186
Brain Research, 172 (1979) 186-190 (~) Elsevier/North-HollandBiomedicalPress
Analysis and quantitative evaluation of the depressive effect of adenosine on evoked potentials in hippocampal slices
PETER SCHUBERT and ULLA MITZDORF Max Planck Institute for Psychiatry, Kraepelinstr. 2, 8000 Miinchen 40 ( G.F.R.)
(Accepted April 19th, 1979)
Adenosine has been shown to reduce spontaneous nerve cell firing in various areas of the brain when applied iontophoretically9 and to depress evoked potentials in slices of the olfactory cortexZ. Thus, the questions arise: may adenosine act as a neuronal signal, and can its effects be elicited at physiological concentrations ? These questions were approached experimentally in the hippocampal system. Here, an activity-related release of adenosine derivatives from afferent axon terminals 11,1z and a selective distribution of nucleotide-splitting enzymes were found which together seem to provide a dynamic control of the local extracellular adenosine levels is. The physiological average concentration is approximately given by the overall tissue content of free adenosine, measured to be 5-10 #M in the brain 1°. In the present study, we tested the effectiveness of such adenosine concentrations by using a hippocampal slice preparation and attempted to specify the site of action. On stimulation of the stratum radiatum afferents (SRA) by single test pulses, extracellular recordings were taken from the somal and/or the dendritic layer of CA1 neurons (see Fig. 1). The observed extracellular responses have been analyzed and identified by Andersen and coworkers 1,2, i.e. the somal response as population spike and the dendritic response as field EPSP. The latter is preceded by a fast potential, the presynaptic volley, which can be used for input control, i.e. for monitoring the number of afferent fibers activatedL In our first group of experiments (n --- t 5), we tested the effect of 1-20/~M adenosine on population spikes previously adjusted in normal medium to a size of about 2 mV. A significant depression of about 25 ?/o a: 7 (S.D.) was already observed in the presence of 2.5 #M adenosine (Fig. 1) reaching about 100~o with 20/~M adenosine. Since such a decrease in the evoked discharge of nerve ceils might be a secondary effect, we studied the influence of adenosine on the generation of synaptie potentials and recorded simultaneously from both, the somal and dendritic layers of CA 1. These experiments (n -- 7) revealed that the adenosine-induced depression of the population spike was always accompanied by a definite reduction in the amplitude of the field EPSP. The reduction can not be attributed to an adenosine effect on afferent fiber activation since it was found to occur at constant input strengths (Fig. 1). This is demonstrated
187
normal
*
Ads
5
FM
+ Ads 2.5/JM
normal
normal
÷ Ads 10 /uM
Fig. 1. Hippocampus slices, (400/~m), prepared from 200 g Sprague-Dawley rats, were maintained in a temperature-controlled (36 °C) chamber 6 and constantly superfused with oxygenated (normal) medium containing 124 mM NaC1, 3.3 mM KC1, 1.25 mM KH2PO4, 2.4 mM MgSO4, 2.5 mM CaC12, 25.7 mM NaHCOa and 10 mM glucose. A bipolar stimulating electrode and two extracellular recording electrodes were located as indicated. In each experiment when stable recordings were obtained, the effect of several concentrations of adenosine was tested, 15 min after the addition to the medium. A: recordings from the somal layer; reversible depression of the population spike by 2.5/~M adenosine. B: recordings from the dendritic layer; middle: reference field EPSP obtained in normal medium; left: depression of the EPSP amplitude by 15-20 % in 5/~M adenosine at constant input strength (unchanged fiber potentials, arrows); right: afferent stimulation has to be considerably increased in the presence of adenosine (enlarged fiber potential, arrow), to reach approximately the amplitude of the reference EPSP. Calibration: 5 msec, 1 mV.
o/
"~E 1"5'
~/a° o
! ~0"
/
a.
•
~
/j
"o ._o
i
//
1.0
o
o
o~O
1.0"
/.
//o
A~
A
3
.t~ e e ~ o
¢o
fiber potential
[mV]
~0
-
B
field
EPSP
[mV]
Fig. 2. A: input-output curves as determined successively in the presence of 5/~M adenosine (O), in normal medium (©), then in 10 #M adenosine (A), again followed by normal medium (A). In the demonstrated experiment, the depression of the field EPSP was 20 % in 5 #M, 80% in 10/~M adenosine and completely reversible. B: same experiment. Relation of field EPSP and evoked population spike in normal medium (©) and in 5 #M adenosine (O).
188 by input-output graphs in which the field EPSPs evoked at various stimulus intensities in the presence and absence of adenosine are related to the afferent volleys (Fig. 2A). Such a quantitative analysis was performed in 5 experiments. When comparing corresponding EPSPs elicited by afferent volleys of the same size, the EPSPs obtained in the presence of 2.5-10/~M adenosine were found in all experiments to be smaller than the corresponding EPSPs obtained in normal medium and the EPSP-saturation value could no longer be reached. The depression of the EPSP-amplitude was concentration-dependent and in the range of 8-12 ~ at 2.5 #M, 15-25 ~ at 5 #M, and 2 5 - 8 0 ~ at 10 /~M adenosine. Since the absolute amount of depression became continuously larger with increasing input volleys (see Fig. 2A) it appears to be proportional to the number of activated fibers. These data are consistent with the interpretation that adenosine exerts its depressive effect by acting at the individual synapse. Supporting evidence that adenosine interferes primarily with the generation of synaptic currents was obtained from a current source density analysis (Fig. 3). This technique allows one to demonstrate rather precisely the size and spatial distribution of currents flowing into and out of the neurons during activation s. Extracellular potentials elicited by radiatum stimulation in the presence and absence of adenosine were recorded along the whole extension of the CA1 neurons and current fluxes were normal medium
0
',5 p M adenosine
i.
i. . . . .
i .2
i2
.3
-
.4
.5 L CA I
jb
.6
mm
I "E f-
I
I
-
'
t 10
msec
._•INK
OURCE
Fig. 3. Localization of adenosine effects by a current source density analysis in the CA 1 area on stimulation of stratum radiatum afferents ($RA). Paired pulse stimulation leading to frequency potentiation was used here to demonstrate both the pure synaptic events together with those accompanying cell firing. Left: in normal medium, large EPSP sinks (inward currents), are evoked in the synaptic region by the first subthreshold pulse (at position 0.25-0.5 ram). The more effective second pulseinitiates an additional high amplitude sink of short durationin thesoma region (at position 0.55-0.65 mm) reflecting the net inward current which flows during the generation of action potentials. Right: in the presenceof 5/~M adenosine, the EPSP sinks are drastically reduced and the somal sinks absent.
189 calculated as previously described 7. In the dendritic layer, where the stimulated afferent fibers terminate, a well defined c u r r e n t sink can be localized, p r o b a b l y representing the t r a n s m i t t e r - m e d i a t e d synaptic inward current. This synaptic current is clearly seen to be depressed in the presence o f adenosine (Fig. 3). The c o n c o m i t a n t depression o f evoked nerve cell firing can be explained as a s e c o n d a r y effect resulting from the observed deficit in synaptic activation. This is s u p p o r t e d by an analysis o f the relation between EPSP a n d p o p u l a t i o n spike (see Fig. 2B). In the presence o f adenosine, the EPSP a m p l i t u d e s necessary for p r o d u c i n g defined p o p u l a t i o n spikes were the same (3 cases) or even slightly smaller (4 cases, Fig. 2B) than in n o r m a l m e d i u m , but in no experiment increased. W e conclude t h a t the basic action o f adenosine is n o t to p r o d u c e a general p o s t s y n a p t i c m e m b r a n e depression a n d decreased n e u r o n a l excitability, but to interfere selectively with the mechanism o f synaptic transmission. Here, also in view o f the r e p o r t e d evidence t h a t adenosine reduces t r a n s m i t t e r release 3,4,14, a presynaptic action has to be considered. Since the a d e n o s i n e - m e d i a t e d depression o f evoked potentials was present at the expected physiological concentrations, quickly reversible, a n d c o n c e n t r a t i o n - d e p e n d ent, adenosine m a y function as a m o d u l a t o r y n e u r o n a l signal exerting a tonic a n d locally adjustable influence on the efficacy o f synaptic transmission. Such a feedback depression by adenosine m a y serve in the h i p p o c a m p u s as an internal stabilizing mechanism for controlling the powerful signal amplification ~ in t h a t system.
1 Andersen, P., Bliss, T. V. P. and Skrede, K. K., Unit analysis of hippocampal population spikes, Exp. Brain Res., 13 (1971)208-221. 2 Andersen, P., Sundberg, S. H., Sveen, D. and Wigstrbm, H., Specific long-lasting potentiation of synaptic transmission in hippocampal slices, Nature (Land.), 266 (1977) 736-737. 3 Clanachan, A. S., Johns, A. and Paton, D. M., Presynaptic inhibitory actions of adenine nucleotides and adenosine on neurotransmission in the rat vas deferens., Neuroseience, 2 (1977) 597-602. 4 Harms, H. H., Wardeh, G. and Mulder, A. H., Adenosine modulates depolarization-induced release of 3H-noradrenaline from slices of rat brain neocortex, Earop. J. Pharmacol., 49 (1978) 305-308. 5 Kuroda, Y., Saito, M. and Kobayashi, K., Concomitant changes in cyclic AMP level and postsynaptic potentials of olfactory cortex slices induced by adenosine derivatives, Brain Research, 109 (1976) 196-201. 6 Lynch, G., Smith, R. L., Browning, M. P. and Deadwyler, S., Evidence for bidirectional dendritic transport of horseradish peroxydase. In G. W. Kreutzberg (Ed.), Advances in Neurology, Vol. 12, Raven Press, New York, 1975, pp. 297-313. 7 Mitzdorf, U. and Singer, W., Prominent excitatory pathways in the ca t visual cortex (A 17 and A 18) : a current source density analysis of electrically evoked potentials, Exp. Brain Res., 33 (1978) 371-394. 8 Nicholson, C. and Freeman, J. A., Theory of current source-density analysis and determination of conductivity tensor for Anuran cerebellum, J. Neurophysiol., 38 (1975) 356-368. 9 Phillis, J. W. and Kostopoulos, G. K., Adenosine as a putative transmitter in the cerebral cortex, Life Sci., 19 (1975) 1085-1094. 10 Rubio, R., Berne, R. M. and Winn, H. R., Production, metabolism and possible functions of adenosine in brain tissue in situ. In Cerebral Vascular Smooth Muscle and its Control, Ciba Found. Syrup., Elsevier, Amsterdam 1978, pp. 355-378. 11 Schubert, P., Lee, K., West, M., Deadwyler, S. and Lynch, G., Stimulation-de!cendent release of :~H-adenosine derivatives from central axon terminals to target neurones, Nature (Lond.), 260 (1976) 541-542. 12 Schubert, P., Rose, G., Lee, K., Lynch, G. and Kreutzberg, G. W., Axonal release and transfer
190 of nucleoside derivatives in the entorhinal-hippocampal system: an autoradiographi¢ study, Brain Research, 134 (i977) 347-352. 13 Schubert, P., Komp, W. and Kreutzberg, G. W., Correlation of 5'-nucleotidaseactivity and selective transnouronal transfer of adenosine in the hippocampus, Brain Research, (1979) in press. 14 Vizi, E. S. and Knoll, J., The inhibitory effect of adenosine and related nucleotidcs onthe release of acetylcholine, Neuroscience, 1 (1976) 391-398.
Brain Research, 172 (1979) 186-190 (~) Elsevier/North-HollandBiomedicalPress
Analysis and quantitative evaluation of the depressive effect of adenosine on evoked potentials in hippocampal slices
PETER SCHUBERT and ULLA MITZDORF Max Planck Institute for Psychiatry, Kraepelinstr. 2, 8000 Miinchen 40 ( G.F.R.)
(Accepted April 19th, 1979)
Adenosine has been shown to reduce spontaneous nerve cell firing in various areas of the brain when applied iontophoretically9 and to depress evoked potentials in slices of the olfactory cortexZ. Thus, the questions arise: may adenosine act as a neuronal signal, and can its effects be elicited at physiological concentrations ? These questions were approached experimentally in the hippocampal system. Here, an activity-related release of adenosine derivatives from afferent axon terminals 11,1z and a selective distribution of nucleotide-splitting enzymes were found which together seem to provide a dynamic control of the local extracellular adenosine levels is. The physiological average concentration is approximately given by the overall tissue content of free adenosine, measured to be 5-10 #M in the brain 1°. In the present study, we tested the effectiveness of such adenosine concentrations by using a hippocampal slice preparation and attempted to specify the site of action. On stimulation of the stratum radiatum afferents (SRA) by single test pulses, extracellular recordings were taken from the somal and/or the dendritic layer of CA1 neurons (see Fig. 1). The observed extracellular responses have been analyzed and identified by Andersen and coworkers 1,2, i.e. the somal response as population spike and the dendritic response as field EPSP. The latter is preceded by a fast potential, the presynaptic volley, which can be used for input control, i.e. for monitoring the number of afferent fibers activatedL In our first group of experiments (n --- t 5), we tested the effect of 1-20/~M adenosine on population spikes previously adjusted in normal medium to a size of about 2 mV. A significant depression of about 25 ?/o a: 7 (S.D.) was already observed in the presence of 2.5 #M adenosine (Fig. 1) reaching about 100~o with 20/~M adenosine. Since such a decrease in the evoked discharge of nerve ceils might be a secondary effect, we studied the influence of adenosine on the generation of synaptie potentials and recorded simultaneously from both, the somal and dendritic layers of CA 1. These experiments (n -- 7) revealed that the adenosine-induced depression of the population spike was always accompanied by a definite reduction in the amplitude of the field EPSP. The reduction can not be attributed to an adenosine effect on afferent fiber activation since it was found to occur at constant input strengths (Fig. 1). This is demonstrated
187
normal
*
Ads
5
FM
+ Ads 2.5/JM
normal
normal
÷ Ads 10 /uM
Fig. 1. Hippocampus slices, (400/~m), prepared from 200 g Sprague-Dawley rats, were maintained in a temperature-controlled (36 °C) chamber 6 and constantly superfused with oxygenated (normal) medium containing 124 mM NaC1, 3.3 mM KC1, 1.25 mM KH2PO4, 2.4 mM MgSO4, 2.5 mM CaC12, 25.7 mM NaHCOa and 10 mM glucose. A bipolar stimulating electrode and two extracellular recording electrodes were located as indicated. In each experiment when stable recordings were obtained, the effect of several concentrations of adenosine was tested, 15 min after the addition to the medium. A: recordings from the somal layer; reversible depression of the population spike by 2.5/~M adenosine. B: recordings from the dendritic layer; middle: reference field EPSP obtained in normal medium; left: depression of the EPSP amplitude by 15-20 % in 5/~M adenosine at constant input strength (unchanged fiber potentials, arrows); right: afferent stimulation has to be considerably increased in the presence of adenosine (enlarged fiber potential, arrow), to reach approximately the amplitude of the reference EPSP. Calibration: 5 msec, 1 mV.
o/
"~E 1"5'
~/a° o
! ~0"
/
a.
•
~
/j
"o ._o
i
//
1.0
o
o
o~O
1.0"
/.
//o
A~
A
3
.t~ e e ~ o
¢o
fiber potential
[mV]
~0
-
B
field
EPSP
[mV]
Fig. 2. A: input-output curves as determined successively in the presence of 5/~M adenosine (O), in normal medium (©), then in 10 #M adenosine (A), again followed by normal medium (A). In the demonstrated experiment, the depression of the field EPSP was 20 % in 5 #M, 80% in 10/~M adenosine and completely reversible. B: same experiment. Relation of field EPSP and evoked population spike in normal medium (©) and in 5 #M adenosine (O).
188 by input-output graphs in which the field EPSPs evoked at various stimulus intensities in the presence and absence of adenosine are related to the afferent volleys (Fig. 2A). Such a quantitative analysis was performed in 5 experiments. When comparing corresponding EPSPs elicited by afferent volleys of the same size, the EPSPs obtained in the presence of 2.5-10/~M adenosine were found in all experiments to be smaller than the corresponding EPSPs obtained in normal medium and the EPSP-saturation value could no longer be reached. The depression of the EPSP-amplitude was concentration-dependent and in the range of 8-12 ~ at 2.5 #M, 15-25 ~ at 5 #M, and 2 5 - 8 0 ~ at 10 /~M adenosine. Since the absolute amount of depression became continuously larger with increasing input volleys (see Fig. 2A) it appears to be proportional to the number of activated fibers. These data are consistent with the interpretation that adenosine exerts its depressive effect by acting at the individual synapse. Supporting evidence that adenosine interferes primarily with the generation of synaptic currents was obtained from a current source density analysis (Fig. 3). This technique allows one to demonstrate rather precisely the size and spatial distribution of currents flowing into and out of the neurons during activation s. Extracellular potentials elicited by radiatum stimulation in the presence and absence of adenosine were recorded along the whole extension of the CA1 neurons and current fluxes were normal medium
0
',5 p M adenosine
i.
i. . . . .
i .2
i2
.3
-
.4
.5 L CA I
jb
.6
mm
I "E f-
I
I
-
'
t 10
msec
._•INK
OURCE
Fig. 3. Localization of adenosine effects by a current source density analysis in the CA 1 area on stimulation of stratum radiatum afferents ($RA). Paired pulse stimulation leading to frequency potentiation was used here to demonstrate both the pure synaptic events together with those accompanying cell firing. Left: in normal medium, large EPSP sinks (inward currents), are evoked in the synaptic region by the first subthreshold pulse (at position 0.25-0.5 ram). The more effective second pulseinitiates an additional high amplitude sink of short durationin thesoma region (at position 0.55-0.65 mm) reflecting the net inward current which flows during the generation of action potentials. Right: in the presenceof 5/~M adenosine, the EPSP sinks are drastically reduced and the somal sinks absent.
189 calculated as previously described 7. In the dendritic layer, where the stimulated afferent fibers terminate, a well defined c u r r e n t sink can be localized, p r o b a b l y representing the t r a n s m i t t e r - m e d i a t e d synaptic inward current. This synaptic current is clearly seen to be depressed in the presence o f adenosine (Fig. 3). The c o n c o m i t a n t depression o f evoked nerve cell firing can be explained as a s e c o n d a r y effect resulting from the observed deficit in synaptic activation. This is s u p p o r t e d by an analysis o f the relation between EPSP a n d p o p u l a t i o n spike (see Fig. 2B). In the presence o f adenosine, the EPSP a m p l i t u d e s necessary for p r o d u c i n g defined p o p u l a t i o n spikes were the same (3 cases) or even slightly smaller (4 cases, Fig. 2B) than in n o r m a l m e d i u m , but in no experiment increased. W e conclude t h a t the basic action o f adenosine is n o t to p r o d u c e a general p o s t s y n a p t i c m e m b r a n e depression a n d decreased n e u r o n a l excitability, but to interfere selectively with the mechanism o f synaptic transmission. Here, also in view o f the r e p o r t e d evidence t h a t adenosine reduces t r a n s m i t t e r release 3,4,14, a presynaptic action has to be considered. Since the a d e n o s i n e - m e d i a t e d depression o f evoked potentials was present at the expected physiological concentrations, quickly reversible, a n d c o n c e n t r a t i o n - d e p e n d ent, adenosine m a y function as a m o d u l a t o r y n e u r o n a l signal exerting a tonic a n d locally adjustable influence on the efficacy o f synaptic transmission. Such a feedback depression by adenosine m a y serve in the h i p p o c a m p u s as an internal stabilizing mechanism for controlling the powerful signal amplification ~ in t h a t system.
1 Andersen, P., Bliss, T. V. P. and Skrede, K. K., Unit analysis of hippocampal population spikes, Exp. Brain Res., 13 (1971)208-221. 2 Andersen, P., Sundberg, S. H., Sveen, D. and Wigstrbm, H., Specific long-lasting potentiation of synaptic transmission in hippocampal slices, Nature (Land.), 266 (1977) 736-737. 3 Clanachan, A. S., Johns, A. and Paton, D. M., Presynaptic inhibitory actions of adenine nucleotides and adenosine on neurotransmission in the rat vas deferens., Neuroseience, 2 (1977) 597-602. 4 Harms, H. H., Wardeh, G. and Mulder, A. H., Adenosine modulates depolarization-induced release of 3H-noradrenaline from slices of rat brain neocortex, Earop. J. Pharmacol., 49 (1978) 305-308. 5 Kuroda, Y., Saito, M. and Kobayashi, K., Concomitant changes in cyclic AMP level and postsynaptic potentials of olfactory cortex slices induced by adenosine derivatives, Brain Research, 109 (1976) 196-201. 6 Lynch, G., Smith, R. L., Browning, M. P. and Deadwyler, S., Evidence for bidirectional dendritic transport of horseradish peroxydase. In G. W. Kreutzberg (Ed.), Advances in Neurology, Vol. 12, Raven Press, New York, 1975, pp. 297-313. 7 Mitzdorf, U. and Singer, W., Prominent excitatory pathways in the ca t visual cortex (A 17 and A 18) : a current source density analysis of electrically evoked potentials, Exp. Brain Res., 33 (1978) 371-394. 8 Nicholson, C. and Freeman, J. A., Theory of current source-density analysis and determination of conductivity tensor for Anuran cerebellum, J. Neurophysiol., 38 (1975) 356-368. 9 Phillis, J. W. and Kostopoulos, G. K., Adenosine as a putative transmitter in the cerebral cortex, Life Sci., 19 (1975) 1085-1094. 10 Rubio, R., Berne, R. M. and Winn, H. R., Production, metabolism and possible functions of adenosine in brain tissue in situ. In Cerebral Vascular Smooth Muscle and its Control, Ciba Found. Syrup., Elsevier, Amsterdam 1978, pp. 355-378. 11 Schubert, P., Lee, K., West, M., Deadwyler, S. and Lynch, G., Stimulation-de!cendent release of :~H-adenosine derivatives from central axon terminals to target neurones, Nature (Lond.), 260 (1976) 541-542. 12 Schubert, P., Rose, G., Lee, K., Lynch, G. and Kreutzberg, G. W., Axonal release and transfer
190 of nucleoside derivatives in the entorhinal-hippocampal system: an autoradiographi¢ study, Brain Research, 134 (i977) 347-352. 13 Schubert, P., Komp, W. and Kreutzberg, G. W., Correlation of 5'-nucleotidaseactivity and selective transnouronal transfer of adenosine in the hippocampus, Brain Research, (1979) in press. 14 Vizi, E. S. and Knoll, J., The inhibitory effect of adenosine and related nucleotidcs onthe release of acetylcholine, Neuroscience, 1 (1976) 391-398.
