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Inhibitory and indirect excitatory effects of dopamine on sympathetic preganglionic neurones in the neonatal rat spinal cord in vitro
Brain Research 818 Ž1999. 397–407
Research report
Inhibitory and indirect excitatory effects of dopamine on sympathetic preganglionic neurones in the neonatal rat spinal cord in vitro Simon J. Gladwell ) , John H. Coote Department of Physiology, School of Medicine, UniÕersity of Birmingham, Edgbaston, Birmingham, B15 2TT, UK Accepted 8 December 1998
Abstract Regions of the thoraco-lumbar spinal cord containing sympathetic preganglionic neurones are rich in dopamine terminals. To determine the influence of this innervation intracellular recordings were made from antidromically identified sympathetic preganglionic neurones in Ž400 mm. transverse neonatal rat spinal cord slices. Dopamine applied by superfusion caused a slow monophasic hyperpolarisation in 46% of sympathetic preganglionic neurones, a slow monophasic depolarisation in 28% of sympathetic preganglionic neurones and a biphasic effect consisting of a slow depolarisation followed by a slow hyperpolarisation or vice-versa in 23% of sympathetic preganglionic neurones. Three percent of sympathetic preganglionic neurones did not respond to the application of dopamine. Low Ca2qrhigh Mg 2q Krebs solution or TTX did not change the resting membrane potential but abolished the slow depolarisation elicited by dopamine, indicating this was synaptic and did not prevent the dopamine induced hyperpolarisation. The dopamine induced slow hyperpolarisation was mimicked by the selective D1 agonists SKF 38393 or SKF 81297-C and blocked by superfusion with the D1 antagonist SCH 23390. It was not prevented by superfusion of the slices with alpha 1 or alpha 2 or beta-adrenoceptor antagonists, whereas the inhibitory or excitatory actions of adrenaline were prevented by alpha 1 or alpha 2 antagonists, respectively. The dopamine induced slow depolarisation occurring in a sub-population of sympathetic preganglionic neurones was mimicked by quinpirole, a D 2 agonist, and blocked by haloperidol, a D 2 antagonist. Haloperidol did not block the dopamine induced hyperpolarisations. Dopamine also induced fast synaptic activity which was mimicked by a D 2 agonist and blocked by haloperidol. D1 agonists did not elicit fast synaptic activity. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Sympathetic preganglionic neurone ŽSPN.; Dopamine ŽDA.; SKF 38393; Spinal cord
1. Introduction There is now considerable evidence that dopamine ŽDA. has a neurotransmitter role in the spinal cord separate from being a precursor to noradrenaline w11,25,29x. The evidence initially depended on the different distribution of DA in microdissected regions of the cat spinal cord compared to other catecholamines w23x and its differential decline compared to noradrenaline following spinal cord transection w35x. More recently DA has been recognised using a specific antibody and shown to be present in nerve terminals throughout the spinal cord w29,40,49,55x These nerve terminals arise from spinally projecting neurones in, the dorsal and posterior hypothalamus w6,7x, the paraventricular nucleus w53,54x and possibly the substantia nigra of the mid brain w10x. ) Corresponding author. [email protected]
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Specific regions of the spinal grey matter are targeted by the DA fibres; laminae III and IV of the dorsal horn, lamina X and the thoracic intermediolateral cell column ŽIML. displaying highest levels, as measured by tyrosine hydroxylase immunoreactivity in rats pre-treated with 6hydroxydopamine Ž6-OHDA. to remove noradrenaline fibres w21x. Of all these regions the IML has the highest density of enzyme, confirming the biochemical estimations of DA w23,3x. This would indicate that the amine has an important influence on sympathetic activity since the IML is where the majority of sympathetic preganglionic neurones ŽSPN. are located w1,30,43–45,52x. The first study to examine this influence used microiontophoresis to eject DA into the vicinity of identified extracellularly recorded SPN in the cat spinal cord in vivo w17x. It was shown that spontaneous or glutamate initiated activity was inhibited by DA. Consistent with this is the recent demonstrations that intrathecal application of DA receptor agonists to the thoracic cord of the rat causes a fall in
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blood pressure and heart rate w42x. In contrast Simon and Schramm w50x found that in acutely spinalised rats the amine increased activity in the renal sympathetic nerve, when it was applied by intrathecal superfusion to the thoracic spinal cord. In view of this conflicting data the functional significance of the DA innervation of sympathetic nerves is unclear. Part of the reason for this is that iontophoresis or intrathecal techniques fail to discriminate between sites of action of a transmitter. We therefore have studied the actions of DA on single SPN recorded intracellularly in slices of rat spinal cord in vitro.
2. Materials and methods 2.1. Spinal cord preparation The preparation of thin transverse slices from neonatal rats has been previously documented by this group w32x. Briefly, Wistar rats Ž8–16 days old. were taken and deeply anaesthetised with diethyl ether and a dorsal laminectomy performed. A 10–15 mm section of middle to upper spinal cord was removed and placed onto moistened filter paper where the dura and pia mater were dissected off. This section was then trimmed to leave a 7–8 mm piece of upper thoracic spinal cord which was placed into a mould filled with agar ŽBDH, 1.5%. which had been preboiled and allowed to cool to 35–408C. The mould was topped up with agar and then immersed in cold Ž48C. artificial cerebrospinal fluid ŽaCSF. to set. The agar block containing the section of cord was glued Žcyanoacetate. to the cutting stage of a Vibroslice ŽCamden Instruments. where 400 mm transverse slices were cut. These slices were transferred to an incubation chamber containing oxygenated Ž95%O 2r5% CO 2 . aCSF at room temperature. The composition of the normal aCSF was ŽmM.: NaCl 127; KCl 1.9; KH 2 PO4 1.2; MgSO4 1.3; NaHCO 3 26; D-Glucose 10; CaCl 2 2.5, pH s 7.4. For low Ca2qrhigh Mg 2q aCSF the following composition was used ŽmM.: NaCl 127; KCl 1.9; KH 2 PO4 1.2; MgSO4 1.3; NaHCO 3 26; D-Glucose 10; CaCl 2 0.2, MgCl 2 5.0, pH s 7.4. The slices were incubated for at least an hour prior to any recordings being made. As required a slice was transferred to the recording chamber and was continually superfused with oxygenated aCSF Ž3 ml miny1 , 318C..
2.2. Electrophysiology Neurones located in the lateral horn were impaled with 3 M potassium acetate filled glass microelectrodes Žtip resistance 45–105 M V .. Signals were amplified using an Axoclamp-2A ŽAxon Instruments. intracellular preamplifier and displayed on two oscilloscopes Ži. a Gould 1604 in refresh mode on a short time base and Žii. a Gould OS4020 in rolling display mode on a long time base. The signal was also directed to, a DC chart recorder ŽGould TA240. for constant recording of membrane potential during intracellular recording, and an FM tape recorder ŽRacal Store 4. for data storage and off-line analysis. SPN were identified by stimulating antidromically the ventral root exit zone with a concentric bipolar stimulating electrode ŽSNE-100, Clark-Electromedical, 1–100 nA 0.02 ms.. Cell input resistance was determined by intracellular negative current injections across the bridge of the preamplifier. 2.3. Chemicals Drugs used in this study were dissolved in aCSF and added to the superfusate in known concentrations. 3-Hydroxytyramine ŽDA. readily oxidised in the aCSF, it was therefore prepared immediately prior to its superfusate application. The following drugs were used. 3-Hydroxytyramine ŽDA, Sigma., adrenaline ŽAdr, Sigma., haloperidol ŽSigma., SKF 38393 ŽŽ".-1-Phenyl-2,3,4,5-tetrahydro-1 H-3-benzazepine-7,8-diol hydrochloride, RBI., quinpirole ŽRBI., SCH-23390 Ž RŽ".-7-Chloro-8-hydroxy-3methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride, RBI., yohimbine ŽSigma., prazosin ŽSigma., propranolol ŽSigma.. SKF 81297-C Ž6-Chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1 H-3-benzazepine hydrobromide. was a gift from SmithKline Beecham. Results are expressed as means " standard deviation ŽS.D..
3. Results 3.1. Electrophysiological characteristics Intracellular recordings of up to 3 h were made from neurones located within the IML region of the spinal cord. Recordings of this duration allowed for a sufficient period
Fig. 1. The actions of dopamine ŽDA. on the resting membrane potential of four different antidromically identified sympathetic preganglionic neurones. Each trace is from a different neurone. ŽA. The application of DA Ž10y4 M. for 30 s, indicated by the arrow, caused a slow monophasic hyperpolarisation. Resting membrane potentials y58 mV. ŽB. The application of DA Ž10y4 M. for 30 s, indicated by the arrow, caused a slow monophasic depolarisation. Resting membrane potentials y62 mV. ŽC. The application of DA Ž10y4 M. for 30 s, indicated by the arrow, caused a biphasic response consisting of a slow depolarisation followed by a slow hyperpolarisation. Resting membrane potentials y57 mV. ŽD. The application of DA Ž10y4 M. for 30 s, indicated by the arrow, caused a biphasic response consisting of a slow hyperpolarisation followed by a slow depolarisation with an increase in synaptic activity Ždemonstrated by a thickening of the trace, arrow. and a train of action potentials. Resting membrane potentials y60 mV.
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of recovery between drug applications to avoid tachyphalaxis. All these neurones were identified as SPN by eliciting an antidromic action potential following stimulation of the ventral root exit zone. Their identification was further confirmed on the basis of characteristic electrophysiological properties described below. In addition some of the intracellular recording electrodes were filled with a Biocytin solution Ž1.5% in 1.5 M potassium methyl sulphate. which was injected into impaled cells by passing a depolarising current Ž1.5 nA, q0.5 nA pulses, 85 ms duration 0.5 Hz for 10 to 20 min.. Subsequent immunocytochemical processing of the slice confirmed the location in the lateral horn and the typical morphology of the neurone as SPN w43x, when viewed with a Zeiss Photomicroscope III w27x. Immediately after intracellular penetration the majority of neurones displayed spontaneous action potential discharge up to a maximum of 20 Hz. In the majority of cases 82 neurones out of a total of 111 Ž82r111. this activity ceased after 1–2 min of stable membrane potential recording; some neurones continued to fire action potentials spontaneously Ž29r111. and some also demonstrated a degree of membrane oscillation Ž24r111. in which a fast depolarising transient is followed by a slower hyperpolarising phase. The remaining SPN were silent but could be induced to fire action potentials by injecting a depolarising current through the recording electrode or by antidromic stimulation via the bipolar electrode in the ventral root exit zone. The 111 SPN had resting membrane potentials in the range y46 to y72 mV Žmean 60.5 " 7.3 mV. and a cell input resistance which was calculated from the voltage deflection of intracellular hyperpolarising pulses of 72 " 27 M V Žrange 21 to 117 M V .. Both spontaneous and antidromically induced action potentials had mean amplitudes of 81 " 8.9 mV Žrange 65–98 mV. and durations of 3.0 " 1.1 ms Žrange 1.3–4.6 ms.. All of the neurones displayed a characteristic distinct after hyperpolarisation following an action potential discharge; this had a mean amplitude of 18 " 9 mV Žrange 8–29 mV. and a mean duration of 98 " 36 ms Žrange 24–148 ms.. 3.2. Actions of dopamine DA was applied by superfusion in concentrations ranging from 10 nM to 10 mM for between 15 s and 45 s at a flow rate of 3 ml miny1 . A submaximal concentration of 100 mM for 30 s was routinely used as application of higher doses yielded no further significant change in membrane potential. On 111 SPN the catecholamine caused either a slow hyperpolarisation Žtime to peak mean s 95 " 32 s, n s 51, Fig. 1A., a slow depolarisation Žtime to peak mean s 63 " 26, n s 31, Fig. 1B, or a biphasic response Ž n s 26. which consisted of either a slow depolarisation followed by a slow hyperpolarisation Ž n s 16, Fig. 1C. or vice versa Ž n s 10, Fig. 1D.. Three neurones showed no change in
membrane potential on the application of DA when the slices were perfused with normal aCSF, but in these a slow hyperpolarisation was revealed when DA was applied in a low Ca2qrhigh Mg 2q aCSF solution. In a sample of 12 neurones in which the effect of DA was a slow monophasic hyperpolarisation at least 2 doses of DA was tested. In these low doses Ž1 mM. elicited a mean change of 4.5 mV whereas higher doses Ž100 mM. caused a maximal change of 10 mV. During DA induced hyperpolarisation of SPN there was a decrease Ž- 23%. in cell input resistance which could be observed when the membrane potential was briefly clamped at resting levels during the long hyperpolarisation. In 10 neurones the membrane potential was gradually made more negative by intracellular injection of current. In these tests the DA Ž) 1 mM. induced slow hyperpolarisation was reduced at more negative potentials and nullified at a membrane potential between y95 mV to y105 mV. No reversal of the slow hyperpolarisation was observed. DA Žor the D 2 agonist quinpirole, both 10y5 M. when applied to the superfusate could induce fast synaptic activity ŽfSA.. This fSA consisted of fast excitatory post synaptic potentials with a rise to peak of 3 ms and duration 16 ms, or fast inhibitory post synaptic potentials, rise to peak 3 ms and duration 13 ms or less commonly a combination of both types. 3.3. Effect of low Ca 2 q r high Mg 2 q tetrodotoxin (TTX)
aCSF and
Superfusion with low Ca2q aCSF ŽCa2q: 0.25 or 0.2 mM, Mg 2q: 5 mM. for 15 min prior to testing with DA had no significant effect on resting membrane potential there being less than a 1% change compared to values in normal aCSF ŽCa2q: 2.6 mM.. However, this procedure abolished the slow depolarisation and fast synaptic activity induced by DA Ž1–100 mM., see Fig. 2Ai; Aii and Bi; Bii, indicating that there was no calcium dependent conductance contributing to the membrane potential. In contrast the slow hyperpolarisation of the SPN induced by DA in normal aCSF was still present in low Ca2q aCSF having a similar duration Žmean 105 " 26 ms. but a slightly increased magnitude Ž8 " 3.1 mV. although this was not significant when compared to test in normal aCSF. As a further test of the synaptic and non-synaptic actions of DA slices were superfused with TTX Ž0.2–0.5 mM.. In three SPN on which this was studied TTX abolished depolarising responses and fast synaptic activity induced by superfusion with DA Ž50 mM. but did not prevent a DA induced slow hyperpolarisation. 3.4. Effect of adrenergic antagonists on the actions of dopamine Earlier studies have shown that noradrenaline w34,56,58x and adrenaline w38x can elicit either a depolarisation or
S.J. Gladwell, J.H. Cooter Brain Research 818 (1999) 397–407 Fig. 2. Characteristics of the DA induced slow depolarisation. ŽAi. DA Ž10y4 M, 30 s. application in superfusate, beginning indicated by arrow, caused a biphasic response consisting of a depolarisation followed by a prolonged hyperpolarisation. ŽAii. Perfusion of the slice with a low Ca2q Ž0.25 mM. aCSF solution for 15 min caused the initial slow depolarisation to be abolished on the reapplication of DA Ž10y4 M, 30 s.. ŽAiii. Slice perfused with normal aCSF. Application of SKF 38393 Ž10y5 M, 30 s., beginning indicated by arrow, caused a slow monophasic hyperpolarisation. ŽAiv. Slice perfused with haloperidol Ž10y5 M, 15 min. before the reapplication of DA Ž10y4 M, 30 s, arrow.. This abolished the initial depolarisation Žcompare with Ai. leaving a slow monophasic hyperpolarisation which is little different from that shown in ŽAii.. Resting membrane potentialsy54 mV. ŽBi. Application of DA Ž10y4 M, 30 s., indicated by arrow, caused a slow monophasic depolarisation with an increase in fast synaptic activity shown as a thickening of the trace. ŽBii. Perfusion of the slice with a low Ca2q Ž0.25 mM. aCSF for 15 min prior to the reapplication of DA Ž10y4 M, 30 s., indicated by the arrow, now caused a slow monophasic hyperpolarisation with no synaptic activity evident. ŽBiii. Perfusion of the slice with normal aCSF containing haloperidol Ž10y5 M, 15 min.. Reapplication of DA Ž10y4 M, 30 s., indicated by the arrow, now caused a slow monophasic hyperpolarisation, the depolarisation and synaptic activity which was evident in ŽBi. is absent. Resting membrane potentialsy57 mV. 401
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hyperpolarisation of SPN. There remains the possibility that the slow membrane events in SPN evoked by DA were due to activation of an adrenergic receptor w36x. This was tested in 6 experiments using specific antagonists. In 10 neurones in which DA Ž100 mM. induced a slow hyperpolarisation Žmean s 9 " 2.8 mV. this was little changed Ž"7%. when DA was applied following superfusion for 15 min with the alpha 1 adrenoceptor antagonist prazosin Ž10 mM.. Similarly, superfusion with the alpha 2 adrenoceptor antagonist yohimibine Ž10 mM. did not block the DA hyperpolarisation Ž8 " 3.2 mV before, 8 " 2.5 mV after, n s 4.. The beta-adrenergic antagonist propranolol Ž10 mM. was also tested in 3 neurones by superfusing the slice for 12–15 min before applying DA Ž100 mM.. This too had no effect on the DA induced hyperpolarisation of the neurone Ž7 " 2.3 mV before, 6 " 2.6 mV after, n s 3.. In contrast, in the same experiments slow depolarisations or slow hyperpolarisations induced by superfusion with adrenaline Ž1–100 mM. were blocked by prazosin or
yohimbine, respectively. A comparison of the effects of adrenaline Ž1–100 mM. and DA Ž1–100 mM. on the same neurone Ž n s 9, Fig. 3A and B. showed that the two catecholamines when superfused separately onto the slice could have the opposite effect, with adrenaline depolarising and DA hyperpolarising the same neurone or in 5 neurones both evoked similar inhibitory effects. There was no recorded instance where DA excited and adrenaline inhibited the same SPN. 3.5. Effects of selectiÕe dopamine agonists and antagonists The actions of two different D 1 receptor agonists were tested in an effort to determine the DA receptor subtype on the membrane of SPN. SKF 38393 and SKF 81297-C were used at concentrations of 100 nM–100 mM Ž30 s bath application.. Both these benzazepines caused membrane hyperpolarisations ŽFig. 2AiiiFig. 4. in all neurones tested Ž n s 23, 6.9 " 1.8 mV.. No D 1 receptor induced fast synaptic activity was recorded from any of the neurones.
Fig. 3. Opposite voltage changes in resting membrane potential of an SPN on application of DA or Adrenaline. ŽA. Application of DA Ž10y4 M, 30 s., indicated by arrow, caused a slow monophasic hyperpolarisation. ŽB. Application of adrenaline Ž10y4 M, 30 s., indicated by arrow, to the same neurone as ŽA. caused a slow monophasic depolarisation. Thickening of the trace indicates fast synaptic activity. Resting membrane potentials y65 mV.
S.J. Gladwell, J.H. Cooter Brain Research 818 (1999) 397–407
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Fig. 4. Pharmacology of the DA induced hyperpolarisation of an SPN. ŽA. Application of DA Ž10y4 M, 30 s., indicated by arrow, caused a slow monophasic hyperpolarisation. ŽB. Application of SKF 38393 Ž10y4 M, 30 s., indicated by arrow, caused a slow monophasic hyperpolarisation. ŽC. Perfusion of the slice with SCH 23390 Ž10y5 M, 15 min. blocked the DA Žarrow, 10y4 M, 30 s. induced hyperpolarisation seen in ŽA.. ŽD. Perfusion of the slice with SCH 23390 Ž10y5 M, 15 min. blocked the SKF 38393 Žarrow, 10y4 M, 30 s. induced hyperpolarisation seen in ŽB.. ŽE. Fifteen minutes after washout of the antagonist SCH 23390 with normal aCSF shows return of the DA Žarrow, 10y4 M, 30 s. induced slow hyperpolarisation originally seen in ŽA., beginning to recover. Resting membrane potentials y62 mV.
The effect of these two D 1 agonists was similar in low Ca2qrhigh Mg 2q aCSF.
An example of a typical response is shown in Fig. 4. Here superfusion with DA Ž100 mM. for 30 s ŽFig. 4A.
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resulted in a slow hyperpolarisation of 13 mV, 300 s duration. Subsequent superfusion with SKF 38393 Ž100 mM. also for 30 s, Fig. 4B resulted in a similar slow hyperpolarisation of slightly less magnitude. Both of these pharmacological effects were abolished if they were applied during superfusion with the D 1 antagonist SCH 23390 Ž10 mM, 15 min. Fig. 4C and D. These effects were always reversible as shown by the action of DA Ž100 mM. which by 15 min was beginning to recover during perfusion with normal aCSF ŽFig. 4E.. The D 2 agonist quinpirole was also studied at a range of concentrations Ž1–100 mM. on 13 neurones. In 4 SPN there was a small reproducible depolarisation Žmean 3.5 mV " 1.1 mV range 2–5 mV. which was completely blocked by the D 2 selective antagonist haloperidol Ž100 nM to 1 mM.. No change in membrane potential was observed in the other 9 neurones. Haloperidol, a D 2 selective antagonist when superfused onto the slice at concentrations ranging from 100 nM to 100 mM blocked the depolarisation induced by DA Ž100 mM. revealing a hyperpolarisation on all neurones tested. When the antagonist was superfused onto a neurone responding biphasically to DA haloperidol removed the slow depolarising component and left the slow hyperpolarisation ŽFig. 2Aiv. and slightly increased its magnitude. When superfused onto neurones displaying a monophasic slow depolarising response to DA Žmean s 8 " 1.9 mV, n s 5., haloperidol completely removed the slow depolarisation and left a membrane hyperpolarisation of 7 " 2.1 mV which previously had not been evident during the control application of DA ŽFig. 2Bi and Biii.. Following recovery from haloperidol a similar effect on the action of DA was observed when superfusing the slices with low Ca2q aCSF. Finally, in a further series of experiments in which DA Ž100 mM. induced a monophasic slow hyperpolarisation, these were augmented during superfusion of haloperidol Ž10 mM., the mean change for 3 neurones being 3 " 1.4 mV.
4. Discussion 4.1. Action of dopamine These results show that DA hyperpolarises the majority of SPN and has revealed a subpopulation of SPN on which it also elicits a slow depolarisation. The more frequently observed increase in negative membrane potential was demonstrated to be a direct effect on the SPN membrane because it persisted in low Ca2q or TTX containing aCSF. Like the other catecholamines, noradrenaline and adrenaline w38,56x, the primary ionic mechanism underlying this inhibitory action of DA most likely is an increase in membrane permeability to Kq ions. Thus DA hyperpolarisation was associated with a decrease in membrane resistance, it was reduced by making the membrane poten-
tial more negative and was nullified at potentials around y100 mV. The inability to reverse the hyperpolarisation at more negative potentials than this may reflect the localisation of receptor sites at the distal dendrites or alternatively other ion conductance’s may be involved. This inability to demonstrate a reversal potential has been reported by several studies on SPN using other putative neurotransmitters in both cat and rat w34,38,56,57x. A direct inhibitory action of DA confirms an earlier interpretation w17x based on a limited in vivo study in the cat whereby DA applied iontophoretically into the vicinity of SPN decreased both spontaneous and glutamate induced action potential discharge. It also accords with the more recent observation that intrathecal application of DA to the rat thoracic spinal cord in vivo results in a bradycardia and depressor response w42x. Furthermore, this direct action of DA explains in part the very profound inhibition of sympathetic activity following intravenous L-DOPA administration in spinal animals which was reported in early studies of the actions of catecholamines on sympathetic neurones w16,41,47,51x. 4.2. Receptor pharmacology In the present study convincing evidence was obtained that the DA hyperpolarisation was mediated by a specific DA receptor and not via other catecholamine receptors. It was shown to be mimicked by the specific D 1 agonist SKF 38393 at a dose level which should be highly selective for the D 1 receptor. Furthermore, the inhibitory effect of DA and the D 1 agonist was reversibly blocked by the D 1 selective antagonist SCH 23390 and not affected by the D 2 antagonist haloperidol. The D 1 antagonist was shown to be working selectively since it did not affect the other synaptic actions of DA neither did it block the action of adrenaline on the SPN. Finally, a 1 , a 2 and b-adrenoceptor antagonists were without effect on this hyperpolarising action of DA. A DA induced depolarisation was observed in a subpopulation of SPN and since this was absent in low Ca2q or TTX containing aCSF it is most likely to be brought about by an interneurone excited by DA. However, there remains the faint possibility that DA could have been acting presynaptically, increasing the release of an unknown transmitter from terminals of neurones whose cell bodies are not located in the spinal cord. If this had been the case D 2 receptors ought to be located on the presynaptic terminals around SPN perikarya. This is not supported by a recent autoradiographic study conducted by us which clearly demonstrated a high density of D 1 receptors but a virtual absence of D 2 receptors. The latter were located in medial regions of the spinal grey matter some distance from SPN. We routinely used low Ca2q aCSF superfusion in the majority of tests because it has been previously shown to be an effective method of synaptically isolating other types of neurone. It is likely that this procedure was
S.J. Gladwell, J.H. Cooter Brain Research 818 (1999) 397–407
acting in a similar way on SPN rather than reducing a calcium dependent potassium conductance of the SPN membrane because there was no evidence that this conductance was activated at resting membrane potential which was similar before and during low Ca2q perfusion. Furthermore, this aCSF was not zero Ca2q and a DA induced depolarisation acting via such a conductance could if anything be increased in low Ca2q. A calcium dependent potassium conductance has been described in SPN which is influenced by catecholamines. This is a voltage dependent conductance observed following an action potential whereby noradrenaline application causes an after depolarisation to appear following a fast hyperpolarisation w56x. The slow hyperpolarisations produced by either noradrenaline or adrenaline Ž a-2 receptor dependent. or slow depolarisation of the SPN membrane mediated by a-1 receptors result from changes in a different potassium conductance w34,38,57x. We therefore believe the evidence strongly points to the depolarising action of DA being on presympathetic interneurones. This action was shown to be mediated via a D 2 receptor since it was mimicked by the specific D 2 agonist quinpirole and blocked selectively by the D 2 antagonist haloperidol and unaffected by the D 1 antagonist. 4.3. Slow depolarisation The DA induced depolarisation with its slow rise and slow decay is unlike anything so far described in SPN which is synaptically induced. This raises the question as to the chemical mediator of this synaptic action. It is unlikely to be dependent on activation of an ionotropic glutamate receptor since it is unaffected by kynurenic acid even at 10y5 M. We have so far not tested for a metabotropic receptor mediation but the rise time and decay of the synaptic potential seem somewhat long for this. The depolarisation does though have the characteristics similar to that induced by Substance P w9,22x. For example the slow rise and slow decay of the response, the induction of cell discharge and changes in cell input resistance as well as the finding that it is only induced in less than half the neurones studied. This latter observation accords with the demonstration that Substance P receptors are only present on a specific group of SPN w9,28,33x. In addition a substantial portion Ž) 30%. of intraspinal Substance P innervation of SPN is independent of supraspinal projections w18,19x. This spinal innervation cannot be due to dorsal root fibres passing directly to SPN since there are no monosynaptic afferent connections with SPN w4,5,13,14,24x. Therefore, there is a possibility that DA was stimulating Substance P presympathetic interneurones lying in lamina V or VII of the spinal grey matter w8,31x. Further studies are in progress to identify the neurotransmitter mediating the synaptic depolarisation of SPN induced by DA.
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4.4. Fast synaptic actiÕity Activation of the D 2 receptor by DA also resulted in the appearance of fast synaptic activity in SPN, both IPSP’s and EPSP’s. Similar events are initiated in SPN when superfusing spinal cord slices with adrenaline, substance P, vasopressin, oxytocin and 5-hydroxytryptamine w20,22, 32,38,48x. This suggests that there is a pool of interneurones in the spinal cord onto which there is a heavy convergence of differently chemically coded supraspinal afferents. The significance of this to regulation of spinal sympathetic network remains to be worked out. Nonetheless, this study of the actions of DA has again revealed that there are two ways by which supraspinal afferents may influence SPN; a direct monosynaptic one and an indirect one dependent on presympathetic interneurones w11,26x. In the case of DA it seems unlikely that it is the same group of phenotypic cells that contribute to both mono- and polysynaptic connections since they initiate quite opposite actions. 4.5. Origin of dopamine pathway There are two groups of DA neurones which project to the sympathetic intermediolateral cell column. These arise from the hypothalamus, one from the paraventricular nucleus ŽPVN. w53,54x and the other from the dorsal-posterior hypothalamus the A11 group of catecholamine cells w6x. Of these the PVN-DA projection is most likely to be monosynaptic since it is labelled following retrograde trans-synaptic transfer of pseudorabies virus into a peripheral target w53x. Furthermore, PVN terminals labelled anterogradely have been shown in close association with SPN w30,39,46x. Therefore activation of a DA-spinal pathway when stimulating neurones in the PVN could explain the sympatho-inhibition and depressor responses. It is of interest that another group of PVN-spinal neurones which release vasopressin has been shown to be sympatho-excitatory w37x. It may therefore be that appropriate activation of different neuronal phenotypes results in the pattern of sympathetic activation, whereby some neurones Žcardiac, splanchnic, adrenal. are excited and some Žrenal. are inhibited as for example occurs in the response to plasma volume expansion w2,12,15x.
Acknowledgements We thank the Medical Research Council for their financial support and Drs. D.I. Lewis and S. Pyner for their helpful discussions.
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dopamine- and adrenaline-containing axons to the innervation of different regions of the spinal cord of the cat, Brain Res. 206 Ž1981. 95–106. D.N. Franz, M.H. Evans, E.R. Perl, Characteristics of viscero-sympathetic reflexes in the spinal cat, Am. J. Physiol. 211 Ž1966. 1292–1298. K. Fuxe, B. Tinner, B. Bjelke, L.F. Agnati, A. Verhofstad, H.G.W. Steinbusch, M. Goldstein, M. Kalia, Monoaminergic and peptidergic innervation of the intermediolateral horn of the spinal cord, Eur. J. Neurosci. 2 Ž1990. 430–450. G.L. Gebber, R.B. McCall, Identification and discharge patterns of spinal sympathetic interneurones, Am. J. Physiol. 231 Ž1976. 722– 733. S.J. Gladwell, Dopamine: A Multifunctional Neuromodulatory Role in Spinal Sympathetic Networks, PhD Thesis, University of Birmingham, 1998. I. Grkovic, C.R. Anderson, Distribution of immunoreactivity for the NK 1 receptor on different subpopulations of sympathetic preganglionic neurons in the rat, J. Comp. Neurol. 374 Ž1996. 376–386. J.C. Holstege, H. Van Dijken, R.M. Buijs, H. Goedknegt, T. Gosens, C.M.H. Bongers, Distribution of dopamine immunoreactivity in the rat, cat and monkey spinal cord, J. Comp. Neurol. 376 Ž1996. 631–652. Y. Hosoya, N. Okado, Y. Sugiura, K. Kohno, Coincidence of ‘ladderlike patterns’ in distributions of monoaminergic terminals and sympathetic preganglionic neurons in the rat spinal cord, Exp. Brain Res. 86 Ž1991. 224–228. S. Joshi, M.A. Levatte, G.A. Dekaban, L.C. Weaver, Identification of spinal interneurones antecedent to adrenal sympathetic preganglionic neurons using trans-synaptic transport of herpes simplex virus type 1, Neuroscience 65 Ž3. Ž1995. 893–903. D.I. Lewis, E. Sermasi, J.H. Coote, Excitatory and indirect inhibitory actions of 5-hydroxytryptamine on sympathetic preganglionic neurones in the neonatal rat spinal cord in vitro, Brain Res. 610 Ž1993. 267–275. I.J. Llewellyn-Smith, C.L. Martin, J.B. Minson, P.M. Pilowsky, L.F. Arnolda, A.I. Basbaum, J.P. Chalmers, Neurokinin-1 receptor-immunoreactive sympathetic preganglionic neurons: target specificity and ultrastructure, Neuroscience 77 Ž1997. 1137–1149. R.C. Ma, N.J. Dun, Norepinephrine depolarizes lateral horn cells of neonatal rat spinal cord in vitro, Neurosci. Lett. 60 Ž1985. 163–168. T. Magnusson, Effect of chronic transection on dopamine, noradrenaline and 5-hydroxytryptamine in the rat spinal cord, NaunynSchmiedeberg’s Arch. Pharmacol. 278 Ž1973. 13–22. R.C. Malenka, R.A. Nicoll, Dopamine decreases the calciumactivated afterhyperpolarization in hippocampal CA1 pyramidal cells, Brain Res. 379 Ž1986. 210–215. S.C. Malpas, J.H. Coote, Role of vasopressin in sympathetic response to paraventricular nucleus stimulation in anaesthetized rats, Am. J. Physiol. 266 Ž1994. R228–R236. T. Miyazaki, J.H. Coote, N.J. Dun, Excitatory and inhibitory effects of epinephrine on neonatal rat sympathetic preganglionic neurons in vitro, Brain Res. 497 Ž1989. 108–116. K. Motawei, S. Pyner, J.H. Coote, Identification of discrete sympathetic functional pathways in the central nervous system, J. Physiol. 483 Ž1995. 101P. P. Mouchet, M. Manier, C. Feuerstein, Immunohistochemical study of the catecholaminergic innervation of the spinal cord of the rat using specific antibodies against dopamine and noradrenaline, J. Chem. Neuroanat. 5 Ž1992. 427–440. R.J. Neumayr, B.D. Hare, D.N. Franz, Evidence for bulbospinal control of sympathetic neurones by monoaminergic pathways, Life Sci. 14 Ž1974. 793–806. G. Pellissier, P. Demenge, Hypotensive and bradycardic effects elicited by spinal dopamine receptor stimulation: effects of D1 and D 2 receptor agonists and antagonists, J. Cardiovasc. Pharmacol. 18 Ž1991. 548–555.
S.J. Gladwell, J.H. Cooter Brain Research 818 (1999) 397–407 w43x S. Pyner, J.H. Coote, Evidence that sympathetic preganglionic neurons are arranged in target-specific columns in the thoracic spinalcord of the rat, J. Comp. Neurol. 342 Ž1994. 15–22. w44x S. Pyner, J.H. Coote, A comparison between the adult-rat and neonate rat of the architecture of sympathetic preganglionic neurons projecting to the superior cervical-ganglion, stellate ganglion and adrenal-medulla, J. Auton. Nerv. Syst. 48 Ž1994. 153–166. w45x S. Pyner, J.H. Coote, Arrangement of dendrites and morphologicalcharacteristics of sympathetic preganglionic neurons projecting to the superior cervical-ganglion and adrenal-medulla in adult cat, J. Auton. Nerv. Syst. 52 Ž1995. 35–41. w46x R.N. Ranson, K. Motawei, S. Pyner, J.H. Coote, The paraventricular nucleus of the hypothalamus sends efferents to the spinal cord of the rat that closely appose sympathetic preganglionic neurones projecting to the stellate ganglion, Exp. Brain Res. 120 Ž1998. 164–172. w47x H. Schmitt, H. Schmitt, S. Fenard, Localisation of the central sympatho-inhibitory action of L-DOPA in dogs and cats, Eur. J. Pharmacol. 22 Ž1969. 212–216. w48x E. Sermasi, J.H. Coote, Oxytocin acts at V1 receptors to excite sympathetic preganglionic neurones in neonate rat spinal cord in vitro, Brain Res. 647 Ž1994. 323–332. w49x M. Shirouzu, T. Anraku, Y. Iwashita, M. Yoshida, A new dopaminergic terminal plexus in the ventral horn of the rat spinal cord: immunohistochemical studies at the light and electron microscopical level, Experimentia 46 Ž1990. 201–204. w50x O.R. Simon, L.P. Schramm, Spinal superfusion of dopamine excites renal sympathetic-nerve activity, Neuropharmacology 22 Ž1983. 287–293.
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w51x J.N. Sinha, J.M. Atkinson, H. Schmitt, Effects of clonidine and L-DOPA on spontaneous and evoked splanchnic nerve discharges, Eur. J. Pharmacol. 23 Ž1973. 113–119. w52x A.M. Strack, W.B. Sawyer, L.M. Marubio, A.D. Loewy, Spinal origin of sympathetic preganglionic neurons in the rat, Brain Res. 455 Ž1988. 187–191. w53x A.M. Strack, W.B. Sawyer, K.B. Platt, A.D. Loewy, CNS cell groups regulating the sympathetic outflow to adrenal gland as revealed by transneuronal cell body labelling with pseudorabies virus, Brain Res. 491 Ž1989. 274–296. w54x L.W. Swanson, P.E. Sawchenko, A. Berod, B.K. Hartman, K.B. ´ Helle, D.E. Vanorden, An immunohistochemical study of the organisation of catecholaminergic cell and terminal fields in the paraventricular and supraoptic nuclei of the hypothalamus, J. Comp. Neurol. 196 Ž1981. 271–285. w55x M. Yoshida, M. Tanaka, Existence of a new dopaminergic terminal plexus in the rat spinal cord: assessment by immunohistochemistry using anti-dopamine serum, Neurosci. Lett. 94 Ž1988. 5–9. w56x M. Yoshimura, C. Polosa, S. Nishi, Noradrenaline modifies sympathetic preganglionic neuron spike and afterpotential, Brain Res. 362 Ž1986. 370–374. w57x M. Yoshimura, C. Polosa, S. Nishi, Electrophysiological properties of sympathetic preganglionic neurons in the cat spinal cord in vitro, Pflugers Arch. Physiol. 406 Ž1986. 91–98. ¨ w58x M. Yoshimura, C. Polosa, S. Nishi, Noradrenaline induces rhythmic bursting in sympathetic preganglionic neurones, Brain Res. 420 Ž1987. 147–151.
Research report
Inhibitory and indirect excitatory effects of dopamine on sympathetic preganglionic neurones in the neonatal rat spinal cord in vitro Simon J. Gladwell ) , John H. Coote Department of Physiology, School of Medicine, UniÕersity of Birmingham, Edgbaston, Birmingham, B15 2TT, UK Accepted 8 December 1998
Abstract Regions of the thoraco-lumbar spinal cord containing sympathetic preganglionic neurones are rich in dopamine terminals. To determine the influence of this innervation intracellular recordings were made from antidromically identified sympathetic preganglionic neurones in Ž400 mm. transverse neonatal rat spinal cord slices. Dopamine applied by superfusion caused a slow monophasic hyperpolarisation in 46% of sympathetic preganglionic neurones, a slow monophasic depolarisation in 28% of sympathetic preganglionic neurones and a biphasic effect consisting of a slow depolarisation followed by a slow hyperpolarisation or vice-versa in 23% of sympathetic preganglionic neurones. Three percent of sympathetic preganglionic neurones did not respond to the application of dopamine. Low Ca2qrhigh Mg 2q Krebs solution or TTX did not change the resting membrane potential but abolished the slow depolarisation elicited by dopamine, indicating this was synaptic and did not prevent the dopamine induced hyperpolarisation. The dopamine induced slow hyperpolarisation was mimicked by the selective D1 agonists SKF 38393 or SKF 81297-C and blocked by superfusion with the D1 antagonist SCH 23390. It was not prevented by superfusion of the slices with alpha 1 or alpha 2 or beta-adrenoceptor antagonists, whereas the inhibitory or excitatory actions of adrenaline were prevented by alpha 1 or alpha 2 antagonists, respectively. The dopamine induced slow depolarisation occurring in a sub-population of sympathetic preganglionic neurones was mimicked by quinpirole, a D 2 agonist, and blocked by haloperidol, a D 2 antagonist. Haloperidol did not block the dopamine induced hyperpolarisations. Dopamine also induced fast synaptic activity which was mimicked by a D 2 agonist and blocked by haloperidol. D1 agonists did not elicit fast synaptic activity. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Sympathetic preganglionic neurone ŽSPN.; Dopamine ŽDA.; SKF 38393; Spinal cord
1. Introduction There is now considerable evidence that dopamine ŽDA. has a neurotransmitter role in the spinal cord separate from being a precursor to noradrenaline w11,25,29x. The evidence initially depended on the different distribution of DA in microdissected regions of the cat spinal cord compared to other catecholamines w23x and its differential decline compared to noradrenaline following spinal cord transection w35x. More recently DA has been recognised using a specific antibody and shown to be present in nerve terminals throughout the spinal cord w29,40,49,55x These nerve terminals arise from spinally projecting neurones in, the dorsal and posterior hypothalamus w6,7x, the paraventricular nucleus w53,54x and possibly the substantia nigra of the mid brain w10x. ) Corresponding author. [email protected]
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Specific regions of the spinal grey matter are targeted by the DA fibres; laminae III and IV of the dorsal horn, lamina X and the thoracic intermediolateral cell column ŽIML. displaying highest levels, as measured by tyrosine hydroxylase immunoreactivity in rats pre-treated with 6hydroxydopamine Ž6-OHDA. to remove noradrenaline fibres w21x. Of all these regions the IML has the highest density of enzyme, confirming the biochemical estimations of DA w23,3x. This would indicate that the amine has an important influence on sympathetic activity since the IML is where the majority of sympathetic preganglionic neurones ŽSPN. are located w1,30,43–45,52x. The first study to examine this influence used microiontophoresis to eject DA into the vicinity of identified extracellularly recorded SPN in the cat spinal cord in vivo w17x. It was shown that spontaneous or glutamate initiated activity was inhibited by DA. Consistent with this is the recent demonstrations that intrathecal application of DA receptor agonists to the thoracic cord of the rat causes a fall in
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 1 3 3 0 - 4
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blood pressure and heart rate w42x. In contrast Simon and Schramm w50x found that in acutely spinalised rats the amine increased activity in the renal sympathetic nerve, when it was applied by intrathecal superfusion to the thoracic spinal cord. In view of this conflicting data the functional significance of the DA innervation of sympathetic nerves is unclear. Part of the reason for this is that iontophoresis or intrathecal techniques fail to discriminate between sites of action of a transmitter. We therefore have studied the actions of DA on single SPN recorded intracellularly in slices of rat spinal cord in vitro.
2. Materials and methods 2.1. Spinal cord preparation The preparation of thin transverse slices from neonatal rats has been previously documented by this group w32x. Briefly, Wistar rats Ž8–16 days old. were taken and deeply anaesthetised with diethyl ether and a dorsal laminectomy performed. A 10–15 mm section of middle to upper spinal cord was removed and placed onto moistened filter paper where the dura and pia mater were dissected off. This section was then trimmed to leave a 7–8 mm piece of upper thoracic spinal cord which was placed into a mould filled with agar ŽBDH, 1.5%. which had been preboiled and allowed to cool to 35–408C. The mould was topped up with agar and then immersed in cold Ž48C. artificial cerebrospinal fluid ŽaCSF. to set. The agar block containing the section of cord was glued Žcyanoacetate. to the cutting stage of a Vibroslice ŽCamden Instruments. where 400 mm transverse slices were cut. These slices were transferred to an incubation chamber containing oxygenated Ž95%O 2r5% CO 2 . aCSF at room temperature. The composition of the normal aCSF was ŽmM.: NaCl 127; KCl 1.9; KH 2 PO4 1.2; MgSO4 1.3; NaHCO 3 26; D-Glucose 10; CaCl 2 2.5, pH s 7.4. For low Ca2qrhigh Mg 2q aCSF the following composition was used ŽmM.: NaCl 127; KCl 1.9; KH 2 PO4 1.2; MgSO4 1.3; NaHCO 3 26; D-Glucose 10; CaCl 2 0.2, MgCl 2 5.0, pH s 7.4. The slices were incubated for at least an hour prior to any recordings being made. As required a slice was transferred to the recording chamber and was continually superfused with oxygenated aCSF Ž3 ml miny1 , 318C..
2.2. Electrophysiology Neurones located in the lateral horn were impaled with 3 M potassium acetate filled glass microelectrodes Žtip resistance 45–105 M V .. Signals were amplified using an Axoclamp-2A ŽAxon Instruments. intracellular preamplifier and displayed on two oscilloscopes Ži. a Gould 1604 in refresh mode on a short time base and Žii. a Gould OS4020 in rolling display mode on a long time base. The signal was also directed to, a DC chart recorder ŽGould TA240. for constant recording of membrane potential during intracellular recording, and an FM tape recorder ŽRacal Store 4. for data storage and off-line analysis. SPN were identified by stimulating antidromically the ventral root exit zone with a concentric bipolar stimulating electrode ŽSNE-100, Clark-Electromedical, 1–100 nA 0.02 ms.. Cell input resistance was determined by intracellular negative current injections across the bridge of the preamplifier. 2.3. Chemicals Drugs used in this study were dissolved in aCSF and added to the superfusate in known concentrations. 3-Hydroxytyramine ŽDA. readily oxidised in the aCSF, it was therefore prepared immediately prior to its superfusate application. The following drugs were used. 3-Hydroxytyramine ŽDA, Sigma., adrenaline ŽAdr, Sigma., haloperidol ŽSigma., SKF 38393 ŽŽ".-1-Phenyl-2,3,4,5-tetrahydro-1 H-3-benzazepine-7,8-diol hydrochloride, RBI., quinpirole ŽRBI., SCH-23390 Ž RŽ".-7-Chloro-8-hydroxy-3methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride, RBI., yohimbine ŽSigma., prazosin ŽSigma., propranolol ŽSigma.. SKF 81297-C Ž6-Chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1 H-3-benzazepine hydrobromide. was a gift from SmithKline Beecham. Results are expressed as means " standard deviation ŽS.D..
3. Results 3.1. Electrophysiological characteristics Intracellular recordings of up to 3 h were made from neurones located within the IML region of the spinal cord. Recordings of this duration allowed for a sufficient period
Fig. 1. The actions of dopamine ŽDA. on the resting membrane potential of four different antidromically identified sympathetic preganglionic neurones. Each trace is from a different neurone. ŽA. The application of DA Ž10y4 M. for 30 s, indicated by the arrow, caused a slow monophasic hyperpolarisation. Resting membrane potentials y58 mV. ŽB. The application of DA Ž10y4 M. for 30 s, indicated by the arrow, caused a slow monophasic depolarisation. Resting membrane potentials y62 mV. ŽC. The application of DA Ž10y4 M. for 30 s, indicated by the arrow, caused a biphasic response consisting of a slow depolarisation followed by a slow hyperpolarisation. Resting membrane potentials y57 mV. ŽD. The application of DA Ž10y4 M. for 30 s, indicated by the arrow, caused a biphasic response consisting of a slow hyperpolarisation followed by a slow depolarisation with an increase in synaptic activity Ždemonstrated by a thickening of the trace, arrow. and a train of action potentials. Resting membrane potentials y60 mV.
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of recovery between drug applications to avoid tachyphalaxis. All these neurones were identified as SPN by eliciting an antidromic action potential following stimulation of the ventral root exit zone. Their identification was further confirmed on the basis of characteristic electrophysiological properties described below. In addition some of the intracellular recording electrodes were filled with a Biocytin solution Ž1.5% in 1.5 M potassium methyl sulphate. which was injected into impaled cells by passing a depolarising current Ž1.5 nA, q0.5 nA pulses, 85 ms duration 0.5 Hz for 10 to 20 min.. Subsequent immunocytochemical processing of the slice confirmed the location in the lateral horn and the typical morphology of the neurone as SPN w43x, when viewed with a Zeiss Photomicroscope III w27x. Immediately after intracellular penetration the majority of neurones displayed spontaneous action potential discharge up to a maximum of 20 Hz. In the majority of cases 82 neurones out of a total of 111 Ž82r111. this activity ceased after 1–2 min of stable membrane potential recording; some neurones continued to fire action potentials spontaneously Ž29r111. and some also demonstrated a degree of membrane oscillation Ž24r111. in which a fast depolarising transient is followed by a slower hyperpolarising phase. The remaining SPN were silent but could be induced to fire action potentials by injecting a depolarising current through the recording electrode or by antidromic stimulation via the bipolar electrode in the ventral root exit zone. The 111 SPN had resting membrane potentials in the range y46 to y72 mV Žmean 60.5 " 7.3 mV. and a cell input resistance which was calculated from the voltage deflection of intracellular hyperpolarising pulses of 72 " 27 M V Žrange 21 to 117 M V .. Both spontaneous and antidromically induced action potentials had mean amplitudes of 81 " 8.9 mV Žrange 65–98 mV. and durations of 3.0 " 1.1 ms Žrange 1.3–4.6 ms.. All of the neurones displayed a characteristic distinct after hyperpolarisation following an action potential discharge; this had a mean amplitude of 18 " 9 mV Žrange 8–29 mV. and a mean duration of 98 " 36 ms Žrange 24–148 ms.. 3.2. Actions of dopamine DA was applied by superfusion in concentrations ranging from 10 nM to 10 mM for between 15 s and 45 s at a flow rate of 3 ml miny1 . A submaximal concentration of 100 mM for 30 s was routinely used as application of higher doses yielded no further significant change in membrane potential. On 111 SPN the catecholamine caused either a slow hyperpolarisation Žtime to peak mean s 95 " 32 s, n s 51, Fig. 1A., a slow depolarisation Žtime to peak mean s 63 " 26, n s 31, Fig. 1B, or a biphasic response Ž n s 26. which consisted of either a slow depolarisation followed by a slow hyperpolarisation Ž n s 16, Fig. 1C. or vice versa Ž n s 10, Fig. 1D.. Three neurones showed no change in
membrane potential on the application of DA when the slices were perfused with normal aCSF, but in these a slow hyperpolarisation was revealed when DA was applied in a low Ca2qrhigh Mg 2q aCSF solution. In a sample of 12 neurones in which the effect of DA was a slow monophasic hyperpolarisation at least 2 doses of DA was tested. In these low doses Ž1 mM. elicited a mean change of 4.5 mV whereas higher doses Ž100 mM. caused a maximal change of 10 mV. During DA induced hyperpolarisation of SPN there was a decrease Ž- 23%. in cell input resistance which could be observed when the membrane potential was briefly clamped at resting levels during the long hyperpolarisation. In 10 neurones the membrane potential was gradually made more negative by intracellular injection of current. In these tests the DA Ž) 1 mM. induced slow hyperpolarisation was reduced at more negative potentials and nullified at a membrane potential between y95 mV to y105 mV. No reversal of the slow hyperpolarisation was observed. DA Žor the D 2 agonist quinpirole, both 10y5 M. when applied to the superfusate could induce fast synaptic activity ŽfSA.. This fSA consisted of fast excitatory post synaptic potentials with a rise to peak of 3 ms and duration 16 ms, or fast inhibitory post synaptic potentials, rise to peak 3 ms and duration 13 ms or less commonly a combination of both types. 3.3. Effect of low Ca 2 q r high Mg 2 q tetrodotoxin (TTX)
aCSF and
Superfusion with low Ca2q aCSF ŽCa2q: 0.25 or 0.2 mM, Mg 2q: 5 mM. for 15 min prior to testing with DA had no significant effect on resting membrane potential there being less than a 1% change compared to values in normal aCSF ŽCa2q: 2.6 mM.. However, this procedure abolished the slow depolarisation and fast synaptic activity induced by DA Ž1–100 mM., see Fig. 2Ai; Aii and Bi; Bii, indicating that there was no calcium dependent conductance contributing to the membrane potential. In contrast the slow hyperpolarisation of the SPN induced by DA in normal aCSF was still present in low Ca2q aCSF having a similar duration Žmean 105 " 26 ms. but a slightly increased magnitude Ž8 " 3.1 mV. although this was not significant when compared to test in normal aCSF. As a further test of the synaptic and non-synaptic actions of DA slices were superfused with TTX Ž0.2–0.5 mM.. In three SPN on which this was studied TTX abolished depolarising responses and fast synaptic activity induced by superfusion with DA Ž50 mM. but did not prevent a DA induced slow hyperpolarisation. 3.4. Effect of adrenergic antagonists on the actions of dopamine Earlier studies have shown that noradrenaline w34,56,58x and adrenaline w38x can elicit either a depolarisation or
S.J. Gladwell, J.H. Cooter Brain Research 818 (1999) 397–407 Fig. 2. Characteristics of the DA induced slow depolarisation. ŽAi. DA Ž10y4 M, 30 s. application in superfusate, beginning indicated by arrow, caused a biphasic response consisting of a depolarisation followed by a prolonged hyperpolarisation. ŽAii. Perfusion of the slice with a low Ca2q Ž0.25 mM. aCSF solution for 15 min caused the initial slow depolarisation to be abolished on the reapplication of DA Ž10y4 M, 30 s.. ŽAiii. Slice perfused with normal aCSF. Application of SKF 38393 Ž10y5 M, 30 s., beginning indicated by arrow, caused a slow monophasic hyperpolarisation. ŽAiv. Slice perfused with haloperidol Ž10y5 M, 15 min. before the reapplication of DA Ž10y4 M, 30 s, arrow.. This abolished the initial depolarisation Žcompare with Ai. leaving a slow monophasic hyperpolarisation which is little different from that shown in ŽAii.. Resting membrane potentialsy54 mV. ŽBi. Application of DA Ž10y4 M, 30 s., indicated by arrow, caused a slow monophasic depolarisation with an increase in fast synaptic activity shown as a thickening of the trace. ŽBii. Perfusion of the slice with a low Ca2q Ž0.25 mM. aCSF for 15 min prior to the reapplication of DA Ž10y4 M, 30 s., indicated by the arrow, now caused a slow monophasic hyperpolarisation with no synaptic activity evident. ŽBiii. Perfusion of the slice with normal aCSF containing haloperidol Ž10y5 M, 15 min.. Reapplication of DA Ž10y4 M, 30 s., indicated by the arrow, now caused a slow monophasic hyperpolarisation, the depolarisation and synaptic activity which was evident in ŽBi. is absent. Resting membrane potentialsy57 mV. 401
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hyperpolarisation of SPN. There remains the possibility that the slow membrane events in SPN evoked by DA were due to activation of an adrenergic receptor w36x. This was tested in 6 experiments using specific antagonists. In 10 neurones in which DA Ž100 mM. induced a slow hyperpolarisation Žmean s 9 " 2.8 mV. this was little changed Ž"7%. when DA was applied following superfusion for 15 min with the alpha 1 adrenoceptor antagonist prazosin Ž10 mM.. Similarly, superfusion with the alpha 2 adrenoceptor antagonist yohimibine Ž10 mM. did not block the DA hyperpolarisation Ž8 " 3.2 mV before, 8 " 2.5 mV after, n s 4.. The beta-adrenergic antagonist propranolol Ž10 mM. was also tested in 3 neurones by superfusing the slice for 12–15 min before applying DA Ž100 mM.. This too had no effect on the DA induced hyperpolarisation of the neurone Ž7 " 2.3 mV before, 6 " 2.6 mV after, n s 3.. In contrast, in the same experiments slow depolarisations or slow hyperpolarisations induced by superfusion with adrenaline Ž1–100 mM. were blocked by prazosin or
yohimbine, respectively. A comparison of the effects of adrenaline Ž1–100 mM. and DA Ž1–100 mM. on the same neurone Ž n s 9, Fig. 3A and B. showed that the two catecholamines when superfused separately onto the slice could have the opposite effect, with adrenaline depolarising and DA hyperpolarising the same neurone or in 5 neurones both evoked similar inhibitory effects. There was no recorded instance where DA excited and adrenaline inhibited the same SPN. 3.5. Effects of selectiÕe dopamine agonists and antagonists The actions of two different D 1 receptor agonists were tested in an effort to determine the DA receptor subtype on the membrane of SPN. SKF 38393 and SKF 81297-C were used at concentrations of 100 nM–100 mM Ž30 s bath application.. Both these benzazepines caused membrane hyperpolarisations ŽFig. 2AiiiFig. 4. in all neurones tested Ž n s 23, 6.9 " 1.8 mV.. No D 1 receptor induced fast synaptic activity was recorded from any of the neurones.
Fig. 3. Opposite voltage changes in resting membrane potential of an SPN on application of DA or Adrenaline. ŽA. Application of DA Ž10y4 M, 30 s., indicated by arrow, caused a slow monophasic hyperpolarisation. ŽB. Application of adrenaline Ž10y4 M, 30 s., indicated by arrow, to the same neurone as ŽA. caused a slow monophasic depolarisation. Thickening of the trace indicates fast synaptic activity. Resting membrane potentials y65 mV.
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Fig. 4. Pharmacology of the DA induced hyperpolarisation of an SPN. ŽA. Application of DA Ž10y4 M, 30 s., indicated by arrow, caused a slow monophasic hyperpolarisation. ŽB. Application of SKF 38393 Ž10y4 M, 30 s., indicated by arrow, caused a slow monophasic hyperpolarisation. ŽC. Perfusion of the slice with SCH 23390 Ž10y5 M, 15 min. blocked the DA Žarrow, 10y4 M, 30 s. induced hyperpolarisation seen in ŽA.. ŽD. Perfusion of the slice with SCH 23390 Ž10y5 M, 15 min. blocked the SKF 38393 Žarrow, 10y4 M, 30 s. induced hyperpolarisation seen in ŽB.. ŽE. Fifteen minutes after washout of the antagonist SCH 23390 with normal aCSF shows return of the DA Žarrow, 10y4 M, 30 s. induced slow hyperpolarisation originally seen in ŽA., beginning to recover. Resting membrane potentials y62 mV.
The effect of these two D 1 agonists was similar in low Ca2qrhigh Mg 2q aCSF.
An example of a typical response is shown in Fig. 4. Here superfusion with DA Ž100 mM. for 30 s ŽFig. 4A.
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resulted in a slow hyperpolarisation of 13 mV, 300 s duration. Subsequent superfusion with SKF 38393 Ž100 mM. also for 30 s, Fig. 4B resulted in a similar slow hyperpolarisation of slightly less magnitude. Both of these pharmacological effects were abolished if they were applied during superfusion with the D 1 antagonist SCH 23390 Ž10 mM, 15 min. Fig. 4C and D. These effects were always reversible as shown by the action of DA Ž100 mM. which by 15 min was beginning to recover during perfusion with normal aCSF ŽFig. 4E.. The D 2 agonist quinpirole was also studied at a range of concentrations Ž1–100 mM. on 13 neurones. In 4 SPN there was a small reproducible depolarisation Žmean 3.5 mV " 1.1 mV range 2–5 mV. which was completely blocked by the D 2 selective antagonist haloperidol Ž100 nM to 1 mM.. No change in membrane potential was observed in the other 9 neurones. Haloperidol, a D 2 selective antagonist when superfused onto the slice at concentrations ranging from 100 nM to 100 mM blocked the depolarisation induced by DA Ž100 mM. revealing a hyperpolarisation on all neurones tested. When the antagonist was superfused onto a neurone responding biphasically to DA haloperidol removed the slow depolarising component and left the slow hyperpolarisation ŽFig. 2Aiv. and slightly increased its magnitude. When superfused onto neurones displaying a monophasic slow depolarising response to DA Žmean s 8 " 1.9 mV, n s 5., haloperidol completely removed the slow depolarisation and left a membrane hyperpolarisation of 7 " 2.1 mV which previously had not been evident during the control application of DA ŽFig. 2Bi and Biii.. Following recovery from haloperidol a similar effect on the action of DA was observed when superfusing the slices with low Ca2q aCSF. Finally, in a further series of experiments in which DA Ž100 mM. induced a monophasic slow hyperpolarisation, these were augmented during superfusion of haloperidol Ž10 mM., the mean change for 3 neurones being 3 " 1.4 mV.
4. Discussion 4.1. Action of dopamine These results show that DA hyperpolarises the majority of SPN and has revealed a subpopulation of SPN on which it also elicits a slow depolarisation. The more frequently observed increase in negative membrane potential was demonstrated to be a direct effect on the SPN membrane because it persisted in low Ca2q or TTX containing aCSF. Like the other catecholamines, noradrenaline and adrenaline w38,56x, the primary ionic mechanism underlying this inhibitory action of DA most likely is an increase in membrane permeability to Kq ions. Thus DA hyperpolarisation was associated with a decrease in membrane resistance, it was reduced by making the membrane poten-
tial more negative and was nullified at potentials around y100 mV. The inability to reverse the hyperpolarisation at more negative potentials than this may reflect the localisation of receptor sites at the distal dendrites or alternatively other ion conductance’s may be involved. This inability to demonstrate a reversal potential has been reported by several studies on SPN using other putative neurotransmitters in both cat and rat w34,38,56,57x. A direct inhibitory action of DA confirms an earlier interpretation w17x based on a limited in vivo study in the cat whereby DA applied iontophoretically into the vicinity of SPN decreased both spontaneous and glutamate induced action potential discharge. It also accords with the more recent observation that intrathecal application of DA to the rat thoracic spinal cord in vivo results in a bradycardia and depressor response w42x. Furthermore, this direct action of DA explains in part the very profound inhibition of sympathetic activity following intravenous L-DOPA administration in spinal animals which was reported in early studies of the actions of catecholamines on sympathetic neurones w16,41,47,51x. 4.2. Receptor pharmacology In the present study convincing evidence was obtained that the DA hyperpolarisation was mediated by a specific DA receptor and not via other catecholamine receptors. It was shown to be mimicked by the specific D 1 agonist SKF 38393 at a dose level which should be highly selective for the D 1 receptor. Furthermore, the inhibitory effect of DA and the D 1 agonist was reversibly blocked by the D 1 selective antagonist SCH 23390 and not affected by the D 2 antagonist haloperidol. The D 1 antagonist was shown to be working selectively since it did not affect the other synaptic actions of DA neither did it block the action of adrenaline on the SPN. Finally, a 1 , a 2 and b-adrenoceptor antagonists were without effect on this hyperpolarising action of DA. A DA induced depolarisation was observed in a subpopulation of SPN and since this was absent in low Ca2q or TTX containing aCSF it is most likely to be brought about by an interneurone excited by DA. However, there remains the faint possibility that DA could have been acting presynaptically, increasing the release of an unknown transmitter from terminals of neurones whose cell bodies are not located in the spinal cord. If this had been the case D 2 receptors ought to be located on the presynaptic terminals around SPN perikarya. This is not supported by a recent autoradiographic study conducted by us which clearly demonstrated a high density of D 1 receptors but a virtual absence of D 2 receptors. The latter were located in medial regions of the spinal grey matter some distance from SPN. We routinely used low Ca2q aCSF superfusion in the majority of tests because it has been previously shown to be an effective method of synaptically isolating other types of neurone. It is likely that this procedure was
S.J. Gladwell, J.H. Cooter Brain Research 818 (1999) 397–407
acting in a similar way on SPN rather than reducing a calcium dependent potassium conductance of the SPN membrane because there was no evidence that this conductance was activated at resting membrane potential which was similar before and during low Ca2q perfusion. Furthermore, this aCSF was not zero Ca2q and a DA induced depolarisation acting via such a conductance could if anything be increased in low Ca2q. A calcium dependent potassium conductance has been described in SPN which is influenced by catecholamines. This is a voltage dependent conductance observed following an action potential whereby noradrenaline application causes an after depolarisation to appear following a fast hyperpolarisation w56x. The slow hyperpolarisations produced by either noradrenaline or adrenaline Ž a-2 receptor dependent. or slow depolarisation of the SPN membrane mediated by a-1 receptors result from changes in a different potassium conductance w34,38,57x. We therefore believe the evidence strongly points to the depolarising action of DA being on presympathetic interneurones. This action was shown to be mediated via a D 2 receptor since it was mimicked by the specific D 2 agonist quinpirole and blocked selectively by the D 2 antagonist haloperidol and unaffected by the D 1 antagonist. 4.3. Slow depolarisation The DA induced depolarisation with its slow rise and slow decay is unlike anything so far described in SPN which is synaptically induced. This raises the question as to the chemical mediator of this synaptic action. It is unlikely to be dependent on activation of an ionotropic glutamate receptor since it is unaffected by kynurenic acid even at 10y5 M. We have so far not tested for a metabotropic receptor mediation but the rise time and decay of the synaptic potential seem somewhat long for this. The depolarisation does though have the characteristics similar to that induced by Substance P w9,22x. For example the slow rise and slow decay of the response, the induction of cell discharge and changes in cell input resistance as well as the finding that it is only induced in less than half the neurones studied. This latter observation accords with the demonstration that Substance P receptors are only present on a specific group of SPN w9,28,33x. In addition a substantial portion Ž) 30%. of intraspinal Substance P innervation of SPN is independent of supraspinal projections w18,19x. This spinal innervation cannot be due to dorsal root fibres passing directly to SPN since there are no monosynaptic afferent connections with SPN w4,5,13,14,24x. Therefore, there is a possibility that DA was stimulating Substance P presympathetic interneurones lying in lamina V or VII of the spinal grey matter w8,31x. Further studies are in progress to identify the neurotransmitter mediating the synaptic depolarisation of SPN induced by DA.
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4.4. Fast synaptic actiÕity Activation of the D 2 receptor by DA also resulted in the appearance of fast synaptic activity in SPN, both IPSP’s and EPSP’s. Similar events are initiated in SPN when superfusing spinal cord slices with adrenaline, substance P, vasopressin, oxytocin and 5-hydroxytryptamine w20,22, 32,38,48x. This suggests that there is a pool of interneurones in the spinal cord onto which there is a heavy convergence of differently chemically coded supraspinal afferents. The significance of this to regulation of spinal sympathetic network remains to be worked out. Nonetheless, this study of the actions of DA has again revealed that there are two ways by which supraspinal afferents may influence SPN; a direct monosynaptic one and an indirect one dependent on presympathetic interneurones w11,26x. In the case of DA it seems unlikely that it is the same group of phenotypic cells that contribute to both mono- and polysynaptic connections since they initiate quite opposite actions. 4.5. Origin of dopamine pathway There are two groups of DA neurones which project to the sympathetic intermediolateral cell column. These arise from the hypothalamus, one from the paraventricular nucleus ŽPVN. w53,54x and the other from the dorsal-posterior hypothalamus the A11 group of catecholamine cells w6x. Of these the PVN-DA projection is most likely to be monosynaptic since it is labelled following retrograde trans-synaptic transfer of pseudorabies virus into a peripheral target w53x. Furthermore, PVN terminals labelled anterogradely have been shown in close association with SPN w30,39,46x. Therefore activation of a DA-spinal pathway when stimulating neurones in the PVN could explain the sympatho-inhibition and depressor responses. It is of interest that another group of PVN-spinal neurones which release vasopressin has been shown to be sympatho-excitatory w37x. It may therefore be that appropriate activation of different neuronal phenotypes results in the pattern of sympathetic activation, whereby some neurones Žcardiac, splanchnic, adrenal. are excited and some Žrenal. are inhibited as for example occurs in the response to plasma volume expansion w2,12,15x.
Acknowledgements We thank the Medical Research Council for their financial support and Drs. D.I. Lewis and S. Pyner for their helpful discussions.
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