termination of the application increased in size (Fig. 4). The initial depolarizing effect was superceded by a repolarization despite ongoing application of GLU. This effect was proportional to the amount of G L U released. In 5 neurones the firing initially induced by G L U disappeared, whereas with current neutralization the neuronal discharge persisted throughout the application. The onset of the depolarizing response was generally slower in the neutralized mode of application. Application of negative current of the same magnitude out from the NaCl-filled barrel showed the same coupling artefact for 'on' and 'off' and had no effect on the membrane potential of the neurone. DISCUSSION Applying G L U to more 'dendritic' regions of spinal motoneurones resulted in a measurable depolarization which occurred earlier than when comparable amounts of the amino acid were administered to more 'somatic' regions. These findings suggest a very specific distribution of the receptive sites for G L U on these neurones, although a different number of receptors or a less sensitive population of receptors on the 'somatic' sites can not be ruled out by these experiments. N o information can be provided as to whether dendritic sites more remote than 350~/~m are also sensitive to
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Fig. 4. Effect of current neutralization upon the time course of GLU-induced depolarization. Superimposed pen-writer recordings of the membrane potential following GLU (100 nA/20 sec) applied with (solid line) and without (dotted line) current neutralization (Cal: 10 mV). The insert illustrates the mechanism. Note: the anionic current during GLU application can enter the cell more easily when GLU has elicited an increase in conductance causing a repolarization in spite of continued drug application (Ag). The slower time course of the depolarizing response with current neutralization is due to the absence of an electric field. The larger response and the longer lasting effect at drug offsetarise most likely from the greater extracellular drug concentration reached without neutralizing current, which draws drug ions into the neutralizing barrel.
102 GLU, because the response to the amounts of G L U applied became too small to allow a clear measurement of the onset of the depolarizing action. Employing identical phoretic currents, the responses evoked by 'dendritic' application were usually smaller. This is most likely due to the fact that a smaller area of the neuronal membrane is influenced by such an application. Although the functional role of dendritically located synapses is well established 1,3,10,12,13,16,17,19, the peripheral location of synapses 6 introduces factors which seriously complicate the current/voltage analyses of ionic mechanisms involved in synaptic transmission 4. Dependent on the site of application, various virtual equilibrium potentials for the drug effect were measured. As described previously 2 rather small amounts of G L U applied 'dendritically' were able to induce a marked depolarization usually not accompanied by a measurable change of the membrane conductance at the (somatic) recording site. In some cases the depolarizations, induced by GLU, led to an increase in membrane resistance, most probably attributable to anomalous rectification18, although no detailed analysis was made of this membrane behavior. Increased doses of G L U usually caused a proportional increase in conductance as previously described z2. In the present study current neutralization through an adjacent NaCl-filled barrel was employed in most neurones studied to avoid artefacts originating from extracellular polarization. Without current neutralization the initially depolarizing action of G L U fades despite ongoing administration. It may be assumed that, due to a locally increased membrane conductance elicited by GLU, the phoretic current (anionic, tip negative) presumably enters the cell more readily and causes this repolarization, superceding the initial depolarizing effect of the amino acid. Such an artifactual displacement of the membrane potential by the application current can only be avoided by current neutralization. Passing positive or negative current through an NaCl-filled barrel before or after a drug test is not an appropriate control because there is no drug-associated conductance increase shunting the phoretic current into the cell. This phenomenon can also be seen when G L U is applied 'dendritically', indicating that the permeability of the postsynaptic membrane at the application site is increased, but these conductance increases are usually not detected by the electrode which is most likely lodged in the soma of the cell. In contrast to GLU, GLY application led to a hyperpolarization associated with an increase in conductanceT,8,15,21 with a clearly shorter delay when it was administered closer to the soma rather than to the dendritic areas. These findings indicate a predominantly 'somatic' location of glycinergic receptive sites. From the comparison of the equilibrium potential of the action of GLY with the reversal potential of directly evoked and recurrent IPSPs it became obvious that the reversal potential of the orthodromically evoked IPSP is close to that measured for electrophoretically applied GLY. The reversal point of the recurrent IPSPs occurred at slightly more hyperpolarized values. Assuming identical inhibitory transmitters it might be speculated that only the receptors mediating direct inhibition are located on the soma, therefore being more readily reached by intracellular current injection. The above results suggest that in cat spinal motoneurones G L U receptive sites
103 are preferentially localized o n p r o x i m a l dendrites in a m a n n e r similar to that reported in P u r k i n j e cells 5 a n d h i p p o c a m p a l cells 20. G L Y receptive sites, o n the other hand, appear to have a more somatic localization. Such a preferential localization of receptive sites complicates the analysis o f the ionic m e c h a n i s m u n d e r l y i n g the actions of electrophoretically applied putative t r a n s m i t t e r s in the m a m m a l i a n CNS. Nevertheless, the data strongly indicate a site-specific role for these two a m i n o acids as n e u r o t r a n s m i t t e r s at these particular synapses. ACKNOWLEDGEMENTS The a u t h o r s are i n d e b t e d to Drs. A. Herz a n d H. D. Lux for valuable c o m m e n t s a n d criticism d u r i n g the p r e p a r a t i o n of this manuscript.
REFERENCES 1 Barrett, J. N. and Crill, W. E., Influence of dendritic location and membrane properties on the effectiveness of synapses on cat motoneurons, J. Physiol. (Lond.), 239 (1974) 325-346. 2 Bernardi, G., Zieglg/insberger, W., Herz, A. and Puil, E. A., Intracellular studies on the action of L-glutamic acid on spinal neurones of the cat, Brain Research, 39 (1972) 523-525. 3 Burke, R. E. and Ten Bruggencate, G., Electrotonic characteristics of alpha motoneurone of varying size, J. Physiol. (Lond.), 212 (1971) 1-20. 4 Calvin, W. H., Dendritic synapses and reversal potentials: theoretical implications of the view from the soma, Exp. Neurol., 24 (1969) 248-264. 5 Chujo, T., Yamada, Y. and Yamamoto, C., Sensitivity of Purkinje cell dendrites to glutamic acid, Exp. Brain Res., 23 (1975) 293-300. 6 Conradi, S., On motoneuron synaptology in adult cats, Acta physiol, scand., Suppl. 332 (1969) 5-85. 7 Curtis, D. R., H6sli, L. and Johnston, G. A. R., Inhibition of spinal neurones by glycine, Nature (Lond.), 215 (1967) 1502-1503. 8 Curtis, D. R., Htisli, L., Johnston, G. A. R. and Johnston, I. H., The hyperpolarisation of spinal motoneurones by glycine and related amino acids, Exp. Brain Res., 5 (1968) 235-258. 9 Curtis, D. R. and Johnston, G. A. R., Amino acid transmitters in the mammalian central nervous system, Ergebn. PhysioL, PhysioL Rev., 69 (1974) 98-188. 10 Edwards, F. R., Redman, S. J. and Walmsley, B., The effect of polarizing currents on unitary Ia excitatory post-synaptic potentials evoked in spinal motoneurones, J. PhysioL (Lond.), 259 (1976) 705-723. 11 Freeman, A. R., Polyfunctional role of glutamic acid in excitatory synaptic transmission, Progr. Neurobiol., 6 (1976). 12 Iansek, R. and Redman, S. J., The amplitude, time course and charge of unitary excitatory post synaptic potentials evoked in spinal motoneurone dendrites, J. PhysioL (Lond.), 234 (1973) 665--668. 13 Jack, J. J. B. and Redman, S. J., An electrical description of the motoneurone and its application to the analysis of synaptic potentials, J. PhysioL (Lond.), 215 (1971) 321-352. 14 Krnjevi6, K., Chemical nature of synaptic neurotransmission in vertebrates, Physiol. Rev., 54 (1974) 418-540. 15 Krnjevi6, K., Puil, E. and Werman, R., GABA and glycine actions on spinal motoneurones, Canad. J. PhysioL PharmacoL, 55 (1977) 658-669. 16 Lux, H. D., Schubert, P. and Kreutzberg, G. W., Direct matching of morphological and electrophysiological data in cat spinal motoneurones. In P. Andersen and J. K. S. Janse (Eds.), Excitatory Synaptic Mechanism, Universitetsforlaget, Oslo, 1970, pp. 189-198. 17 Lux, H. D. and Schubert, P., Some aspects of the electroanatomy of dendrites. In G. W. Kreutzberg (Ed.), Advance in Neurology, Vol. 12, Raven Press, New York, 1975, pp. 29-44. 18 Nelson, P. G. and Frank, K., Anomalous rectification in cat spinal motoneurones and the effect of polarizing currents on the excitatory postsynaptic potential, J. NeurophysioL, 130 (1967) 10971113.
104 19 Rail, W., Burke, R. E., Smith, T. G., Nelson, P. G. and Frank, K., Dendritic location of synapses and possible mechanisms for the monosynaptic EPSP in motoneurons, J. Neurophysiol., 30 (1967) 1169-1193. 20 Schwartzkroin, P. A. and Andersen, P., Glutamic acid sensitivity of dendrites in hippocampal slices in vitro. In G. W. Kreutzberg (Ed.), Advances in Neurology, Fol. 12, Raven Press, New York, 1975, pp. 45-51. 21 Werman, R., Davidoff, R. A. and Aprison, M. H., Inhibition of motoneurones by iontophoresis of glycine, Nature (Lond.), 214 (1967) 681-683. 22 Zieglg~insberger,W. and Puil, E. A., Actions of glutamic acid on spinal neurons, Exp. Brain Res., 17 (1973) 35-49.
