Brain Research, 525 (1990) 181-188
181
Elsevier BRES 15769
Research Reports
Sympathetic preganglionic neurones in neonatal rat spinal cord in vitro: electrophysiological characteristics and the effects of selective excitatory amino acid receptor agonists David Spanswick and Stephen D. Logan Department of Physiology, The Medical School, Birmingham University, Birmingham (U. K.) (Accepted 20 February 1990)
Key words: Sympathetic preganglionic neuron; Rat spinal cord slice; Burst firing; Kainate; N-Methyl-D-aspartate; Quisqualate; Inhibitory postsynaptic potential
Intracellular recordings were made from 52 lateral horn neurones in thin slices of neonatal rat thoracolumbar spinal cord. Of these neurones 12 were spontaneously active and the remainder silent. A number of these cells could be activated antidromically by stimulation of ventral roots. The conduction velocity of the antidromic potential was estimated to be 0.9-2 m/s which is within the range reported for axons of sympathetic preganglionic neurones (SPNs). The membrane properties of antidromically identified SPNs were similar to other lateral horn neurones included in this study and comparable to those reported for SPNs by others. Spontaneous burst firing was recorded in 3 neurones and activity in a further 5 neurones was characterized by the discharge of an action potential followed by an afterhyperpolarization potential (AHP) of peak amplitude 3-13 mV and duration 0.5-4 s. The AHP had an initial fast component (fAHP) which was sensitive to the potassium channel blocker tetraethylammonium (TEA), and a second slower component (sAHP) which was both sensitive to extracellular calcium and TEA. The effects of the selective excitatory amino acid receptor agonists N-methyI-D-aspartate (NMDA), kainate and quisqualate were investigated by superfusion of the agonists, at known concentrations (100 nM to 100 ~M). These agonists induced concentration-dependent depolarizations which were primarily associated with a reduction in neuronal input resistance. NMDA-indueed depolarizations were potentiated in the absence of magnesium. In a number of neurones NMDA, kainate and quisqualate (1-50 ~M) induced, in addition to a depolarizing response, the discharge of small (1.5-6.5 mV), brief (7-20 ms) IPSPs which were reversed just below resting membrane potential at -64 mV. These results suggest that both NMDA and non-NMDA receptors may have an important role in the excitation of SPNs and of inhibitory interneurones presynaptic to SPNs. INTRODUCTION Sympathetic preganglionic neurones (SPNs) are utilized in cardiovascular control and are probably the major site of integration of sympathetic nerve activity in the CNS 5. The majority of SPNs are located in the intermediolateral nucleus (IML) of the thoracolumbar spinal cord 1'15,33,38,40. Investigations into the chemical nature of synaptic transmission in the I M L have shown that excitatory amino acids ( E A A s ) excite SPNs in vivo 3"6'13 and in vitro in adult cat spinal cord slice preparation 34. Recently it has been suggested that E A A s may mediate fast synaptic transmission in SPNs, in the neonate rat spinal cord slice, and that the receptor responsible is the N-methyl-o-aspartate ( N M D A ) subtype 27. In addition to this, inhibitory postsynaptic potentials (IPSPs), which may be mediated by glycine or a glycine-like substance, have been reported to occur spontaneously and in the presence of N M D A , suggesting that inhibitory interneurones, presynaptic to SPNs, are
activated through N M D A receptors, and further, indirect evidence was provided for the existence of an inhibitory pathway impinging on SPNs which is analogous to the Renshaw cell circuit of ventral horn motoneurones 9. Electrophysiologicai, biochemical and pharmacological studies have shown that at least 3 types of E A A receptors exist, these being defined by the selective agonists N M D A , kainate and quisqualate 25,42. We have utilized the isolated thoracolumbar spinal cord slice preparation to investigate the effects of E A A s on lateral horn cells including neurones identified as SPNs by antidromic stimulation of ventral rootlets. We report, for the first time, spontaneous burst firing in lateral horn neurones, and also a long-lasting, two-component afterhyperpolarization potential which is similar to that reported for the SPN in the adult cat in vitro 44. We further show that in addition to N M D A , the selective E A A receptor agonists kainate and quisqualate excite SPNs and give rise also to IPSPs suggesting that n o n - N M D A receptors may have an important role in excitation of SPNs and of inhibitory
Correspondence: S.D. Logan, Department'of Physiology, The Medical School, Birmingham University, Birmingham B15 2Tr, U.K. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
182 i n t e r n e u r o n e s p r e s y n a p t i c to SPNs. S o m e of these results h a v e b e e n p r e s e n t e d p r e v i o u s l y in abstract f o r m 41.
MATERIALS AND METHODS Sprague-Dawley rats 9-22 days old were employed in this study. The procedure used to obtain spinal cord slices from neonatal rats was similar to that described by other investigators 17'32. The animals were anaesthetized with ether and a laminectomy performed to reveal the spinal cord. Following taminectomy, a 1-1.5 cm section of the thoracolumbar spinal cord was excised and placed in cold (4 °C), aerated (95% 02, 5% CO2) artificial cerebrospinal fluid (ACSF). The pia mater was removed and the spinal segment cut with a razor blade to form one or two approximately 5 mm sections. The sections were fixed to an agar block and the tissue-agar assembly in turn fixed to a petri-dish with cyanoacrylic glue (Superglue). The petri-dish was secured to the cutting chamber of a modified Oxford Vibratome and filled with cold (4 °C) aerated ACSF. Several 300-450/~m slices of spinal cord were cut, care being taken to ensure that some slices were removed with intact ventral rootlets. The first slice was generally discarded and the remaining slices transferred to an incubation chamber where they were maintained in aerated ACSF at 25 °C for at least 1 h prior to recording. One slice was transferred to the recording chamber and continuously superfused at a rate of 3 ml/min with an ACSF solution of the following composition (mM): NaCI 127, KC1 1.9, KH2PO 4 1.2, CaCI 2 1.2, MgSO 4 1.3, NaHCO 3 26, glucose 10, pH 7.4. The solution was saturated with 95% 02, 5% CO 2 and the temperature maintained at 34 + 0.5 °C. Intracellular recordings were obtained from neurones located in the lateral horn of the thoracolumbar spinal cord slices with glass microelectrodes, backfiiled with 3 M potassium acetate, of impedance 60-150 M~2. Stable intracellular recordings were obtained from neurones for up to 6 h. A high impedance bridge amplifier (WPI 'Chopper' SVC2000) was employed to inject current through the microelectrode and to record signals which were displayed on an oscilloscope (Tektronix 5113). Recordings were also directed to the following data storage facilities: a chart recorder (Gould RS3200);
A
an X - Y plotter (Gould Colourwriter) via a digital storage oscilloscope (Gould 1425); a tape recorder (Racal Store 4) via an oscilloscope (Tektronix 502A) with an amplification (× 100) facility. Electrical stimulation of the ventral roots was achieved via a concentric bipolar electrode positioned close to the rootlets. Drugs were dissolved in ACSF to a known concentration and applied to the slice by superfusion at a rate of 3 ml/min. The following agents were employed: NMDA, kainic acid, quisqualic acid, tetraethylammonium chloride (TEA) (Sigma). The following salts were also used: NaCI, KCI, NaHCO> D-glucose (Fisons); MgSO~, KH2PO ~ CaCI2.6H20 (BDH).
RESULTS
Membrane properties Stable i n t r a c e l l u l a r r e c o r d i n g s w e r e o b t a i n e d f r o m 52 n e u r o n e s l o c a t e d in the lateral h o r n o f t h o r a c o l u m b a r spinal c o r d slices. O f t h e s e n e u r o n e s , 12 w e r e s p o n t a n e ously active and
the r e m a i n d e r
silent.
Lateral
horn
n e u r o n e s had resting m e m b r a n e p o t e n t i a l s o f - 6 0
+ 6
m V ( m e a n + S . D . ) with a r a n g e o f - 5 0 to - 7 8 mV. T h e input resistance, m e a s u r e d as the v o l t a g e d e f l e c t i o n i n d u c e d by h y p e r p o l a r i z i n g r e c t a n g u l a r c u r r e n t pulses ( 0 . 0 5 - 0 . 5 n A , 100-200 ms), was 110 + 24 Mr2 with a r a n g e of 7 5 - 1 6 0 MI2 (n = 21) (see Fig. 1 A , B ) . A c t i o n p o t e n t i a l s ( b o t h s p o n t a n e o u s a n d electrically e v o k e d by direct d e p o l a r i z i n g c u r r e n t i n j e c t i o n ) had a p e a k amplitude, m e a s u r e d f r o m t h r e s h o l d of 58 + 8 m V with a r a n g e of 4 6 - 7 5 m V and d u r a t i o n 2.8 + 0.6 ms with a r a n g e of 1 . 8 - 3 . 6 ms (Fig. 1C). S t i m u l a t i o n of v e n t r a l roots e v o k e d an a l l - o r - n o n e spike p o t e n t i a l in 5 of 8 lateral h o r n n e u r o n e s tested (Fig. 1D). T h e c o n d u c t i o n v e l o c i t y of
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_..A ~ Fig. 1. Passive membrane properties and action potentials evoked in rat sympathetic preganglionic neurons (SPNs). A: eleetrotonic potentials evoked in a neurone. B: plot of voltage-current relations of neurone A. C: action potentials evoked by a depolarizing current pulse. D: antidromic spike induced by stimulation of ventral rootlets. The conduction velocity was calculated to be 1.1 m/s. E: antidromicaUy evoked action potentials showing the IS and SD components of the spike. High frequency stimulation and hyperpolarization of the neurone abolishes the SD component leaving only the IS response.
183 these axons was estimated to be 1.3 + 0.2 m/s (mean +_ S.E.M.) with a range of 0.9-2 m/s. At low-frequency stimulation (0.2-1 Hz) a spike potential was always evoked. At higher frequency stimulation, up to 10 Hz, a pronounced inflexion in the rising phase of the antidromic action potential was apparent (Fig. 1E). This is generally attributed to antidromic invasion of the initial segment (IS) and the soma-dendritic region (SD) of the neurone 4. The SD component of the action potential was abolished by membrane hyperpolarization. The membrane properties of antidromically identified SPNs were similar to other lateral horn neurones included in this study, and comparable to those reported by other investigators for lateral horn neurones 17'1s and for identified SPNs 1°.
Spontaneous activity Burst firing. Spontaneous burst firing was recorded in 3 lateral horn neurones. Bursts were characterized by a slow depolarizing shift, with superimposed action potentials, followed by a relatively rapid hyperpolarization before the start of the next burst (see Fig. 2A). The frequency of bursting in these 3 neurones was 0.4, 1.0 and 1.6 Hz. The number of spikes per burst varied between 7 and 13. Superfusion of medium containing low calcium reduced the duration of the burst, the number of action potentials per burst, the amplitude and duration of the interburst hyperpolarization and increased the frequency of bursting (Fig. 2B2). A 2 x normal calcium-containing medium (2.4 mM) maintained burst firing, augmented the amplitude and duration of the interburst hyperpolarization and the overshoot of action potentials associated with the bursts (Fig. 2B3). Action potentials also increased in duration in the presence of 2× normal calcium and an increase in synaptic activity was apparent. Superfusion of T E A (30 mM) reduced the neurones to discharging action potentials in triplets and doublets. Bursting was restored by returning to a normal calciumcontaining medium (1.2 mM). Afterhyperpolarization potential (AHP). Spontaneous activity recorded in 4 lateral horn neurones (two identified as SPNs by the appearance of an antidromic spike following stimulation of ventral rootlets) was characterized by the discharge of a single action potential followed by a long-lasting AHP (Fig. 3A). The AHP comprised of an early fast component (fAHP) followed by a late slow component (sAHP) (Fig. 3A1). The AHP was associated with a reduction in the neuronal input resistance, the most marked reduction being associated with the early phase of the AHP (Fig. 3A2) , and had maximal amplitude of 3-13 mV and duration 0.5-4 s, respectively. The frequency of action potential discharge in these neurones
ranged from 0 to 0.3 Hz. Superfusion of a low calciumcontaining medium reversibly abolished the late component AHP and increased the frequency of action potential discharge, up to as much as 1 Hz (Fig. 3B2). Addition of a 2 x normal calcium-containing medium (2.4 mM calcium chloride) reduced the frequency of action potential discharge to 0-0.1 Hz, and augmented the amplitude and duration of the late component AHP (Fig. 3B3). Superfusion of the potassium channel blocker T E A (30 mM) or TEA in a low calcium-containing ACSF abolished both the early and late components of the AHP (Fig. 3B4, B5). In one neurone a long-duration AHP was induced by superfusion of a low magnesium-containing ACSE A further feature of these neurones was that following bursts of 3-5 action potentials a large amplitude, long duration, hyperpolarization was evoked with a delay between the termination of the last action potential and the onset of the hyperpolarization (Fig. 3A3).
A
0.4s B
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] 20my ( 2.4mM )
8s
Fig. 2. Spontaneous burst firing in lateral horn neurones. A: spontaneous bursting recorded in one neurone. B: the effects of calcium on burst firing in a lateral horn neurone. Bl: spontaneous burst firing in normal C a 2+ (1.2 mM). Bz: superfusion of nominally zero Ca2+-containingsolution for 2 min reduced the burst duration and interburst interval amplitude and duration. The frequency of bursting also increased in nominally zero calcium. The peak amplitude of spike discharge in this and subsequent figures were truncated due to the limited frequency response of the pen recorder. Ba: superfusion of a 2x normal calcium (2.4 mM)-containing solution augmented the burst duration and interburst amplitude and duration. The frequency of bursting decreased in high calcium solution.
184
Excitatory amino acids The actions of the selective E A A agonists N M D A , kainate and quisqualate on lateral horn neurones, including 5 neurones identified antidromically as SPNs, was investigated by superfusion of the agonists, at known concentrations (100 nM to 100yM), in the medium bathing the slice.
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1.3mM Magnesium
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3 2 x N o r m a l C a l c i u m ( 2 , 4 mM )
ilpprox. 40 s
lll!lllllal!!JHi!!l!!H!!!ll 5 TEA ( 3 0 mM ) + Low Calcium ( 0 mM ) _.J 20 mV
J 20mV 10s Fig. 4. Effects of excitatory amino acids (EAAs) on a lateral horn neurone and an identified SPN. A: depolarizaton induced by quisqualate (20 pM) superfused for 2 s to an identified SPN. Repeated hyperpolarizing rectangular current pulses (0.1 hA, 200 ms) were used to evoke hyperpolarizing electrotonic potentials (downward deflections of the trace) which indicate neuronal input resistance. Quisqualate-induced depolarization at high concentrations was accompanied by intense neuronal discharge. The peak amplitude of spike discharge in this and other figures were truncated due to the limited frequency response of the pen recorder. B: the effects of NMDA and the antagonist action of magnesium on NMDA-induced responses. BI: depolarization induced by NMDA (100 #M) superfused for 8 s to a lateral horn neurone. The response was associated with small increases and decreases in neuronal input resistance and action potential discharge. B2: the effect of the same concentration of NMDA in the absence of magnesium from the bathing medium. Depolarization of the neurone was accompanied by intense neuronal discharge and primarily a reduction in neuronal input resistance. C: depolarization induced by kainate (50 pM) superfused for 2 s to an identified SPN. Depolarization was prolonged, accompanied by intense neuronal discharge, and a reduction in neuronal input resistance in the early phase of the response.
15s
Fig. 3. The effects of calcium and the potassium channel blocker TEA on the AHP. A: the hyperpolarization following the discharge of a single (A 1 and A2) and a burst of 3 (A3) action potentials. At: demonstration of two-component AHP. Note the AHP has an initial fast component (fAHP) and a second slower prolonged phase (sAHP). A2: an AHP with associated conductance change. A3: large amplitude, long duration hyperpolarization following a burst of action potentials. Note also the delay following the last action potential and the onset of the hyperpolarization. B: records from two lateral horn neurones including one identified SPN showing the effects of calcium and TEA on the AHP. Bt: control showing spontaneous action potentials followed by an AHP. B2: superfusion of nominally zero calcium-containing ACSF abolished the sAHP and increased the rate of action potential firing. B3: superfusion of 2x normal calcium-containing ACSF (2.4 mM calcium chloride) restored and augmented the amplitude and duration of the sAHP. The rate of firing decreased in 2x normal calcium-containing ACSE B& superfusion of TEA (30 mM) reduced both the fAHP and sAHP and increased the rate of firing. Bs: superfusion of TEA and nominally zero calcium-containing ACSF reduced both components of the AHP. Action potentials in these figures were truncated due to the limited frequency response of the pen recorder.
Quisqualate (100 nM to 50 g M ) was superfused for 2-30 s to 10 lateral horn neurones, including 5 identified SPNs. Quisqualate induced c o n c e n t r a t i o n - d e p e n d e n t depolarizing responses in all cells investigated. T h e response was rapid in onset and offset (i.e. a response was observed within a few seconds of the drug reaching the bath, and the time course of the response, allowing for dilution effects, closely p a r a l l e l e d that of the drug application), and at higher concentrations (10-50 p M ) was associated with a reduction in the neuronal input resistance (Fig. 4A). D e p o l a r i z a t i o n in all of these cells reached threshold and gave rise to the discharge of action potentials in the presence of quisqualate at 10-50 g M . N M D A (1-100 p M ) was superfused for 2-30 s to 16 lateral horn neurones, including 5 identified SPNs. N M D A induced d o s e - d e p e n d e n t depolarizing responses
185 A
•
Quis IOJuM 30s
•
NMDA IO~uM30s
C
I^^-IV •
Keln lOjJM30s
D I Control
2 Qulsqualete onset 3 NMDA onset 4 Kainate onset
JlOmV 50ms
5 Kainate offset
Fig. 5. EAA-induced IPSPs in an identified SPN. A: superfusion of quisqualate (10/zM) for 30 s evoked a depolarization and IPSPs primarily in the onset and peak of the depolarizing response. B: superfusion of NMDA (10/~M) for 30 s also evoked a depolarizing response and IPSPs. C: kainate (10/~M) superfused for 30 s induced a depolarizing response and IPSPs which were primarily associated with the offset of the depolarizing response. D: IPSPs evoked in the presence of EAAs, shown on an expanded time scale. The arrows 1-5 shown on traces A, B and C correspond with traces 1-5 shown in D. DI: control wash. D2: IPSPs evoked during the onset of the depolarizing response induced by quisqualate (A). D3: IPSPs evoked during the onset of the depolarizing response to NMDA (B). Few IPSPs were evoked during the onset of the kainateinduced (C) depolarizing response (D4) but were apparent during the peak and offset of the response (Ds).
in 14 of the neurones, including the 5 identified SPNs, and had no effect on two neurones, either in the presence or absence of magnesium. NMDA-induced responses were rapid in onset and offset and potentiated in the absence of magnesium from the bathing medium (see Fig. 4-B1,2). At low concentrations (1-10/zM) NMDAinduced responses were associated with small increases or no measurable change in neuronal input resistance. At higher concentrations (20-50 pM), or in the absence of magnesium from the bathing medium the response was associated with a reduction in neuronal input resistance, and reached threshold giving rise to action potential discharge. Kainate (1-50/~M) was superfused for 2-30 s to 12
lateral horn neurones, including 5 identified SPNs. Kainate induced dose-dependent depolarizing responses in all cells investigated. The response in 8 neurones was rapid in onset and offset. In 3 neurones high concentrations of kainate (20-50/zM for 2-10 s) induced depolarizing responses which were rapid in onset and long lasting (Fig. 4C). High concentrations of kainate (10-50 /zM) induced depolarizing responses which reached threshold giving rise to vigorous action potential discharge. Kainate-induced responses were associated with a reduction in neuronal input resistance.
Excitatory amino acids and IPSPs Superfusion of N M D A , kainate and quisqualate (1-50 /~M) to 3, 2 and 5 lateral horn neurones, respectively, induced, in addition to a depolarizing response, the discharge of IPSPs (Fig. 5). Of these lateral horn neurones two were identified as SPNs, these neurones both responding to all 3 agonists. The IPSPs were fast and small with a peak amplitude ranging between 1.5 and 6.5 mV and duration 7-20 ms. IPSPs smaller in amplitude than 1.5 mV were ignored as this was close to the noise level of the system. These IPSPs were blocked by strychnine (0.5-5 pM) and in 3 neurones including one identified SPN reversed at membrane potentials o f - 6 2 , -64 and -65 mV, respectively. A further feature of this type of response was that in the presence of N M D A and quisqualate (1-10 pM) IPSPs evoked in the depolarizing response could be associated with the onset, peak and offset of the response (see Fig. 5A, B, D E and D3). However, IPSPs evoked in the presence of kainate (1-10 pM) were not as apparent in the onset of the depolarizing response as with the other two agonists, but associated with the peak and offset of the response (see Fig. 5C, D4 and D5). DISCUSSION The main observation made in this study is that lateral horn cells are excited by the selective E A A receptor agonists N M D A , kainate and quisqualate and further that spinal interneurones impinging on lateral horn cells may also be activated through these 3 E A A receptor subtypes. The type of lateral horn cells recorded from is difficult to establish in the slice preparation. However, of the 8 out of 52 lateral horn neurones tested, 5 could be activated antidromically by stimulation of ventral roots. These neurones had conduction velocities of about 1 m/s, within the range reported for SPNs by other investigators in the slice preparation 1° and in the intact animal TM. Spontaneous burst firing was recorded in 3 lateral horn neurones. The frequency of bursting was augmented and the interburst interval duration and amplitude, and the
186 duration of action potentials were reduced in low calcium solution. In 2x normal calcium-containing solution the reverse effects were observed. Bursting was also reduced by the K+-channel blocker TEA. These results imply that the bursting behaviour is dependent upon calcium and potassium conductances. This is the first report of spontaneous burst firing in lateral horn neurones or putative SPNs and raises the interesting possibility that a subpopulation of these neurones may be capable of this type of behaviour. There have been no reports of burst firing in intact preparations, but regular low-frequency firing has been reported in isolated deafferented fragments of thoracic spinal cord 2°'36, and it has been suggested that this type of activity could be due to pacemaker activity. Furthermore, recent investigations have shown noradrenaline to induce rhythmic bursting in SPNs in the adult cat spinal cord slice preparation 43. It may be, therefore, that under certain conditions, SPNs adopt a burst firing pattern of behaviour. Spontaneous activity recorded in 5 lateral horn neurones, including two identified SPNs, was characterized by the discharge of one or occasionally several action potentials followed by a two-component AHP. The A H P comprised of an initial fast component (fAHP) which was sensitive to the potassium channel blocker T E A and insensitive to calcium, and a second slow component (sAHP) which was both T E A and calcium sensitive. The f A H P would therefore appear to be mediated by potassium and the sAHP by calcium-activated potassium conductances. These observations are similar to those reported for the A H P following an action potential in SPNs of the adult cat in vitro 44 and for other central and peripheral neurones 16'26"31. Superfusion of low calciumcontaining ACSF abolished the sAHP and gave rise to an increased rate of spontaneous action potential discharge suggesting that the sAHP is important in the control of the rate of firing in these neurones. In relation to this in SPNs in vivo there is a period of silence 21 which is associated with a hyperpolarization lasting between 40 and 500 ms 7'8'12'24. The hyperpolarization observed in SPNs in vitro in the cat spinal cord slice is considerably longer, around 2.8 s44. In addition to this long-lasting AHP, on occasions bursts of two or more action potentials gave rise to a long-lasting large amplitude hyperpolarization which was immediately preceded by a delay between the last action potential and the onset of the hyperpolarization. Although the most likely explanation for this would be an increase in the Ca2+-sensitive sAHP coupled with a reduction or complete abolition of the f A H P we cannot rule out the possibility that this delay may represent a synaptic delay and hence a synaptic component which could be analogous to Renshaw inhibition 11'~. Such a Renshaw-type circuit has been sug-
gested to exist in SPNs in the neonate rat spinal cord slice in vitro `) and in hemisected rat spinal cord preparations 23. In 5 lateral horn neurones, including two identified SPNs, the selective E A A agonist N M D A induced, in addition to a depolarizing response, small (1.5-6.5 mV), brief (7-20 ms) IPSPs, which reverse at around -64 mV and are blocked by strychnine. This is in agreement with a recent study of SPNs where strychnine-sensitive chloride mediated IPSPs similar to those described above were evoked in the presence of N M D A 9. These authors attributed this type of response to the activation of inhibitory interneurones, which are presynaptic to SPNs, by NMDA. In studies carried out here we show that in addition to NMDA, the selective non-NMDA receptor agonists kainate and quisqualate also activate 'unitary' IPSPs suggesting that both non-NMDA and N M D A receptors may activate inhibitory spinal interneurones. One further interesting observation is that IPSPs activated in the presence of N M D A and quisqualate occur mainly during the onset and peak of the depolarizing response whereas IPSPs activated in the presence of kainate occur mainly at the peak and offset of the depolarizing response. This suggests that kainate receptors may have either a more indirect action on inhibitory interneurones than do N M D A and quisqualate, or the kainate receptor is situated at a site distant from N M D A and quisqualate receptors which may be coiocalized on inhibitory spinal interneurones. This would be in agreement with receptor autoradiographic studies in other parts of the CNS which have shown N M D A sites 2s and quisqualate sites 3°'37 to be codistributed whereas kainate sites tend to have a dissimilar distribution and complement N M D A sites 29. N M D A excited 14 of 16 lateral horn neurones including 3 identified SPNs. NMDA-mediated depolarizing responses were potentiated in magnesium-free solution suggesting that the N M D A channel of lateral horn neurones and SPNs is gated by magnesium ions. This is in agreement with other studies carried out on SPNs in the neonate rat spinal cord slice preparation 9"2v and on other central neurones 2'19'22'35. The major observation made in studies carried out here is that in addition to NMDA, the selective non-NMDA agonists kainate and quisqualate also excited all lateral horn neurones, including 3 and 5 identified SPNs, respectively. These agonists either induced silent neurones to discharge action potentials or increased the firing rate of spontaneously active neurones, and the response was generally associated with a reduction of the neuronal input resistance. These findings raise the possibility that in addition to N M D A receptors, which have been suggested to mediate fast synaptic transmission in these neurones in the neonate rat
187 through a n o n - N M D A , kainate or quisqualate receptor 34.
spinal cord slice preparation 27, n o n - N M D A receptors may have an important role also in exciting SPNs. In
Acknowledgements. We are indebted to the Friedreich's Ataxia Group (U.K.) for financialsupport. We thank Professor J.H. Coote for helpful discussionsand for reading the manuscript and Dr. D.I. Lewis for help in preliminary investigations.
relation to this, investigations carried out in SPNs in the adult cat spinal cord slice provide indirect evidence that a fast excitatory postsynaptic potential may be mediated REFERENCES
19 MacDonald,J.F., Porietis, A.V. and Wojtowicz, J.M., LAspartic acid induces a region of negative slope conductance in the current-voltage relationship of cultured spinal cord neurons, Brain Research, 237 (1982) 248-253. 20 Mannard, A. and Polosa, C., Analysis of background firing of single sympathetic preganglionic neurons of cat cervical nerve, J. Neurophysiol., 36 (1973) 398-408. 21 Mannard, A., Rajchgot, P. and Polosa, C., Effet of postimpulse depression on background firing of sympathetic preganglionic neurons, Brain Research, 126 (1977) 243-261. 22 Mayer, M.L., Westbrook, G.L. and Guthrie, P.B., Voltagedependent block by Mg2+ of NMDA responses in spinal cord neurones, Nature, 309 (1984) 261-263. 23 McKenna, K.E. and Schramm, L.P., Mechanisms mediating the sympathetic silent period: studies in the isolated spinal cord of the neonatal rat, Brain Research, 329 (1985) 233-240. 24 McLachlan, E.M. and Hirst, G.D., Some properties of preganglionic neurones in the upper thoracic spinal cord of the cat, J. Neurophysiol., 43 (1980) 1251-1265. 25 McLennan, H., Receptors for the excitatory amino acids in the mammalian central nervous system, Prog. Neurobiol., 20 (1983) 251-271. 26 Meech, R.W., Calcium-dependent potassium activation in nervous tissues, Ann. Rev. Biophys. Bioeng., 7 (1978) 1-18. 27 Mo, N. and Dun, N.J., Excitatory postsynaptic potentials in neonatal rat sympathetic preganglionic neurons: possible mediation by NMDA receptors, Neurosci. Lett., 77 (1987) 327-332. 28 Monaghan, D.T. and Cotman, C.W., Distribution of N-methylD-aspartate-sensitive L-3H-glutamate binding in rat brain as determined by quantitative autoradiography, J. Neurosci., 5 (1985) 2909-2919. 29 Monaghan, D.T. and Cotman, C.W., Distribution of [3H]kainic acid binding sites in rat CNS as determined by autoradiography, Brain Research, 252 (1982) 91-100. 30 Monaghan, D.T., Yao, D. and Cotman, C.W., Distribution of [3H]AMPA binding sites in rat brain as determined by quantitative autoradiography, Brain Research, 324 (1984) 160-164. 31 Morita, K., North, R.A. and Tokimasa, T., The calciumactivated potassium conductance in guinea-pig myenteric neutones, J. Physiol., 329 (1982) 341-354. 32 Murase, K. and Randir, M., Electrophysiological properties of rat spinal dorsal horn neurones in vitro: calcium-dependent action potentials, J. Physiol., 334 (1983) 141-153. 33 Murata, Y., Shibata, H. and Chiba, T., A correlative quantitative study comparing the nerve fibres in the cervical sympathetic trunk and locus of the somata from which they originate in the rat, J. Auton. Nerv. Syst., 6 (1982) 323-333. 34 Nishi, S., Yoshimura, M. and Polosa, C., Synaptic potentials and putative transmitter actions in sympathetic preganglionic neutones. In E Calaresu, J. Ciriello and C. Polosa (Eds.), Organization of the Autonomic Nervous System: Central and Peripheral Mechanisms, Liss, New York, 1987, pp. 15-26. 35 Nowak, L., Bregestowski, P., Ascher, P., Herbert, A. and Prochiantz, A., Magnesium gates glutamate-activated channels in mouse central neurones, Nature, 307 (1984) 462-465. 36 Polosa, C., Spontaneous activity of sympathetic preganglionic neurons, Can. J. Physiol. Pharmacol., 46 (1968) 887-896. 37 Rainbow, T.C., Wieczorek, C.M. and Halpain, S., Quantitative autoradiography of binding sites for [3H]-AMPA, a structural analogue of glutamic acid, Brain Research, 309 (1984) 173-177. 38 Rando, T.A., Bowers, C.W. and Zigmond, R.E., Localization of neurons in the rat spinal cord which project to the superior
1 Andersen, C.R., McLachlan, E.M. and Srb-Christie, O., Distribution of sympathetic preganglionic neurons and monoaminergic nerve terminals in the spinal cord of the rat, J. Comp. Neurol., 283 (1989) 269-284. 2 Ault, B., Evans, R.H., Francis, A.A., Oakes, D.J. and Watkins, J.C., Selective depression of excitatory amino acid induced depolarizations by magnesium ions in isolated spinal cord preparations, J. Physiol., 307 (1980) 413-428. 3 Backman, S.B. and Henry, J.L., Effects of glutamate and aspartate on sympathetic preganglionic neurons in the upper thoracic intermediolateral nucleus of the cat, Brain Research, 277 (1983) 370-374. 4 Brock, L.G., Coombs, J.S. and Eccles, J.C., Intracellular recording from antidromically activated motoneurones, J. Physiol., 122 (1953) 429-461. 5 Coote, J.H., The organisation of cardiovascular neurons in the spinal cord, Rev. Physiol. Biochem. Pharmacol., 110 (1988) 147-285. 6 Coote, J.H., McLeod, V.H., Fleetwood-Walker, S. and Gilbey, M.P., The response of individual sympathetic preganglionic neurones to microiontophoretically applied endogenous monoamines, Brain Research, 215 (1981) 135-145. 7 Coote, J.H. and Westbury, D.R., Intracellular recordings from sympathetic preganglionic neurones, Neurosci. Lett., 15 (1979) 171-175. 8 Dembowsky, K., Czachurski, J. and Seller, H., An intracellular study of the synaptic input to sympathetic preganglionic neurones of the third thoracic segment of the cat, J. Auton. Nerv. Syst., 13 (1985) 201-244. 9 Dun, N.J. and Mo, N., Inhibitory postsynaptic potentials in neonatal rat sympathetic preganglionic neurones in vitro, J. Physiol., 410 (1989) 267-281. 10 Dun, N.J. and Mo, N., In vitro effects of substance P on neonatal rat sympathetic preganglionic neurones, J. Physiol., 399 (1988) 321-333. 11 Eccles, J.C., Fatt, E and Koketsu, K., Cholinergic and inhibitory synapses in a pathway from motor-axon collaterals to motoneurones, J. Physiol., 126 (1954) 524-562. 12 Fernandez de Molena, A., Kuno, M. and Perl, E.R., Antidromically evoked responses from sympathetic preganglionic neutones, J. Physiol., 180 (1965) 321-335. 13 Gilbey, M.E, Coote, J.H., Fleetwood-Walker, S. and Peterson, D.E, The influence of the paraventriculo-spinal pathway, and oxytocin and vasopressin on sympathetic preganglionic neurones, Brain Research, 251 (1982) 283-290. 14 Gilbey, M.P., Peterson, D.E and Coote, J.H., Some characteristics of sympathetic preganglionic neurones in the rat, Brain Research, 241 (1982) 43-48. 15 Hancock, M.B. and Peveto, C.A., Preganglionic neurons in the sacral spinal cord of the rat: an HRP study, Neurosci. Lett., 11 (1979) 1-5. 16 Hotson, J.R. and Prince, D.A., A calcium-activated hyperpolarization follows repetitive firing in hippocampal neurons, J. Neurophysiol., 43 (1980) 409-419. 17 Ma, R.C. and Dun, N.J., Excitation of lateral horn neurons of the neonatal rat spinal cord by 5-hydroxytryptamine, Develop. Brain Res., 24 (1986) 89-98. 18 Ma, R.C. and Dun, N.J., Vasopressin depolarises lateral horn cells of the neonatal rat spinal cord in vitro, Brain Research, 348 (1985) 36-43.
•
i
188 cervical ganglion, J. Comp. Neurol., 196 (1981) 73-83. 39 Renshaw, B., Influence of discharge of motoneurones upon excitation of neighbouring motoneurones, J. Neurophysiol., 4 (1941) 167-183. 40 Schramm, L.P., Adair, J.R., Stribling, J.M. and Gray, L.P., Preganglionic innervation of the adrenal gland of the rat: a study using horseradish peroxidase, Exp. Neurol., 49 (1975) 540-553. 41 Spanswick, D., Lewis, D.I., Coote, J.C. and Logan, S.D., Sympathetic preganglionic neurones in the rat thoracic spinal cord slice, Neurosci. Lett., Suppl. 32 (1988) 535.
42 Watkins. J.C. and Olverman, H.J., Agonists and antagonists for excitatory amino acid receptors, Trends Neurosci., III (1987) 265-272. 43 Yoshimura, M., Potosa, C. and Nishi, S., Noradrenaline induces rhythmic bursting in sympathetic preganglionic neurons, Brain Research, 420 (1987) 147-151. 44 Yoshimura, M., Polosa, C. and Nishi, S., Afterhyperpolarization mechanisms in cat sympathetic preganglionic neuron in vitro, J. Neurophysiol., 55 (1986) 1234-1246.
181
Elsevier BRES 15769
Research Reports
Sympathetic preganglionic neurones in neonatal rat spinal cord in vitro: electrophysiological characteristics and the effects of selective excitatory amino acid receptor agonists David Spanswick and Stephen D. Logan Department of Physiology, The Medical School, Birmingham University, Birmingham (U. K.) (Accepted 20 February 1990)
Key words: Sympathetic preganglionic neuron; Rat spinal cord slice; Burst firing; Kainate; N-Methyl-D-aspartate; Quisqualate; Inhibitory postsynaptic potential
Intracellular recordings were made from 52 lateral horn neurones in thin slices of neonatal rat thoracolumbar spinal cord. Of these neurones 12 were spontaneously active and the remainder silent. A number of these cells could be activated antidromically by stimulation of ventral roots. The conduction velocity of the antidromic potential was estimated to be 0.9-2 m/s which is within the range reported for axons of sympathetic preganglionic neurones (SPNs). The membrane properties of antidromically identified SPNs were similar to other lateral horn neurones included in this study and comparable to those reported for SPNs by others. Spontaneous burst firing was recorded in 3 neurones and activity in a further 5 neurones was characterized by the discharge of an action potential followed by an afterhyperpolarization potential (AHP) of peak amplitude 3-13 mV and duration 0.5-4 s. The AHP had an initial fast component (fAHP) which was sensitive to the potassium channel blocker tetraethylammonium (TEA), and a second slower component (sAHP) which was both sensitive to extracellular calcium and TEA. The effects of the selective excitatory amino acid receptor agonists N-methyI-D-aspartate (NMDA), kainate and quisqualate were investigated by superfusion of the agonists, at known concentrations (100 nM to 100 ~M). These agonists induced concentration-dependent depolarizations which were primarily associated with a reduction in neuronal input resistance. NMDA-indueed depolarizations were potentiated in the absence of magnesium. In a number of neurones NMDA, kainate and quisqualate (1-50 ~M) induced, in addition to a depolarizing response, the discharge of small (1.5-6.5 mV), brief (7-20 ms) IPSPs which were reversed just below resting membrane potential at -64 mV. These results suggest that both NMDA and non-NMDA receptors may have an important role in the excitation of SPNs and of inhibitory interneurones presynaptic to SPNs. INTRODUCTION Sympathetic preganglionic neurones (SPNs) are utilized in cardiovascular control and are probably the major site of integration of sympathetic nerve activity in the CNS 5. The majority of SPNs are located in the intermediolateral nucleus (IML) of the thoracolumbar spinal cord 1'15,33,38,40. Investigations into the chemical nature of synaptic transmission in the I M L have shown that excitatory amino acids ( E A A s ) excite SPNs in vivo 3"6'13 and in vitro in adult cat spinal cord slice preparation 34. Recently it has been suggested that E A A s may mediate fast synaptic transmission in SPNs, in the neonate rat spinal cord slice, and that the receptor responsible is the N-methyl-o-aspartate ( N M D A ) subtype 27. In addition to this, inhibitory postsynaptic potentials (IPSPs), which may be mediated by glycine or a glycine-like substance, have been reported to occur spontaneously and in the presence of N M D A , suggesting that inhibitory interneurones, presynaptic to SPNs, are
activated through N M D A receptors, and further, indirect evidence was provided for the existence of an inhibitory pathway impinging on SPNs which is analogous to the Renshaw cell circuit of ventral horn motoneurones 9. Electrophysiologicai, biochemical and pharmacological studies have shown that at least 3 types of E A A receptors exist, these being defined by the selective agonists N M D A , kainate and quisqualate 25,42. We have utilized the isolated thoracolumbar spinal cord slice preparation to investigate the effects of E A A s on lateral horn cells including neurones identified as SPNs by antidromic stimulation of ventral rootlets. We report, for the first time, spontaneous burst firing in lateral horn neurones, and also a long-lasting, two-component afterhyperpolarization potential which is similar to that reported for the SPN in the adult cat in vitro 44. We further show that in addition to N M D A , the selective E A A receptor agonists kainate and quisqualate excite SPNs and give rise also to IPSPs suggesting that n o n - N M D A receptors may have an important role in excitation of SPNs and of inhibitory
Correspondence: S.D. Logan, Department'of Physiology, The Medical School, Birmingham University, Birmingham B15 2Tr, U.K. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
182 i n t e r n e u r o n e s p r e s y n a p t i c to SPNs. S o m e of these results h a v e b e e n p r e s e n t e d p r e v i o u s l y in abstract f o r m 41.
MATERIALS AND METHODS Sprague-Dawley rats 9-22 days old were employed in this study. The procedure used to obtain spinal cord slices from neonatal rats was similar to that described by other investigators 17'32. The animals were anaesthetized with ether and a laminectomy performed to reveal the spinal cord. Following taminectomy, a 1-1.5 cm section of the thoracolumbar spinal cord was excised and placed in cold (4 °C), aerated (95% 02, 5% CO2) artificial cerebrospinal fluid (ACSF). The pia mater was removed and the spinal segment cut with a razor blade to form one or two approximately 5 mm sections. The sections were fixed to an agar block and the tissue-agar assembly in turn fixed to a petri-dish with cyanoacrylic glue (Superglue). The petri-dish was secured to the cutting chamber of a modified Oxford Vibratome and filled with cold (4 °C) aerated ACSF. Several 300-450/~m slices of spinal cord were cut, care being taken to ensure that some slices were removed with intact ventral rootlets. The first slice was generally discarded and the remaining slices transferred to an incubation chamber where they were maintained in aerated ACSF at 25 °C for at least 1 h prior to recording. One slice was transferred to the recording chamber and continuously superfused at a rate of 3 ml/min with an ACSF solution of the following composition (mM): NaCI 127, KC1 1.9, KH2PO 4 1.2, CaCI 2 1.2, MgSO 4 1.3, NaHCO 3 26, glucose 10, pH 7.4. The solution was saturated with 95% 02, 5% CO 2 and the temperature maintained at 34 + 0.5 °C. Intracellular recordings were obtained from neurones located in the lateral horn of the thoracolumbar spinal cord slices with glass microelectrodes, backfiiled with 3 M potassium acetate, of impedance 60-150 M~2. Stable intracellular recordings were obtained from neurones for up to 6 h. A high impedance bridge amplifier (WPI 'Chopper' SVC2000) was employed to inject current through the microelectrode and to record signals which were displayed on an oscilloscope (Tektronix 5113). Recordings were also directed to the following data storage facilities: a chart recorder (Gould RS3200);
A
an X - Y plotter (Gould Colourwriter) via a digital storage oscilloscope (Gould 1425); a tape recorder (Racal Store 4) via an oscilloscope (Tektronix 502A) with an amplification (× 100) facility. Electrical stimulation of the ventral roots was achieved via a concentric bipolar electrode positioned close to the rootlets. Drugs were dissolved in ACSF to a known concentration and applied to the slice by superfusion at a rate of 3 ml/min. The following agents were employed: NMDA, kainic acid, quisqualic acid, tetraethylammonium chloride (TEA) (Sigma). The following salts were also used: NaCI, KCI, NaHCO> D-glucose (Fisons); MgSO~, KH2PO ~ CaCI2.6H20 (BDH).
RESULTS
Membrane properties Stable i n t r a c e l l u l a r r e c o r d i n g s w e r e o b t a i n e d f r o m 52 n e u r o n e s l o c a t e d in the lateral h o r n o f t h o r a c o l u m b a r spinal c o r d slices. O f t h e s e n e u r o n e s , 12 w e r e s p o n t a n e ously active and
the r e m a i n d e r
silent.
Lateral
horn
n e u r o n e s had resting m e m b r a n e p o t e n t i a l s o f - 6 0
+ 6
m V ( m e a n + S . D . ) with a r a n g e o f - 5 0 to - 7 8 mV. T h e input resistance, m e a s u r e d as the v o l t a g e d e f l e c t i o n i n d u c e d by h y p e r p o l a r i z i n g r e c t a n g u l a r c u r r e n t pulses ( 0 . 0 5 - 0 . 5 n A , 100-200 ms), was 110 + 24 Mr2 with a r a n g e of 7 5 - 1 6 0 MI2 (n = 21) (see Fig. 1 A , B ) . A c t i o n p o t e n t i a l s ( b o t h s p o n t a n e o u s a n d electrically e v o k e d by direct d e p o l a r i z i n g c u r r e n t i n j e c t i o n ) had a p e a k amplitude, m e a s u r e d f r o m t h r e s h o l d of 58 + 8 m V with a r a n g e of 4 6 - 7 5 m V and d u r a t i o n 2.8 + 0.6 ms with a r a n g e of 1 . 8 - 3 . 6 ms (Fig. 1C). S t i m u l a t i o n of v e n t r a l roots e v o k e d an a l l - o r - n o n e spike p o t e n t i a l in 5 of 8 lateral h o r n n e u r o n e s tested (Fig. 1D). T h e c o n d u c t i o n v e l o c i t y of
B tm -sl~v
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_..A ~ Fig. 1. Passive membrane properties and action potentials evoked in rat sympathetic preganglionic neurons (SPNs). A: eleetrotonic potentials evoked in a neurone. B: plot of voltage-current relations of neurone A. C: action potentials evoked by a depolarizing current pulse. D: antidromic spike induced by stimulation of ventral rootlets. The conduction velocity was calculated to be 1.1 m/s. E: antidromicaUy evoked action potentials showing the IS and SD components of the spike. High frequency stimulation and hyperpolarization of the neurone abolishes the SD component leaving only the IS response.
183 these axons was estimated to be 1.3 + 0.2 m/s (mean +_ S.E.M.) with a range of 0.9-2 m/s. At low-frequency stimulation (0.2-1 Hz) a spike potential was always evoked. At higher frequency stimulation, up to 10 Hz, a pronounced inflexion in the rising phase of the antidromic action potential was apparent (Fig. 1E). This is generally attributed to antidromic invasion of the initial segment (IS) and the soma-dendritic region (SD) of the neurone 4. The SD component of the action potential was abolished by membrane hyperpolarization. The membrane properties of antidromically identified SPNs were similar to other lateral horn neurones included in this study, and comparable to those reported by other investigators for lateral horn neurones 17'1s and for identified SPNs 1°.
Spontaneous activity Burst firing. Spontaneous burst firing was recorded in 3 lateral horn neurones. Bursts were characterized by a slow depolarizing shift, with superimposed action potentials, followed by a relatively rapid hyperpolarization before the start of the next burst (see Fig. 2A). The frequency of bursting in these 3 neurones was 0.4, 1.0 and 1.6 Hz. The number of spikes per burst varied between 7 and 13. Superfusion of medium containing low calcium reduced the duration of the burst, the number of action potentials per burst, the amplitude and duration of the interburst hyperpolarization and increased the frequency of bursting (Fig. 2B2). A 2 x normal calcium-containing medium (2.4 mM) maintained burst firing, augmented the amplitude and duration of the interburst hyperpolarization and the overshoot of action potentials associated with the bursts (Fig. 2B3). Action potentials also increased in duration in the presence of 2× normal calcium and an increase in synaptic activity was apparent. Superfusion of T E A (30 mM) reduced the neurones to discharging action potentials in triplets and doublets. Bursting was restored by returning to a normal calciumcontaining medium (1.2 mM). Afterhyperpolarization potential (AHP). Spontaneous activity recorded in 4 lateral horn neurones (two identified as SPNs by the appearance of an antidromic spike following stimulation of ventral rootlets) was characterized by the discharge of a single action potential followed by a long-lasting AHP (Fig. 3A). The AHP comprised of an early fast component (fAHP) followed by a late slow component (sAHP) (Fig. 3A1). The AHP was associated with a reduction in the neuronal input resistance, the most marked reduction being associated with the early phase of the AHP (Fig. 3A2) , and had maximal amplitude of 3-13 mV and duration 0.5-4 s, respectively. The frequency of action potential discharge in these neurones
ranged from 0 to 0.3 Hz. Superfusion of a low calciumcontaining medium reversibly abolished the late component AHP and increased the frequency of action potential discharge, up to as much as 1 Hz (Fig. 3B2). Addition of a 2 x normal calcium-containing medium (2.4 mM calcium chloride) reduced the frequency of action potential discharge to 0-0.1 Hz, and augmented the amplitude and duration of the late component AHP (Fig. 3B3). Superfusion of the potassium channel blocker T E A (30 mM) or TEA in a low calcium-containing ACSF abolished both the early and late components of the AHP (Fig. 3B4, B5). In one neurone a long-duration AHP was induced by superfusion of a low magnesium-containing ACSE A further feature of these neurones was that following bursts of 3-5 action potentials a large amplitude, long duration, hyperpolarization was evoked with a delay between the termination of the last action potential and the onset of the hyperpolarization (Fig. 3A3).
A
0.4s B
J2ornv
~) 2 Low calcium ( OmM )
] 20my ( 2.4mM )
8s
Fig. 2. Spontaneous burst firing in lateral horn neurones. A: spontaneous bursting recorded in one neurone. B: the effects of calcium on burst firing in a lateral horn neurone. Bl: spontaneous burst firing in normal C a 2+ (1.2 mM). Bz: superfusion of nominally zero Ca2+-containingsolution for 2 min reduced the burst duration and interburst interval amplitude and duration. The frequency of bursting also increased in nominally zero calcium. The peak amplitude of spike discharge in this and subsequent figures were truncated due to the limited frequency response of the pen recorder. Ba: superfusion of a 2x normal calcium (2.4 mM)-containing solution augmented the burst duration and interburst amplitude and duration. The frequency of bursting decreased in high calcium solution.
184
Excitatory amino acids The actions of the selective E A A agonists N M D A , kainate and quisqualate on lateral horn neurones, including 5 neurones identified antidromically as SPNs, was investigated by superfusion of the agonists, at known concentrations (100 nM to 100yM), in the medium bathing the slice.
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1.3mM Magnesium
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3 2 x N o r m a l C a l c i u m ( 2 , 4 mM )
ilpprox. 40 s
lll!lllllal!!JHi!!l!!H!!!ll 5 TEA ( 3 0 mM ) + Low Calcium ( 0 mM ) _.J 20 mV
J 20mV 10s Fig. 4. Effects of excitatory amino acids (EAAs) on a lateral horn neurone and an identified SPN. A: depolarizaton induced by quisqualate (20 pM) superfused for 2 s to an identified SPN. Repeated hyperpolarizing rectangular current pulses (0.1 hA, 200 ms) were used to evoke hyperpolarizing electrotonic potentials (downward deflections of the trace) which indicate neuronal input resistance. Quisqualate-induced depolarization at high concentrations was accompanied by intense neuronal discharge. The peak amplitude of spike discharge in this and other figures were truncated due to the limited frequency response of the pen recorder. B: the effects of NMDA and the antagonist action of magnesium on NMDA-induced responses. BI: depolarization induced by NMDA (100 #M) superfused for 8 s to a lateral horn neurone. The response was associated with small increases and decreases in neuronal input resistance and action potential discharge. B2: the effect of the same concentration of NMDA in the absence of magnesium from the bathing medium. Depolarization of the neurone was accompanied by intense neuronal discharge and primarily a reduction in neuronal input resistance. C: depolarization induced by kainate (50 pM) superfused for 2 s to an identified SPN. Depolarization was prolonged, accompanied by intense neuronal discharge, and a reduction in neuronal input resistance in the early phase of the response.
15s
Fig. 3. The effects of calcium and the potassium channel blocker TEA on the AHP. A: the hyperpolarization following the discharge of a single (A 1 and A2) and a burst of 3 (A3) action potentials. At: demonstration of two-component AHP. Note the AHP has an initial fast component (fAHP) and a second slower prolonged phase (sAHP). A2: an AHP with associated conductance change. A3: large amplitude, long duration hyperpolarization following a burst of action potentials. Note also the delay following the last action potential and the onset of the hyperpolarization. B: records from two lateral horn neurones including one identified SPN showing the effects of calcium and TEA on the AHP. Bt: control showing spontaneous action potentials followed by an AHP. B2: superfusion of nominally zero calcium-containing ACSF abolished the sAHP and increased the rate of action potential firing. B3: superfusion of 2x normal calcium-containing ACSF (2.4 mM calcium chloride) restored and augmented the amplitude and duration of the sAHP. The rate of firing decreased in 2x normal calcium-containing ACSE B& superfusion of TEA (30 mM) reduced both the fAHP and sAHP and increased the rate of firing. Bs: superfusion of TEA and nominally zero calcium-containing ACSF reduced both components of the AHP. Action potentials in these figures were truncated due to the limited frequency response of the pen recorder.
Quisqualate (100 nM to 50 g M ) was superfused for 2-30 s to 10 lateral horn neurones, including 5 identified SPNs. Quisqualate induced c o n c e n t r a t i o n - d e p e n d e n t depolarizing responses in all cells investigated. T h e response was rapid in onset and offset (i.e. a response was observed within a few seconds of the drug reaching the bath, and the time course of the response, allowing for dilution effects, closely p a r a l l e l e d that of the drug application), and at higher concentrations (10-50 p M ) was associated with a reduction in the neuronal input resistance (Fig. 4A). D e p o l a r i z a t i o n in all of these cells reached threshold and gave rise to the discharge of action potentials in the presence of quisqualate at 10-50 g M . N M D A (1-100 p M ) was superfused for 2-30 s to 16 lateral horn neurones, including 5 identified SPNs. N M D A induced d o s e - d e p e n d e n t depolarizing responses
185 A
•
Quis IOJuM 30s
•
NMDA IO~uM30s
C
I^^-IV •
Keln lOjJM30s
D I Control
2 Qulsqualete onset 3 NMDA onset 4 Kainate onset
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5 Kainate offset
Fig. 5. EAA-induced IPSPs in an identified SPN. A: superfusion of quisqualate (10/zM) for 30 s evoked a depolarization and IPSPs primarily in the onset and peak of the depolarizing response. B: superfusion of NMDA (10/~M) for 30 s also evoked a depolarizing response and IPSPs. C: kainate (10/~M) superfused for 30 s induced a depolarizing response and IPSPs which were primarily associated with the offset of the depolarizing response. D: IPSPs evoked in the presence of EAAs, shown on an expanded time scale. The arrows 1-5 shown on traces A, B and C correspond with traces 1-5 shown in D. DI: control wash. D2: IPSPs evoked during the onset of the depolarizing response induced by quisqualate (A). D3: IPSPs evoked during the onset of the depolarizing response to NMDA (B). Few IPSPs were evoked during the onset of the kainateinduced (C) depolarizing response (D4) but were apparent during the peak and offset of the response (Ds).
in 14 of the neurones, including the 5 identified SPNs, and had no effect on two neurones, either in the presence or absence of magnesium. NMDA-induced responses were rapid in onset and offset and potentiated in the absence of magnesium from the bathing medium (see Fig. 4-B1,2). At low concentrations (1-10/zM) NMDAinduced responses were associated with small increases or no measurable change in neuronal input resistance. At higher concentrations (20-50 pM), or in the absence of magnesium from the bathing medium the response was associated with a reduction in neuronal input resistance, and reached threshold giving rise to action potential discharge. Kainate (1-50/~M) was superfused for 2-30 s to 12
lateral horn neurones, including 5 identified SPNs. Kainate induced dose-dependent depolarizing responses in all cells investigated. The response in 8 neurones was rapid in onset and offset. In 3 neurones high concentrations of kainate (20-50/zM for 2-10 s) induced depolarizing responses which were rapid in onset and long lasting (Fig. 4C). High concentrations of kainate (10-50 /zM) induced depolarizing responses which reached threshold giving rise to vigorous action potential discharge. Kainate-induced responses were associated with a reduction in neuronal input resistance.
Excitatory amino acids and IPSPs Superfusion of N M D A , kainate and quisqualate (1-50 /~M) to 3, 2 and 5 lateral horn neurones, respectively, induced, in addition to a depolarizing response, the discharge of IPSPs (Fig. 5). Of these lateral horn neurones two were identified as SPNs, these neurones both responding to all 3 agonists. The IPSPs were fast and small with a peak amplitude ranging between 1.5 and 6.5 mV and duration 7-20 ms. IPSPs smaller in amplitude than 1.5 mV were ignored as this was close to the noise level of the system. These IPSPs were blocked by strychnine (0.5-5 pM) and in 3 neurones including one identified SPN reversed at membrane potentials o f - 6 2 , -64 and -65 mV, respectively. A further feature of this type of response was that in the presence of N M D A and quisqualate (1-10 pM) IPSPs evoked in the depolarizing response could be associated with the onset, peak and offset of the response (see Fig. 5A, B, D E and D3). However, IPSPs evoked in the presence of kainate (1-10 pM) were not as apparent in the onset of the depolarizing response as with the other two agonists, but associated with the peak and offset of the response (see Fig. 5C, D4 and D5). DISCUSSION The main observation made in this study is that lateral horn cells are excited by the selective E A A receptor agonists N M D A , kainate and quisqualate and further that spinal interneurones impinging on lateral horn cells may also be activated through these 3 E A A receptor subtypes. The type of lateral horn cells recorded from is difficult to establish in the slice preparation. However, of the 8 out of 52 lateral horn neurones tested, 5 could be activated antidromically by stimulation of ventral roots. These neurones had conduction velocities of about 1 m/s, within the range reported for SPNs by other investigators in the slice preparation 1° and in the intact animal TM. Spontaneous burst firing was recorded in 3 lateral horn neurones. The frequency of bursting was augmented and the interburst interval duration and amplitude, and the
186 duration of action potentials were reduced in low calcium solution. In 2x normal calcium-containing solution the reverse effects were observed. Bursting was also reduced by the K+-channel blocker TEA. These results imply that the bursting behaviour is dependent upon calcium and potassium conductances. This is the first report of spontaneous burst firing in lateral horn neurones or putative SPNs and raises the interesting possibility that a subpopulation of these neurones may be capable of this type of behaviour. There have been no reports of burst firing in intact preparations, but regular low-frequency firing has been reported in isolated deafferented fragments of thoracic spinal cord 2°'36, and it has been suggested that this type of activity could be due to pacemaker activity. Furthermore, recent investigations have shown noradrenaline to induce rhythmic bursting in SPNs in the adult cat spinal cord slice preparation 43. It may be, therefore, that under certain conditions, SPNs adopt a burst firing pattern of behaviour. Spontaneous activity recorded in 5 lateral horn neurones, including two identified SPNs, was characterized by the discharge of one or occasionally several action potentials followed by a two-component AHP. The A H P comprised of an initial fast component (fAHP) which was sensitive to the potassium channel blocker T E A and insensitive to calcium, and a second slow component (sAHP) which was both T E A and calcium sensitive. The f A H P would therefore appear to be mediated by potassium and the sAHP by calcium-activated potassium conductances. These observations are similar to those reported for the A H P following an action potential in SPNs of the adult cat in vitro 44 and for other central and peripheral neurones 16'26"31. Superfusion of low calciumcontaining ACSF abolished the sAHP and gave rise to an increased rate of spontaneous action potential discharge suggesting that the sAHP is important in the control of the rate of firing in these neurones. In relation to this in SPNs in vivo there is a period of silence 21 which is associated with a hyperpolarization lasting between 40 and 500 ms 7'8'12'24. The hyperpolarization observed in SPNs in vitro in the cat spinal cord slice is considerably longer, around 2.8 s44. In addition to this long-lasting AHP, on occasions bursts of two or more action potentials gave rise to a long-lasting large amplitude hyperpolarization which was immediately preceded by a delay between the last action potential and the onset of the hyperpolarization. Although the most likely explanation for this would be an increase in the Ca2+-sensitive sAHP coupled with a reduction or complete abolition of the f A H P we cannot rule out the possibility that this delay may represent a synaptic delay and hence a synaptic component which could be analogous to Renshaw inhibition 11'~. Such a Renshaw-type circuit has been sug-
gested to exist in SPNs in the neonate rat spinal cord slice in vitro `) and in hemisected rat spinal cord preparations 23. In 5 lateral horn neurones, including two identified SPNs, the selective E A A agonist N M D A induced, in addition to a depolarizing response, small (1.5-6.5 mV), brief (7-20 ms) IPSPs, which reverse at around -64 mV and are blocked by strychnine. This is in agreement with a recent study of SPNs where strychnine-sensitive chloride mediated IPSPs similar to those described above were evoked in the presence of N M D A 9. These authors attributed this type of response to the activation of inhibitory interneurones, which are presynaptic to SPNs, by NMDA. In studies carried out here we show that in addition to NMDA, the selective non-NMDA receptor agonists kainate and quisqualate also activate 'unitary' IPSPs suggesting that both non-NMDA and N M D A receptors may activate inhibitory spinal interneurones. One further interesting observation is that IPSPs activated in the presence of N M D A and quisqualate occur mainly during the onset and peak of the depolarizing response whereas IPSPs activated in the presence of kainate occur mainly at the peak and offset of the depolarizing response. This suggests that kainate receptors may have either a more indirect action on inhibitory interneurones than do N M D A and quisqualate, or the kainate receptor is situated at a site distant from N M D A and quisqualate receptors which may be coiocalized on inhibitory spinal interneurones. This would be in agreement with receptor autoradiographic studies in other parts of the CNS which have shown N M D A sites 2s and quisqualate sites 3°'37 to be codistributed whereas kainate sites tend to have a dissimilar distribution and complement N M D A sites 29. N M D A excited 14 of 16 lateral horn neurones including 3 identified SPNs. NMDA-mediated depolarizing responses were potentiated in magnesium-free solution suggesting that the N M D A channel of lateral horn neurones and SPNs is gated by magnesium ions. This is in agreement with other studies carried out on SPNs in the neonate rat spinal cord slice preparation 9"2v and on other central neurones 2'19'22'35. The major observation made in studies carried out here is that in addition to NMDA, the selective non-NMDA agonists kainate and quisqualate also excited all lateral horn neurones, including 3 and 5 identified SPNs, respectively. These agonists either induced silent neurones to discharge action potentials or increased the firing rate of spontaneously active neurones, and the response was generally associated with a reduction of the neuronal input resistance. These findings raise the possibility that in addition to N M D A receptors, which have been suggested to mediate fast synaptic transmission in these neurones in the neonate rat
187 through a n o n - N M D A , kainate or quisqualate receptor 34.
spinal cord slice preparation 27, n o n - N M D A receptors may have an important role also in exciting SPNs. In
Acknowledgements. We are indebted to the Friedreich's Ataxia Group (U.K.) for financialsupport. We thank Professor J.H. Coote for helpful discussionsand for reading the manuscript and Dr. D.I. Lewis for help in preliminary investigations.
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