Reticulospinal neurones activate excitatory amino acid receptors

Brain Research, 408 (1987) 321-325 Elsevier

321

BRE 22162

Reticulospinal neurones activate excitatory amino acid receptors J.T. Buchanan, L. Brodin, N. Dale* and S. Grillner The Nobel Institute for Neurophysiology, Karolinska Institutet, Stockholm (Sweden) (Accepted 16 December 1986)

Key words: Reticulospinal neuron; Motoneuron; Excitatory postsynaptic potential; Excitatory amino acid receptor; Lamprey

Paired intracellular recordings were used to study the monosynaptic excitatory postsynaptic potentials (EPSP) in lamprey motoneurones evoked by stimulation of single reticulospinal MOiler and Mauthner cells. The chemical component of the synaptic potentials was depressed by bath application of the non-selective excitatory amino acid antagonists kynurenic acid and cis-2,3-piperidine dicarboxylate. The N-methyl-D-aspartate (NMDA) antagonists Mg2÷ and 2-amino-5-phosphonovalerate caused a selective depression of a late component of the EPSP. Thus, fast-conducting reticulospinal neurones appear to release an excitatory amino acid acting at both NMDA and non-NMDA receptors. Direct reticulospinal control of motoneurones is found throughout the vertebrate series. From primitive cyclostomes to primates, certain fast-conducting reticulospinal neurones produce monosynaptic excitatory postsynaptic potentials (EPSPs) in spinal motoneurones t6'24'25. The transmitter mediating this excitation has not yet been identified. The lamprey, a cyclostome, is advantageous for detailed studies of reticulospinal mechanisms since it has large reticulospinal neurones, the Miiller and Mauthner cells, which can be individually identified in the in vitro brain-spinal cord preparation 26. These fast-conducting neurones excite spinal motoneurones and interneurones via mixed electrotonic-chemical synapses 7'25. Accumulating evidence suggests that excitatory amino acids ( E A A ) play an important role in mediating fast excitation in the central nervous system (for reviews see refs. 15, 21, 29). However, only a few studies have been conducted at the level of single pre- and postsynaptic cell pairs 4'9'13'17. Excitatory amino acids such as glutamate can activate 3 different types of postsynaptic receptors in m a m m a l s as well as in amphibians and cyclostomes 12'13'21'29, referred to as N-methyl-D-aspartate ( N M D A ) , kainate and quis-

qualate receptors 29. We have now studied the synapses between Miiller/Mauthner cells and spinal motoneurones using paired intracellular recordings and bath application of the non-selective E A A antagonists cis-2,3-piperidine dicarboxylate 29 ( P D A ) and kynurenic acid 23 ( K Y A C ) , and the selective N M D A antagonists 2-amino-5-phosphonovalerate (APV) and Mg 2+ (refs. 1, 21, 29). The results have been reported in abstract form 1°. Adult silver lampreys (Ichthyomyzon unicuspis) were anaesthetized with MS-222 (Sandoz) and their bodies were transected behind the gill region. The brain and spinal cord were either isolated or left resting on the basal parts of the cranium and on the notochord 25 and were mounted in the recording chamber containing magnesium-free physiological solution (in mM: NaC1 91, KC1 2.1, CaC! 2 2.6 or CaC12 5.0, N a H C O 3 20, glucose 4) kept at 7 - 9 °C. In the experiments with intra-axonal stimulation, isolated pieces of spinal cord 10-15 segments long were used. Motoneurones were identified by their ventral root spikes recorded with a suction electrode. All motoneurones included in the study had a resting m e m b r a n e potential of at least - 5 0 mV and an action potential of at

* Present address: Center for Neurobiology and Behavior, Columbia University, 722 West 168th Street, New York, NY 10032, U.S.A. Correspondence: S. Grillner, The Nobel Institute for Neurophysiology, Karolinska Instituter, Box 60400, S-104 01 Stockholm, Sweden.

322 least 60 mV. After obtaining a stable intracellular recording of a motoneurone, an identified reticulospinal MOiler ceil, Mauthner cell, or MOiler axon was then impaled with a second microelectrode. The MOiler and Mauthner cell bodies were identified visually by their size, shape and position within the brainstem 26 (Fig. 2). MOiler axons in the isolated spinal cord were identified by their ventromedial location and fast conduction velocity a6 ( > 3 m/s at 9 °C), and most were probably from mesencephalic MOiler cells. While maintaining the intracellular recordings of the pre- and postsynaptic cells, various test solutions were perfused into the recording chamber (3 ml/min into a 6 ml bath) and washed out. The presynaptic cell was usually stimulated 10 times at 0.1 Hz during each of the control and test solutions. The EPSPs were recorded on tape and later averaged. In the case of Fig. 2 where 4 different presynaptic cells were tested on the same motoneurone, the presynaptic cells were each reimpaled under visual control for each test. As previously reported 25, the EPSPs from MOiler and Mauthner cells to motoneurones usually consisted of both an electrotonic and a chemical component (Fig. 1An, MOiler axon to a motoneurone). The electrotonic component was constant in amplitude and time course and persisted in calcium-free physiological solution (Fig. lAd), whereas the chemical component fluctuated slightly in amplitude and disappeared after removal of calcium. To test if the chemical component of these EPSPs involves an activation of E A A receptors, the bath was perfused with 2 mM P D A which is an antagonist of N M D A , kainate, and to some extent quisqualate12"29. The chemical component was almost abolished (Fig. l a b ) and then recovered after washout of the P D A (Fig. 1Ac.~). Fig. 1B shows the time course of the depression of the EPSP, first after application of P D A and then following removal of calcium, The brain-spinal cord preparation was used to stimulate single identified reticulospinal cells. In Fig. 2A, perfusion with 2 mM K Y A C , which is an antagonist of N M D A , kainate and quisqualate 23, depressed the chemical component of the EPSPs evoked in a motoneurone from the reticulospinal Mfiller cells B 2 and B 4 and from the contralateral Mauthner cell. There was no change in the E P S P from B3, but no chemical component appeared to be present in this

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Fig. 1. The effect of EAA antagonists on the EPSPs elicited in a motoneurone by a reticulospinal Miiller axon. A: superimposed EPSPs recorded in a motoneurone during 0.1 Hz intracellular stimulation of a Mtaller axon in an isolated spinal cord. The site of axon stimulation was 7 segments (14 mm) from the motoneurone. The EPSP consists of an electrotonic component followed by a larger chemical component. Representative EPSPs at the time points (a-e) in B are shown. At lower right, the EPSP was first recorded in MgE+-freesolution and then the bath was perfused with physiological solution containing 1.8 mM Mg2+. The electrotonic component and the peak of the chemical component were unaffected by the addition of 1.8 mM Mg2+, but the duration of the EPSP was shortened. The lower right traces are averages of 8 sweeps. B: plot of the mean amplitudes (n = 10 for each point) and 1 S.D. of the EPSPs dur, ing the course of the experiment. A 10-rain perfusion of 2 mM c/s-2,3-piperidine dicarboxylate (PDA) depressed the chemical component (b). After 20 min of washout, there was partial recovery (c). Then perfusion of calcium-free solution With 4 mM Mg2+ abolished the chemical component to reveal the electrotonic component in isolation (d). A return to normal physiological solution restored the chemical component to its original value (e).

323 cal EPSPs tested were depressed by P D A or K Y A C , (n = 14, and includes the MOiler cells I t, B2_ 4 and the Mauthner cell). N M D A is selectively antagonized by A P V and by Mg 2+ (refs. 1, 21, 29). In 44% of the tests (8/18), bath application of 0.1 m M A P V or 1.8 m M Mg 2+ de-

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Fig. 2. Effect of EAA antagonists on the EPSPs evoked in one spinal motoneurone from 4 identified reticulospinal neurones. A: averages of EPSPs (n = 8, 0.1 Hz repetition rate) in a motoneurone evoked by intracellular stimulation of the bulbar Miiller ceils B2_4 and the contralateral Mauthner cell (Mth). Locations of the cells in the brainstem are indicated in the schematic drawing to the right. After obtaining control records, the bath was perfused with 2 mM KYAC and the various brainstem cells were re-impaled and re-tested. The EPSPs from B2, B4 and Mth, but not Bs, showed a clear depression and recovery. This particular B3 EPSP, however, had no observable chemical component since perfusion with calcium-free solution did not alter the EPSP. B: the averaged control EPSP (n = 32, 0.2 Hz stimulation rate) from a Mauthner cell to an unidentified spinal neurone recorded in Mg2+-free solution is superimposed upon the averaged EPSP after perfusion of 0.1 mM APV. There was a depression of the late component without affecting the early component. After 15 sin of washout, the EPSP recovered to its control size. An identical effect was observed after perfusion with a solution containing 1.8 mM Mg2÷. C: for the same EPSP as in B, perfusion with 2 mM KYAC depressed both the early and the late components of the EPSP.

particular B3-motoneurone pair since the EPSP was unchanged in calcium-free solution. As demonstrated in Fig. 2A, different MOiler cells elicited EPSPs of different sizes in a single motoneurone. It was also observed that a single MOiler cell produced different-sized EPSPs in different motoneurones, Regardless of size, however, all reticulospinal chemi-

pressed a late component of the EPSP without affecting either the electrotonic component or the peak of the early chemical component (Fig. 1A, lower right, and Fig. 2B). However, after A P V or Mg 2÷ application in 50% of the cases (9/18), the resting m e m b r a n e potential of the postsynaptic cell hyperpolarized and the amplitude of both the electrotonic and the chemical components of the EPSP increased indicating that the resting input impedance of the cell had increased. These effects were accompanied by a decrease of a previously high level of spontaneous synaptic activity which presumably led to the above effects. Due to these changes, the EPSPs in these cases did not lend themselves to a direct comparison before and after A P V or Mg 2+ and were thus not further considered. Three types of unitary EPSPs have been described previously in the lamprey and the frog: pure N M D A receptor-mediated, pure kainate receptor-mediated, and mixed EPSPs due to an activation of both receptor types 12"13. The reticulospinal chemical EPSPs reported here appear to be of the mixed type since a late component was depressed by A P V and Mg 2÷ and an early component by P D A and K Y A C . Since P D A is a poor antagonist of quisqualate in the lamprey ~2, the early phase of the chemical synaptic potential is probably mediated mainly by kainate receptors. The late phase is most likely mediated by N M D A receptors 1"21'29 Although the N M D A component is not seen at the resting m e m b r a n e potential in a normal physiological environment, it will confer voltage-dependent properties to the postsynaptic cell 2°-22 and thereby increase its sensitivity to other synaptic input and p r o m o t e an oscillatory behaviour 14'21'27'28 which may be of particular importance in the control of locomotion 5"27"28. Since E A A s can induce locomotion in the lamprey spinal cord 15, it seems likely that reticulospinal cells acting on E A A receptors play an important role in the initiation and control of locomotion. MOiler cells fire action potentials during Iocomotor activity 18, and they probably synapse upon spihal neurones of the locomotor network since repetitive stimulation of single MOiler cells changes the

324 rate of l o c o m o t o r activity s.

cal e v i d e n c e for s l o w e r - c o n d u c t i n g reticulospinal sys-

T h e t r a n s m i t t e r acting at E A A receptors is possibly an acidic a m i n o acid. E x p e r i m e n t s with a m i n o

tems, one c o n t a i n i n g 5 - H T (ref. 3) a n d a n o t h e r cont a i n i n g a C C K - l i k e p e p t i d e 2 although these n e u r o n e s

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This study was s u p p o r t e d by the Swedish Medical

fast reticulospinal n e u r o n e s of higher v e r t e b r a t e s

R e s e a r c h C o u n c i l , project n u m b e r 3026. J . T . B . was

w o u l d also use E A A t r a n s m i s s i o n ~6'24. i n l a m p r e y as in m a m m a l s ILl9 we have f o u n d i m m u n o h i s t o c h e m i -

s u p p o r t e d by N I H p o s t d o c t o r a l fellowship 5 F32 NS07314.

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325 neurons and nerve cells in the spinal cord of the sea lamprey, J. Comp. Neurol., 154 (1974) 207-223. 26 Rovainen, C.M., MOiler cells, 'Mauthner' cells and other identified reticulospinal neurons in the lamprey. In D. Faber and H. Korn (Eds.), Neurobiology of the Mauthner Cell, Raven Press, New York, 1978, pp. 245-269. 27 Sigvardt, K.A., Grillner, S., Wall6n, P. and Van Dongen, P.A.M., Activation of NMDA receptors elicits fictive Iocomotion and bistable membrane properties in the lamprey

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