Brain Research, 513 (1990) 149-151 Elsevier
149
BRES 24019
Evidence that NMDA receptors contribute to synaptic function in the guinea pig medial vestibular nucleus Paul F. Smith 1, Cynthia L. Darlington 2 and John I. Hubbard 2 Departments of 1Psychology and 2physiology and the Neuroscience Research Centre, University of Otago, Dunedin (New Zealand) (Accepted 5 December 1989) Key words: Vestibular nucleus; N-MethyI-D-aspartate Single medial vestibular nucleus neurons were recorded from guinea pig brainstem slices in vitro while superfusing with the selective N-methyl-D-aspartate (NMDA) antagonists, MK801 and CPP. The majority of neurons tested showed a decrease in firing rate in response to these NMDA antagonists, suggesting that NMDA receptors may contribute to the resting activity of MVN neurons. The N-methyl-D-aspartate (NMDA) receptor is a type of glutamate receptor which has been implicated in many different types of CNS plasticity, in particular long-term potentiation in the hippocampus and neocortex 1. However, binding studies show that N M D A receptors also exist in some subcortical areas of the CNS 11, and physiological studies suggest that in at least some of these areas the N M D A receptor contributes to normal synaptic function7,12,15. There is increasing evidence that excitatory amino acids are one of the major classes of neurotransmitter used in synapses in the brainstem vestibular nucleus: biochemical studies suggest that the neurotransmitter used by the VIIIth nerve is glutamate or aspartate 14, that there are glutamate binding sites in all 4 vestibular nuclei 17 and specifically N M D A binding sites in the medial vestibular nuclei (MVN) 11. However while it has been demonstrated physiologically that the N M D A receptor contributes to normal synaptic function in the vestibular nucleus of the frog 7, there is little evidence to suggest this is the case in mammalian species 8. Here we report that the potent and specific N M D A receptor/ channel antagonists (3-((+)-2-carboxypiperazin-4-yl)propyl-l-phosphonic acid) (CPP) 5 and ({(+)-5-methyl10,11-dihydro-5H-dibenzo- [a,d]-cyclohepten-5,10-imine maleate}) (MK801) 18, when applied to guinea pig brainstem slices in vitro using superfusion, produce a decrease in the firing rate of the majority of medial vestibular nucleus (MVN) neurons tested. Six labyrinthine-intact guinea pigs were etherized and their brainstems quickly removed to chilled artificial cerebrospinal fluid (ACSF). Coronal slices (600-1000/~m thick) were cut by hand with a chilled razor blade,
incubated in a slice chamber and superfused with a standard ACSF (in mM): NaCI 126.0, KCI 5.0, KH2PO 4 1.25, MgSO 4 1.3, N a H C O 3 26.0, glucose 10.0, CaCI 2 2.5, Phenol red 0.05% 9. The ACSF was continuously bubbled with 95% 0 2 and 5% CO 2 and maintained at a pH of 7.4 and a temperature of 35-37 °C 9. Single neurons within the MVN were recorded extracellularly with glass microelectrodes (4-8 M~2 impedance) filled with 2 M NaCI and Fast green FCF dye, to facilitate visualization of the electrode position. The MVN could be identified by its proximity to the sulcus limitans when looking at the dorsal aspect of the brainstem during brainstem dissection and by its proximity to the IVth ventricle when looking at the coronal slice in the slice chamber. Baseline firing rate was recorded while the neuron was superfused with the ACSF solution (control), then a second ACSF solution (test solution) containing either MK801 (10 -6 M) or CPP (10 -8 M) was turned on, followed by a return to the control solution. Some slices were also superfused with dopamine (DA) dissolved in ACSF (10 -8 M), used as a control substance to determine if the addition of any substance to the ACSF would produce the same effect as the N M D A antagonists. The test solution was normally turned on for 4 min (bath turnover time was 1.5 min), except where the cell stopped firing altogether, in which case the control solution was turned on again immediately. Firing frequency was analysed on-line using an Ortec time-histogram analyser and histograms were plotted using an Apple IIe microcomputer. Firing rate was considered to have increased or decreased from baseline when a change of greater than or equal to 20% occurred 2. Eight out of 10 MVN neurons tested with 10-6 M
Correspondence: P.F. Smith, Department of Psychology, University of Otago, Dunedin, New Zealand. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
150
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MK801 showed a decrease in firing rate from baseline, 1 showed an increase, and 1 showed no change (see Fig. 1). The average percentage decrease from baseline was 59.1 (+ 30.2 S.D.)% (n = 8). Of 10 MVN neurons tested with 10-8 M CPP, 6 showed a decrease in firing rate and 4 showed no change (see Fig. 1). The average percentage decrease from baseline was 46.0 (+ 21.4)% (n = 6). Fig. 2 shows representative examples of the effect of MK801 and CPP on MVN neurons. Of 5 MVN neurons tested with 10-8 M D A , none showed a 20% or greater change from baseline. These results suggest that the N M D A receptor contributes to the resting activity of guinea pig MVN neurons in vitro. Our previous studies on brainstem slices using high Mg 2+ A C S F indicate that a large component of the activity recorded from MVN neurons in vitro is synaptically generated 2"3, therefore it is probable that the N M D A receptor is one of the r e c e p t o r types on MVN neurons which contributes to this synaptic activity. Since in the present study the entire coronal slice was superfused with the N M D A antagonist, the possibility cannot be excluded that the changes in firing rate observed in MVN neurons were due not to the blockade of N M D A receptors on M V N neurons themselves, but to effects on N M D A receptors in other areas of the slice. However, this possibility seems unlikely since binding studies suggest that the reticular formation beneath the vestibular nuclei has few N M D A receptors11; although the inferior olive has a density of N M D A receptors which is comparable to the MVN ]1, this structure was present only on the more caudal slices that we used. Physiological evidence that N M D A receptors contribute to normal synaptic function in the vestibular nucleus was first presented by Knopfei 7, using the frog. Recently, Lewis et al. 8 have reported that N M D A , iontophoresed onto MVN neurons in the rat brainstem slice in vitro, causes depolarization which is blocked by the N M D A antagonist D-APV. Behavioral evidence presented by De
u
100 200 3;0 4;0 TIME (SEC)
CPP
Fig. 1. Percent of medial vestibular nucleus (MVN) neurons tested showing greater than or equal to 20% increase or decrease in firing, or no change in response to 10-6 M MK801 (n = 10) or 10-s M CPP (n = 10).
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.......,-.-...
=,..~===•..,=. •
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4;0 6;0 TIME (SEC) Fig. 2. Examples of the effect of 10 -6 M MK801 (A) or 10 8 M CPP (B) on resting activity for a single medial vestibular nucleus neuron. Points represent successive 10 s bins of average resting activity for the one neuron. Arrows indicate the onset of the test solution and the return to the control solution (C).
Waele et al. ]9 also suggests that N M D A receptors may be active in the normal vestibular nucleus: unilateral infusion of D-APV into the vestibular nucleus of labyrinthineintact guinea pigs induced postural and ocular motor symptoms similar to unilateral labyrinthectomy. There is now considerable evidence that the N M D A receptor contributes to normal synaptic function in other areas of the CNS, such as the visual cortex 4J°, thalamus ~s and spinal cord 12'16. In some of these cases the N M D A receptor appears to contribute to normal sensory processing4,]ojs,16. With respect to the function of N M D A receptors in the vestibular nucleus, it is interesting to note that in many CNS neurons N M D A and glycine receptors are codistributed and that glycine receptor activation may increase the affinity of the N M D A receptor for glutamate 6. Since there is evidence that brainstem commissural inhibition between the bilateral MVN is partially mediated by glyeine 13, it may be that N M D A and glycine receptors are co-distributed on MVN neurons and that the balance of activity between these two receptor types is important for the integration of input from the two labyrinths. This research was supported by the New Zealand Medical Research Council and the Neurological Foundation of New Zealand.
151
1 Cotman, C.W. and Iversen, L.L., Excitatory amino acids in the brain-focus on NMDA receptors, Trends Neurosci., 10 (1987) 263-265. 2 Darlington, C.L. and Smith, P.E, Neuropharmacological investigations of vestibular compensation. In A. Berthoz, W. Graf and P.P. Vidal (Eds.), The Head-Neck Sensory Motor System, Wiley, Chichester, in press. 3 Darlington, C.L., Smith, P.E and Hubbard, J.I., Neuronal activity in the guinea pig medial vestibular nucleus in vitro following chronic unilateral labyrinthectomy, Neurosci. Left., 105 (1989) 143-148. 4 Fox, K., Sato, H. and Daw, N., The location and function of NMDA receptors in cat and kitten visual cortex, J. Neurosci., 9 (1989) 2443-2454. 5 Harris, E.W., Ganong, A.H., Monaghan, D.T., Watkins, J.C. and Cotman, C.W., Action of (3-(+-)-2-carhoxypiperazin-4yl)-propyl-l-phosphonic acid) (CPP): a new and highly potent antagonist of the N-methyl-o-aspartate receptor in the hippocampus, Brain Research, 382 (1986) 174-177. 6 Johnson, J.W. and Ascher, P., Glycine potentiates the NMDA response in cultured mouse brain neurons, Nature (Lond.), 325 (1987) 529-531. 7 Knopfel, T., Evidence for N-methyl-o-aspartic acid mediated modulation of the comrnissural input to central vestibular neurons of the frog, Brain Research, 426 (1987) 212-224. 8 Lewis, M.R., Phelan, K.D., Shinnick-Gallagher, E and Gallagher, J.E, Primary afferent excitatory transmission recorded intracellulary in vitro from rat medial vestibular neurons, Synapse, 3 (1989) 149-153. 9 Llinas, R. and Sugimori, M., Electrophysiological properties of in vitro Purkinje cell somata in mammalian cerebellar slices, J. Physiol. (Lond.), 305 (1980) 171-195. 10 Miller, K.D., Chapman, B. and Stryker, M.P., Visual responses
in adult cat visual cortex depend on N-methyl-D-aspartate receptors, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 5183-5187. 11 Monaghan, D.T. and Cotman, C.W., Distribution of N-methylo-aspartate sensitive L-[3H]glutamate binding sites in rat brain, J. Neurosci., 5 (1985) 2909-2919. 12 Polc, P., NMDA receptors mediate background and excessive activity of y motoneurons in the spinal cord, Eur. J. Pharmacol., 144 (1987) 113-118. 13 Precht, W., Schwindt, P.C. and Baker, R., Removal of vestibular commissural inhibition by antagonists of GABA and glycine, Brain Research, 62 (1973) 222-226. 14 Raymond, J., Nieoullon, A., Dememes, D. and Sans, A., Evidence for glutamate as a neurotransmitter in the cat vestibular nerve: radioautographic and biochemical studies, Exp. Brain Res., 56 (1984) 523-531. 15 Salt, T.E., Mediation of thalamic sensory input by both NMDA receptors and non-NMDA receptors, Nature (Lond.), 322 (1986) 263-265. 16 Stein, ES.G. and Schild, C.E, N-methyl-D-aspartate antagonist applied to the spinal cord hindlimb enlargement reduces the amplitude of flexion reflex in the turtle, Brain Research, 479 (1989) 379-383. 17 Touati, J., Raymond, J. and Dememes, D., Quantitative autoradiographic characterization of L-3H glutamate binding sites in rat vestibular nuclei, Exp. Brain Res., 76 (1989) 646-650. 18 Wong, E.H.E, Kemp, J.A., Priestley, T., Knight, A.R. and Woodruff, G.N., The anticonvulsant MK801 is a potent Nmethyl-D-aspartate antagonist, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 7104-7108. 19 De Waele, C., Vihert, N., Baudrirnont, M. and Vidal, P.E, NMDA receptors contribute to the resting discharge of the vestibular neurons in the normal and hemilabyrinthectomized guinea pig, Exp. Brain Res., in press.
149
BRES 24019
Evidence that NMDA receptors contribute to synaptic function in the guinea pig medial vestibular nucleus Paul F. Smith 1, Cynthia L. Darlington 2 and John I. Hubbard 2 Departments of 1Psychology and 2physiology and the Neuroscience Research Centre, University of Otago, Dunedin (New Zealand) (Accepted 5 December 1989) Key words: Vestibular nucleus; N-MethyI-D-aspartate Single medial vestibular nucleus neurons were recorded from guinea pig brainstem slices in vitro while superfusing with the selective N-methyl-D-aspartate (NMDA) antagonists, MK801 and CPP. The majority of neurons tested showed a decrease in firing rate in response to these NMDA antagonists, suggesting that NMDA receptors may contribute to the resting activity of MVN neurons. The N-methyl-D-aspartate (NMDA) receptor is a type of glutamate receptor which has been implicated in many different types of CNS plasticity, in particular long-term potentiation in the hippocampus and neocortex 1. However, binding studies show that N M D A receptors also exist in some subcortical areas of the CNS 11, and physiological studies suggest that in at least some of these areas the N M D A receptor contributes to normal synaptic function7,12,15. There is increasing evidence that excitatory amino acids are one of the major classes of neurotransmitter used in synapses in the brainstem vestibular nucleus: biochemical studies suggest that the neurotransmitter used by the VIIIth nerve is glutamate or aspartate 14, that there are glutamate binding sites in all 4 vestibular nuclei 17 and specifically N M D A binding sites in the medial vestibular nuclei (MVN) 11. However while it has been demonstrated physiologically that the N M D A receptor contributes to normal synaptic function in the vestibular nucleus of the frog 7, there is little evidence to suggest this is the case in mammalian species 8. Here we report that the potent and specific N M D A receptor/ channel antagonists (3-((+)-2-carboxypiperazin-4-yl)propyl-l-phosphonic acid) (CPP) 5 and ({(+)-5-methyl10,11-dihydro-5H-dibenzo- [a,d]-cyclohepten-5,10-imine maleate}) (MK801) 18, when applied to guinea pig brainstem slices in vitro using superfusion, produce a decrease in the firing rate of the majority of medial vestibular nucleus (MVN) neurons tested. Six labyrinthine-intact guinea pigs were etherized and their brainstems quickly removed to chilled artificial cerebrospinal fluid (ACSF). Coronal slices (600-1000/~m thick) were cut by hand with a chilled razor blade,
incubated in a slice chamber and superfused with a standard ACSF (in mM): NaCI 126.0, KCI 5.0, KH2PO 4 1.25, MgSO 4 1.3, N a H C O 3 26.0, glucose 10.0, CaCI 2 2.5, Phenol red 0.05% 9. The ACSF was continuously bubbled with 95% 0 2 and 5% CO 2 and maintained at a pH of 7.4 and a temperature of 35-37 °C 9. Single neurons within the MVN were recorded extracellularly with glass microelectrodes (4-8 M~2 impedance) filled with 2 M NaCI and Fast green FCF dye, to facilitate visualization of the electrode position. The MVN could be identified by its proximity to the sulcus limitans when looking at the dorsal aspect of the brainstem during brainstem dissection and by its proximity to the IVth ventricle when looking at the coronal slice in the slice chamber. Baseline firing rate was recorded while the neuron was superfused with the ACSF solution (control), then a second ACSF solution (test solution) containing either MK801 (10 -6 M) or CPP (10 -8 M) was turned on, followed by a return to the control solution. Some slices were also superfused with dopamine (DA) dissolved in ACSF (10 -8 M), used as a control substance to determine if the addition of any substance to the ACSF would produce the same effect as the N M D A antagonists. The test solution was normally turned on for 4 min (bath turnover time was 1.5 min), except where the cell stopped firing altogether, in which case the control solution was turned on again immediately. Firing frequency was analysed on-line using an Ortec time-histogram analyser and histograms were plotted using an Apple IIe microcomputer. Firing rate was considered to have increased or decreased from baseline when a change of greater than or equal to 20% occurred 2. Eight out of 10 MVN neurons tested with 10-6 M
Correspondence: P.F. Smith, Department of Psychology, University of Otago, Dunedin, New Zealand. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
150
loo t
n
8O
decrease irl¢-,rA~ ~ A
15
6o iii Z
u_ 0
20"
I
MK801
C
UJ 09 UJ
40
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5
20 o
,."
0
MK801
"
B
-
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500 60~
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L~ LU 03 LU
MK801 showed a decrease in firing rate from baseline, 1 showed an increase, and 1 showed no change (see Fig. 1). The average percentage decrease from baseline was 59.1 (+ 30.2 S.D.)% (n = 8). Of 10 MVN neurons tested with 10-8 M CPP, 6 showed a decrease in firing rate and 4 showed no change (see Fig. 1). The average percentage decrease from baseline was 46.0 (+ 21.4)% (n = 6). Fig. 2 shows representative examples of the effect of MK801 and CPP on MVN neurons. Of 5 MVN neurons tested with 10-8 M D A , none showed a 20% or greater change from baseline. These results suggest that the N M D A receptor contributes to the resting activity of guinea pig MVN neurons in vitro. Our previous studies on brainstem slices using high Mg 2+ A C S F indicate that a large component of the activity recorded from MVN neurons in vitro is synaptically generated 2"3, therefore it is probable that the N M D A receptor is one of the r e c e p t o r types on MVN neurons which contributes to this synaptic activity. Since in the present study the entire coronal slice was superfused with the N M D A antagonist, the possibility cannot be excluded that the changes in firing rate observed in MVN neurons were due not to the blockade of N M D A receptors on M V N neurons themselves, but to effects on N M D A receptors in other areas of the slice. However, this possibility seems unlikely since binding studies suggest that the reticular formation beneath the vestibular nuclei has few N M D A receptors11; although the inferior olive has a density of N M D A receptors which is comparable to the MVN ]1, this structure was present only on the more caudal slices that we used. Physiological evidence that N M D A receptors contribute to normal synaptic function in the vestibular nucleus was first presented by Knopfei 7, using the frog. Recently, Lewis et al. 8 have reported that N M D A , iontophoresed onto MVN neurons in the rat brainstem slice in vitro, causes depolarization which is blocked by the N M D A antagonist D-APV. Behavioral evidence presented by De
u
100 200 3;0 4;0 TIME (SEC)
CPP
Fig. 1. Percent of medial vestibular nucleus (MVN) neurons tested showing greater than or equal to 20% increase or decrease in firing, or no change in response to 10-6 M MK801 (n = 10) or 10-s M CPP (n = 10).
•
Q. 03
2" 0
.......,-.-...
=,..~===•..,=. •
4
0
"%'~"" ""~" " ""~" "-
2;0
4;0 6;0 TIME (SEC) Fig. 2. Examples of the effect of 10 -6 M MK801 (A) or 10 8 M CPP (B) on resting activity for a single medial vestibular nucleus neuron. Points represent successive 10 s bins of average resting activity for the one neuron. Arrows indicate the onset of the test solution and the return to the control solution (C).
Waele et al. ]9 also suggests that N M D A receptors may be active in the normal vestibular nucleus: unilateral infusion of D-APV into the vestibular nucleus of labyrinthineintact guinea pigs induced postural and ocular motor symptoms similar to unilateral labyrinthectomy. There is now considerable evidence that the N M D A receptor contributes to normal synaptic function in other areas of the CNS, such as the visual cortex 4J°, thalamus ~s and spinal cord 12'16. In some of these cases the N M D A receptor appears to contribute to normal sensory processing4,]ojs,16. With respect to the function of N M D A receptors in the vestibular nucleus, it is interesting to note that in many CNS neurons N M D A and glycine receptors are codistributed and that glycine receptor activation may increase the affinity of the N M D A receptor for glutamate 6. Since there is evidence that brainstem commissural inhibition between the bilateral MVN is partially mediated by glyeine 13, it may be that N M D A and glycine receptors are co-distributed on MVN neurons and that the balance of activity between these two receptor types is important for the integration of input from the two labyrinths. This research was supported by the New Zealand Medical Research Council and the Neurological Foundation of New Zealand.
151
1 Cotman, C.W. and Iversen, L.L., Excitatory amino acids in the brain-focus on NMDA receptors, Trends Neurosci., 10 (1987) 263-265. 2 Darlington, C.L. and Smith, P.E, Neuropharmacological investigations of vestibular compensation. In A. Berthoz, W. Graf and P.P. Vidal (Eds.), The Head-Neck Sensory Motor System, Wiley, Chichester, in press. 3 Darlington, C.L., Smith, P.E and Hubbard, J.I., Neuronal activity in the guinea pig medial vestibular nucleus in vitro following chronic unilateral labyrinthectomy, Neurosci. Left., 105 (1989) 143-148. 4 Fox, K., Sato, H. and Daw, N., The location and function of NMDA receptors in cat and kitten visual cortex, J. Neurosci., 9 (1989) 2443-2454. 5 Harris, E.W., Ganong, A.H., Monaghan, D.T., Watkins, J.C. and Cotman, C.W., Action of (3-(+-)-2-carhoxypiperazin-4yl)-propyl-l-phosphonic acid) (CPP): a new and highly potent antagonist of the N-methyl-o-aspartate receptor in the hippocampus, Brain Research, 382 (1986) 174-177. 6 Johnson, J.W. and Ascher, P., Glycine potentiates the NMDA response in cultured mouse brain neurons, Nature (Lond.), 325 (1987) 529-531. 7 Knopfel, T., Evidence for N-methyl-o-aspartic acid mediated modulation of the comrnissural input to central vestibular neurons of the frog, Brain Research, 426 (1987) 212-224. 8 Lewis, M.R., Phelan, K.D., Shinnick-Gallagher, E and Gallagher, J.E, Primary afferent excitatory transmission recorded intracellulary in vitro from rat medial vestibular neurons, Synapse, 3 (1989) 149-153. 9 Llinas, R. and Sugimori, M., Electrophysiological properties of in vitro Purkinje cell somata in mammalian cerebellar slices, J. Physiol. (Lond.), 305 (1980) 171-195. 10 Miller, K.D., Chapman, B. and Stryker, M.P., Visual responses
in adult cat visual cortex depend on N-methyl-D-aspartate receptors, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 5183-5187. 11 Monaghan, D.T. and Cotman, C.W., Distribution of N-methylo-aspartate sensitive L-[3H]glutamate binding sites in rat brain, J. Neurosci., 5 (1985) 2909-2919. 12 Polc, P., NMDA receptors mediate background and excessive activity of y motoneurons in the spinal cord, Eur. J. Pharmacol., 144 (1987) 113-118. 13 Precht, W., Schwindt, P.C. and Baker, R., Removal of vestibular commissural inhibition by antagonists of GABA and glycine, Brain Research, 62 (1973) 222-226. 14 Raymond, J., Nieoullon, A., Dememes, D. and Sans, A., Evidence for glutamate as a neurotransmitter in the cat vestibular nerve: radioautographic and biochemical studies, Exp. Brain Res., 56 (1984) 523-531. 15 Salt, T.E., Mediation of thalamic sensory input by both NMDA receptors and non-NMDA receptors, Nature (Lond.), 322 (1986) 263-265. 16 Stein, ES.G. and Schild, C.E, N-methyl-D-aspartate antagonist applied to the spinal cord hindlimb enlargement reduces the amplitude of flexion reflex in the turtle, Brain Research, 479 (1989) 379-383. 17 Touati, J., Raymond, J. and Dememes, D., Quantitative autoradiographic characterization of L-3H glutamate binding sites in rat vestibular nuclei, Exp. Brain Res., 76 (1989) 646-650. 18 Wong, E.H.E, Kemp, J.A., Priestley, T., Knight, A.R. and Woodruff, G.N., The anticonvulsant MK801 is a potent Nmethyl-D-aspartate antagonist, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 7104-7108. 19 De Waele, C., Vihert, N., Baudrirnont, M. and Vidal, P.E, NMDA receptors contribute to the resting discharge of the vestibular neurons in the normal and hemilabyrinthectomized guinea pig, Exp. Brain Res., in press.
