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Effects of bioamines and peptides on neurones in the ventral nucleus of trapezoid body and rostral periolivary regions of the rat superior olivary complex: an in vitro investigation
Hearing Research 106 (1997) 20^28
E¡ects of bioamines and peptides on neurones in the ventral nucleus of trapezoid body and rostral periolivary regions of the rat superior olivary complex: an in vitro investigation Xueyong Wang, Donald Robertson * The Auditory Laboratory, Department of Physiology, The University of Western Australia, Nedlands, WA 6907, Australia
Received 25 June 1996; revised 12 November 1996; accepted 22 November 1996
Abstract
Intracellular microelectrode recordings were made from single neurones of the ventral nucleus of trapezoid body and rostral periolivary regions in the rat auditory brainstem, using in vitro slice techniques. Bath application was used to examine the effects of putative neurotransmitters and neuromodulators on cell responses to constant depolarizing current pulsse. Noradrenaline exerted excitatory effects (increased firing rate) that were probably mediated by K-receptors, whereas inhibitory effects (decreased firing rate) were probably mediated by L-receptors. Serotonin also produced either excitatory or inhibitory effects in different cells. Of the neuroactive peptides, substance P and enkephalin were especially potent. Substance P was found to be exclusively excitatory and enkephalin was exclusively inhibitory. Choleycystokinin exerted either inhibitory or excitatory effects in a small percentage of cells. Somatostatin had only very weak or non-existent effects. These effects were able to be elicited under conditions of synaptic blockade, indicating they they were mediated by direct action on the cells in question. Most effects on firing rate were accompanied by either depolarization or hyperpolarization of the resting membrane potential although in many cases this change in membrane potential was small. Changes in cell access resistance were also relatively difficult to detect, but in the case of both noradrenaline and substance P, clear increases in cell access resistance were recorded in a number of cells. These could be obtained in the presence of tetrodotoxin, again indicating a direct action of these substances rather than an indirect action mediated via synaptic connections. Although the exact mechanisms of action remain to be investigated in each case, it is clear that neurones in this region of the auditory brainstem are potentially subject to a wide variety of modulatory influences that could be important in auditory processing. Keywords :
Peptide
Auditory brainstem; Superior olivary complex; Rat; Brain slice; Neurotransmitter; Neuromodulator; Bioamine;
1. Introduction
* Corresponding author. Fax: +61 (9) 380 1025; e-mail: [email protected] AHP, afterhyperpolarisation; AVCN, anteroventral cochlear nucleus; cAMP, cyclicAMP; CCK, cholecystokinin; CN, cochlear nucleus; DAG, diacylglycerol; ENK, enkephalin; 5-HT, serotonin; IC, inferior colliculus; IP3, inositol trisphosphate; LSO, lateral superior olivary nucleus; MSO, medial superior olivary nucleus; MNTB, medial nucleus of trapezoid body; NE, noradrenaline; PVCN, posteroventral cochlear nucleus ; RPO, rostral periolivary zone; SOM, somatostatin; SP, substance P; SPN, superior paraolivary nucleus; SOC, superior olivary complex; TTX, tetrodotoxin; VNLL, ventral nucleus of the lateral lemniscus; VNTB, ventral nucleus of trapezoid body Abbreviations :
The mammalian superior olivary complex (SOC) is an important centre for the integration of ascending auditory information, receiving inputs from the anteroventral (AVCN) and posteroventral (PVCN) cochlear nuclei and projecting to the lateral lemniscal nuclei and the inferior colliculus (IC) (see Irvine, 1986; Helfert et al., 1991 for reviews). This region is also the target of descending pathways emanating from the IC and possibly elsewhere (Vetter et al., 1993 ; Thompson and Thompson, 1993 ; Klepper and Herbert, 1991; Harvey et al., 1993) and is itself the source of descending e¡erent systems that terminate in the cochlear nucleus and
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auditory pathway (Warr and Beck, 1996), the VNTB and RPO are targets of descending pathways from the IC (Thompson and Thompson, 1993; Vetter et al., 1993) and are a major source of the descending pathways to the cochlear nucleus (CN) and cochlea (Warr, 1992). Thus any physiological action of the substances tested could have far-reaching implications for both the central processing of auditory information and the functioning of the peripheral receptor organ. 2. Methods
Fig. 1. Schematic representation of the anatomy of rat brainstem at the level of the SOC. Upper section is more caudal than lower section. The VNTB and RPO from which recordings were made are shown as stippled areas.
on the peripheral receptor cells and primary sensory neurones of the cochlea (Warr, 1992; Winter et al., 1989; Sheri¡ and Henderson, 1994; Spangler et al., 1987; Brown, 1993; Zook and Kuwabara, 1993). There is emerging evidence for the functional role of classical excitatory and inhibitory amino acid neurotransmitters such as acetylcholine, glutamate, glycine and GABA in the SOC (Robertson, 1996; Grothe and Sanes, 1993; Wu and Kelly, 1991, 1995; Caspary et al., 1985; Davies and Owen, 1985; Morley et al., 1985; Potashner et al., 1985; Schwartz, 1985; Vater, 1995; Ross et al., 1995). However, immunocytochemical and in-situ hybridization studies indicate a wide variety of other neurotransmitters and neuromodulators are present, either within intrinsic neurones of the region or as putative transmitters/modulators used by its ascending and descending inputs (Ho¡man et al., 1993; Ryan et al., 1991; Harvey et al., 1993; Wynne et al., 1995; Wynne and Robertson, 1996). It was, therefore, the purpose of the present study to investigate the action of some of these non-amino acid transmitters and neuromodulators on cells in the SOC. To investigate the e¡ects of these substances, we used a slice preparation of the rat auditory brainstem. We investigated the action of two bioamines; noradrenaline (NE) and serotonin (5-HT) and a number of neuroactive peptides in a region of the rat SOC that has so far received little attention: the ventral nucleus of the trapezoid body (VNTB) and the associated rostral periolivary zone (RPO) (cf., Osen et al., 1984). The VNTB is a thin sheet of cells lying ventral to the main SOC. The RPO is more or less continuous with the VNTB and is a zone of sparsely distributed neurones lying sandwiched between the rostral edge of the LSO and ventro-caudal limit of the ventral nucleus of the lateral lemniscus (VNLL). Some authors (Vetter et al., 1993) consider the RPO and VNTB as one continuous structure. As well as containing neurones of the ascending
Slices were prepared from 99 PVG/c and 7 Wistar rats of either sex, ranging in age from 3.5 weeks to about 8 weeks postnatal, using methods described in detail previously (Robertson, 1996). All procedures conformed to the Code of Practice of the National Health and Medical Research Council of Australia and were approved by an institutional Animal Ethics Experimentation Committee. The composition of the ¢nal bathing £uid was 130 mM NaCl, 3 mM KCl, 1.2 mM KH2PO4 , 20 mM NaHCO3 , 2.4 mM CaCl2 , 1.3 mM MgSO4, 10 mM. The solution was continuously bubbled with 95% O2 and 5% CO2 and the ¢nal pH was 7.4. Selected slices containing the VNTB and RPO were mounted in the recording chamber and were continuously perfused on both sides at £ow rates of 4^6 ml/min. For the present studies, the temperature of the superfusate was constant at 25³C, in order to eliminate perturbations in temperature during solution changes. Cell viability and recording stability did not appear to be compromised by this procedure and general cell properties were in conformity with those reported in an earlier study (Robertson, 1996). The gross morphology of the brainstem slices is illustrated schematically in Fig. 1. Single neurones in VNTB and RPO were impaled using glass microelectrodes made from borosilicate ¢lament glass 1.2 mm OD (Clark Electromedical) using a Brown Flaming P-87 puller. Electrodes were ¢lled either with 2 M potassium acetate, or 2 M potassium acetate with 3^4% biocytin Table 1 Summary of e¡ects of bioamines and peptides on single cells in VNTB/RPO Drug tested Concentration (WM) Number of cells (+) (3) (0) NE 0.1^20 83 7 25 5-HT 5 ^20 22 3 15 SP 0.1^10 27 0 16 ENK 1 ^30 0 12 2 CCK 0.1^0.5 3 4 18 SOM 0.1^0.5 1 0 14 Excitatory e¡ects designated as (+), inhibitory e¡ects as (3) and no clear e¡ect (0).
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hydrochloride (Sigma). Electrode DC resistances measured in situ ranged from 90 to 150 M6. The microelectrode was advanced through the slice using a Narashige WR-90 micromanipulator, and the cell membrane potentials were recorded with an Axoclamp 2B ampli¢er, Digidata interface and pClamp (ver. 6.0) software. The pClamp software was also used to specify the timing and amplitude of current pulses delivered through the recording microelectrode. Estimates of cell access resistance were obtained by applying 0.1 nA hyperpolarizing current pulses (50 ms duration) and carefully adjusting the capacity compensation and bridge balance to remove, as far as possible, the contribution of electrode resistance. The change in membrane potential was measured at the end of the hyperpolarizing pulse. For drug applications during single cell recording, known concentrations of the drugs were added to the superfusate, without interruption of the £ow through the chamber. Drug solutions were freshly prepared for each experiment and were continuously bubbled with 95% O2 and 5% CO2 before use. The solutions were held in a series of reservoirs that could be switched to connect them to the inlet to the perfusion chamber. In the typical experimental protocol, the DC value of membrane potential, the cell access resistance, the rate of spontaneous action potentials, and the number of action potentials in response to a standard depolarizing pulse were all recorded before, during and after the test solution was introduced into the recording chamber. The depolarizing current pulse used was 250 ms in duration repeated every 10 s. Drugs were applied for periods ranging from 10 to 40 s before switching back to control solution. The chemicals used were: noradrenaline (NE: Sigma), serotonin (5-HT: Sigma), substance P
Fig. 2. Typical baseline data from a single neurone in VNTB. A,B: reconstruction of biocytin-¢lled cell showing location within VNTB. A: Low-power reconstruction; bottom: at higher power. Scale bar=1 mm in (A) and 100 Wm in (B). C: Responses of same cell in (A) and (B) to a current injection series. Note repetitive discharge and spike shape typical of AHP2 category (Robertson, 1996). Vertical scale bar in (C): 40 mV; horizontal scale bar: 20 ms. Resting potential of cell was 374 mV. See Abbreviation List.
Fig. 3. Results from two di¡erent cells illustrating methods of measurement and showing typical excitatory and inhibitory e¡ects of noradrenaline. Upper two panels in each half of ¢gure show samples of access resistance and resting membrane potential. Note signi¢cant changes in membrane potential. Middle panels show plots of number of number of action potentials evoked by standard depolarizing pulses of 0.05 nA. Arrows indicate time of switching of bathing solution. Records below are digitized traces of original recordings of membrane potential showing samples taken at the times indicated by the numbers. Note depolarization and increased ¢ring rate in one case and hyperpolarization and cessation of ¢ring in other case. Fluctuations in action potential height are artifacts caused by lowered digital sampling rate that was used to store long records. Real action potential amplitude was stable throughout experiment.
(SP: synthetic acetate salt, Sigma), cholecystokinin (CCK: fragment 26^33 amide, CCK-8 sulphated, Sigma), enkephalin (ENK: synthetic acetate salt of leucine enkephalin, Sigma) and somatostatin (SOM: synthetic, Sigma). Non-NMDA receptors were blocked by CNQX (Tocris Neuramin) and action potentials were blocked by tetrodotoxin (TTX: Sigma). For noradrenergic receptors, the selective blockers propranolol and phentolamine and the agonist phenylephrine were used. For SP receptors, the selective agonists used were Senkitide (succinyl^Asp6 MePhe8)^SP for NK3 receptors and (Cys3 6 Tyr8Pro9)^SP for NK1 receptors (Auspep). In a number of experiments, synaptic transmission was ;
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lier description (Robertson, 1996) in which it was reported that there was a strong tendancy for AHP1 cells to have ¢ne rapidly tapering aspinous dendrites and axonal arborization apparently limited to the SOC, whereas AHP2 cells possessed thick spiny dendrites and had at least one axonal branch that ascended dorsally to exit from the SOC. The one aberrant cell in the biocytin-¢lled sample was located within the nearby MNTB. This cell showed the single onset spike and marked recti¢cation in response to current injection that is characteristic of the principal cells of this nucleus (Banks and Smith, 1992). Fig. 4. Illustration of e¡ects of three di¡erent drugs on one cell. Record is interrupted between each drug application as indicated by broken bars on time axis. Upper two panels: Cell access resistance and resting membrane potential. Lower panel: Number of action potentials evoked by standard depolarising current pulse. Note signi¢cant excitatory e¡ects of NE, SP and inhibitory e¡ect of ENK.
blocked by omitting CaCl2 from the bathing medium and in some cases also adding 4 mM MgCl2. For morphological identi¢cation, cells were injected with biocytin via the recording microelectrode using 100 ms depolarizing pulses at a rate of 5/s. Histological processing and cell reconstruction were carried out using standard techniques described elsewhere (Robertson, 1996). 3. Results
3.1. General cell properties
Fig. 2 shows typical recordings obtained from a cell that was injected with biocytin. Also shown in the ¢gure is the histological reconstruction showing the cell's location and morphology. The cell was clearly located within the VNTB/RPO. Its spike shape was typical of the type that has been classi¢ed previously as AHP2 (Robertson, 1996) and it showed morphological features consistent with previous descriptions for this physiological category, viz. numerous thick, slowly tapering spinous dendrites and an axon that ascended dorsally out of the SOC. Of the total sample of 233 cells, 40 were injected with biocytin and all except one of the cells that were retrieved in subsequent histological processing were found to be located within the VNTB/RPO. On the basis of their action potential shape, 50% of these ¢lled cells fell into the category de¢ned as AHP1 (Robertson, 1996; and see Fig. 7), 43% were classi¢ed as AHP2 and 7% were AHP3. A detailed description of the detailed morphology of the biocytin-¢lled cells is beyond the scope of the present paper. However, the present sample con¢rmed an ear-
3.2. Responses to neurotransmitters/modulators
When applied to the bath in micromolar or submicromolar concentrations, all but one of the putative neurotransmitters and neuromodulators tested showed e¡ects on cell excitability. The e¡ects were recorded as either an increase or decrease in the action potential rate evoked by a constant depolarizing current pulse. Accompanying the ¢ring rate change, there was usually a detectable change in resting membrane potential, either a depolarization or hyperpolarization depending on whether the e¡ect was excitatory or inhibitory. All e¡ects on excitability were reversible after washing out the drug (Fig. 3). Some substances were found to be multipotent, able to exert either excitatory or inhibitory e¡ects in di¡erent cells, whereas other substances were found to be exclusively excitatory or inhibitory in all cells in which they produced any detectable e¡ect. Noradrenaline was investigated most intensively. It produced either inhibitory or excitatory e¡ects in di¡erent cells (Fig. 3). Concentrations ranging from 1 to 10 WM were tested and the type of response (excitatory or inhibitory) in any one cell did not appear to be dependent on concentration over this range. The e¡ects of NE were tested in 115 cells, with 83 showing clear excitatory e¡ects, 7 being clearly inhibited and 25 failing to exhibit either a clear increase or decrease in excitability. The excitatory e¡ects typically consisted of a small depolarization of the resting membrane potential (mean change: 3.3 þ 0.7 mV in 1 WM NE), and a clear increase in the number of action potentials evoked by a test depolarizing pulse delivered through the recording microelectrode. The rate of action potentials evoked inTable 2 Summary of results testing cell responses to more than one drug Drugs tested Cells tested Cells responding to all NE and CCK 20 3 NE and SP 21 15 NE and 5-HT 28 18 NE and ENK 9 6 ENK, NE and SP 7 4 5-HT, NE and CCK 10 1
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creased by 5 times or more in some instances, with an average increase of 482 þ86% in 10 WM NE. In some cells, the depolarization evoked by NE application was large enough to also cause spontaneous ¢ring of the cells in addition to an increase in the action potential rate during the test current pulse. Inhibitory e¡ects of NE consisted of a hyperpolarization and a decrease in the number of action potentials produced by a standard depolarizing current pulse. The average hyperpolarization produced was 4.3 þ 0.9 mV, and the action potential ¢ring rate during the test pulse usually fell to zero during this period. Strong excitatory or inhibitory e¡ects were also observed in response to 5-HT application. Forty cells in the VNTB/RPO were tested with this substance, of which 22 showed excitation, 3 showed inhibition and 15 showed equivocal or absent changes. As for NE, inhibitory e¡ects were accompanied by depolarizations or hyperpolarization of the resting membrane potential. For the excitatory e¡ects, the mean depolarization was 3.7 þ 0.6 mV and the mean increase in action potential ¢ring rate above control was 362 þ 45% in 10 WM 5-HT. All but one of the neuroactive peptides tested was found to be active in VNTB and RPO neurones. ENK had only inhibitory e¡ects which were apparent in 12 of the 14 cell tested. No clear e¡ect was seen in the remaining cells. The decrease in excitability observed with enkephalin was accompanied by a small hyperpolarization of the resting membrane potential (mean: change
Fig. 5. Results from one cell showing e¡ects of L-blocker propranolol and illustrating persistent e¡ect of noradrenaline in low calcium solution. Upper two panels: Cell access resistance and resting membrane potential. Lower panel: Number of action potentials evoked by standard depolarising current pulse. Time axis interrupted as shown by broken bars. Open bar above time axis: Period of application of 2.5 WM propranolol. Note reversible elimination of inhibitory e¡ect of 1 WM noradrenaline (NE1). Cross-hatched bar: Period of application of 0 Ca2 solution. Note increased excitability caused by low calcium solution, but persistent inhibitory e¡ect 5 WM noradrenaline (NE5).
Fig. 6. Illustration in one cell of excitatory e¡ects of 1^5 WM noradrenaline (NE1,5,2.5) and 1 WM K-agonist phenylephrine (PL1), and blockage of excitatory e¡ect of 2.5 WM NE by K-blocker phentolamine at 2.5 WM. Open bar above time axis: Period of application of phentolamine. Time axis interrupted as shown by broken bars.
1.5 þ 0.2 mV). Forty-two cells were tested for e¡ects of SP and in 27 of these there was a strong excitatory e¡ect with a clear depolarization of the resting membrane potential (mean change: 2.1 þ 0.5 mV for 1 WM SP) being evident during the period of increased action potential ¢ring to the test depolarizing current pulse. The mean increase in ¢ring rate observed was 377 þ73%. The other cells showed no obvious change in change in excitability. CCK was e¡ective on only a relatively small proportion of the cells tested, but was active at concentrations as low as 0.2 WM. When tested in 25 cells CCK was found to increase excitability in 3 and decrease it in 4. Excitatory e¡ects of CCK were accompanied by a small depolarizing change in resting potential (mean change: 2.7 þ 1.3 mV), whereas inhibitory e¡ects were associated with a clear hyperpolarization (4.9 þ 0.4 mV). The least e¡ective substance tested was SOM. Of 15 cells tested only one cell showed any change of excitability (a weak excitatory e¡ect). Table 1 summarizes the results obtained for all substances. In a total of 95 cells the responses to more than one substance were tested. It was a common observation that one cell could be responsive to more than one of the neuroactive substances tested. Fig. 4 shows a typical example, and the results for all cells tested are summarized in Table 2. 3.3. Blockage of synaptic transmission
The e¡ects described above could be elicited under conditions designed to block chemical synaptic trans-
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Fig. 7. Illustration of spike shape varieties found in four di¡erent cells. The ¢rst example on the left is of a cell classi¢ed as AHP1, with a single, slow deep afterhyperpolarisation. The remaining examples are of cells classi¢ed as AHP2, possessing a rapid early component to the afterhyperpolarisation that varied in prominence.
mission. In 14 cells the e¡ects of NE, SP and ENK were investigated before, during and after calcium was omitted from the bathing medium. The interpretation of these experiments was somewhat complicated by the fact that the low calcium solution itself usually caused a signi¢cant increase in ¢ring of the cell. Nevertheless, clear e¡ects of applied NE (9 cells), SP (3 cells) and ENK (1 cell) could be seen during the period of perfusion of the chamber with low calcium solution. Inhibitory e¡ects were relatively easy to demonstrate in such cases as illustrated in Fig. 5. In the case of excitatory actions, clear additional increases in ¢ring that seemed to be due to the applied drug were seen, but reversibility was poor until the normal levels of calcium were restored. Other experiments were carried out in which TTX was included in the bathing medium, resulting in the complete blockade of all action potentials. Under these conditions, NE (2 cells), and SP (1 cell) were observed to cause a signi¢cant reversible depolarizations of the cells as was the case without TTX present. In addition, signi¢cant reversible increases in cell access resistance were also measured for both NE and SP in the absence of action potentials. The e¡ects of SP were also tested in two cells in the presence of 5 WM CNQX, a selective blocker of nonNMDA type glutamate receptors. In both cases the e¡ect of SP persisted despite this selective blockade of synaptic transmission. 3.4. Selective antagonists and agonists
In the case of NE and SP, various antagonists and agonists were also investigated. The excitatory e¡ects of NE were blocked in 6 out of 7 cells by the K-receptor blocker phentolamine at concentrations ranging from 1^20 WM. Fig. 6 shows an example of this result in which the excitatory e¡ect of 2.5 WM NE was blocked in the presence of 2.5 WM phentolamine. In 12 out of a further 13 cells, it was consistently found that the Kreceptor agonist phenylephrine, at concentrations from
25
1^5 WM elicited similar excitatory e¡ects to NE (Fig. 6). Inhibitory e¡ects of NE were totally blocked by the Lreceptor blocker propranolol (1^20 WM) in all three cells tested (Fig. 5). In one cell, 1 WM phenylephrine was applied to a cell in which action potential generation was blocked by including TTX in the bathing medium. The phenylephrine produced a depolarisation that was similar in magnitude to that elicited by 1 WM NE, although the time course of the depolarisation with phenylephrine was longer. In cells in which SP elicited excitatory responses the e¡ect of two selective SP receptor agonists were also tested. All three cells tested with 0.1^1.0 WM of the NK1 agonist showed excitatory e¡ects that were comparable to those seen with SP. All three cells tested with the NK3 agonist failed to show any change in excitability for the same range of concentrations. 3.5. Relationship to spike shape
Cells in VNTB/RPO have been classi¢ed according to their spike shape and it has been suggested that such spike shape categories may correspond to di¡erent functional classes (Robertson, 1996). It was therefore of interest to know if these di¡erent spike shape categories showed a di¡erential sensitivity to di¡erent drugs, since this might indicate that di¡erent functional classes are subject to synaptic in£uence from di¡erent neurotransmitter systems. Fig. 7 illustrates typical examples of spike shapes found in the present study. Essentially, the scheme of Robertson (1996) was con¢rmed, in that at least two basic spike categories, dubbed AHP1, AHP2 could be identi¢ed. AHP1 cells showed a single slow, deep, bowl-shaped afterhyperpolarisation, whereas AHP2 cells showed an early rapid component to their afterhyperpolarisation, with a second, slower component showing variation in amplitude, shape and time course in di¡erent cells. A third and more rare category (AHP3) was identi¢ed in an earlier study (AHP3) in which the second component was more prominent and clearly separable from the ¢rst rapid component. Such cells were also seen in the present study but their numbers were too small for any meaningful analysis of drug e¡ects. In the present sample, 118 cells were classi¢ed as AHP1 and 89 as AHP2. Although not all cells in each category were responsive, examples were found in both groups that responded to at least one of the basic ¢ve test substances listed in Table 1. However, some di¡erences were apparent in the susceptibility of the di¡erent cell types to di¡erent transmitters. It was found for example, that AHP1 and AHP2 cells were equally likely to be responsive to NE (82% and 77% respectively; M2 test, P s 0.9), whereas AHP1 cells tended to be more likely to show excitatory responses to 5-HT than were AHP2 cells (82% and 50% respec-
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tively; M2 test, P 6 0.1). The most striking di¡erence was found in the case of SP which was e¡ective in all 22 AHP2 cells tested, whereas only 5 out of 17 (29%) of AHP1 cells tested showed any change in excitability to this transmitter (M2 test, P 6 0.001). 4. Discussion
The present study shows that the bioamines NE and 5-HT, and a number of peptides found to be neuroactive in a variety of other systems, have signi¢cant e¡ects on excitability of neurones in the VNTB/RPO of the auditory brainstem. The possibility therefore exists that these substances may have a direct neurotransmitter role in these regions. Altenatively, or in addition, these substances may exert a modulatory in£uence on the actions of other transmitters utilised by the regions' inputs and/or intrinsic neuronal circuitry. The only substance we tested that did not show substantial e¡ects on neuronal excitability in VNTB/RPO was SOM, although it has been reported to have e¡ects elsewhere in the nervous system (for review see Epelbaum, 1986). These e¡ects were found using intracellular microelectrode recording from single cells in slices, and they consisted of membrane potential changes and clear increases or decreases in the number of action potentials evoked by the cells in response to ¢xed amplitude depolarizing current pulses delivered through the recording microelectrode. The e¡ects found were not caused by some non-speci¢c artifact related to the physical act of changing the bathing solution because (1) not all drug solutions produced the same e¡ect in the one cell, (2) changing over to an identical control solution caused no e¡ect, (3) the e¡ects were related to the concentration of drug applied, (4) e¡ects were reversible on washing out the test solutions, and (5) speci¢c antagonists blocked the e¡ects of various drugs. In terms of the cellular site of action, we believe that the results support the view that the e¡ects are mediated, in large part, by a direct action of the neuroactive substances on the cells from which recording were being made. However, another possibility that cannot be totally ruled out is that the drugs were acting pre-synaptically to evoke transmitter release and that this could indirectly modulate excitability. Although we carried out a number of experiments which indicated that synaptic transmission is not required for the e¡ects observed, these experiments su¡ered from a number of limitations. The increase in cell excitability caused by low calcium solutions tended to mask excitatory drug e¡ects and prolonged the recovery. Similarly, the results with CNQX indicated that glutamergic non-NMDA receptor pathways do not need to be activated for the e¡ects to be observed, but they provide no information about the possible involvement of presynaptic pathways
using other transmitters. The experiments with TTX also are consistent with a direct action of NE and SP, but they too do not rule out the possibility of a modulation of pre-synaptic transmitter release that is independent of voltage-gated sodium channels. In other neuronal systems, it is known that NE, 5HT, ENK, SP and CCK exert e¡ects at synapses by acting on receptors that are linked to a variety of second-messenger intracellular pathways. Examples have been reported of the involvement of the inositol trisphosphate (IP3), diacylglycerol (DAG) cyclicAMP and Ca2 -regulated pathways. These second messenger pathways act to modulate the state of a variety of ion channels and so produce a wide variety of post-synaptic changes in membrane conductance, excitability and ¢ring patterns (see Hille, 1992; Shepherd, 1994 for reviews). Further levels of complexity arise from the fact that many of these substances can also act presynaptically to modulate transmitter release and that there is frequent co-existence of and possible interactions between the di¡erent transmitter/modulator systems (see, for example Noble et al., 1993). Thus, the failure to ¢nd a direct action of SOM in the present study does not rule out other possible roles for this peptide that we have not investigated. Further work will be required before it can be stated whether the precise mechanisms of action of all these substances in the VNTB/RPO are similar to those which have been reported elsewhere. The existence of neuronal sensitivity to these substances does not, of itself, provide evidence that they have a role in regulating cell excitability in the VNTB/ RPO under in vivo conditions. Further evidence would consist of the anatomical demonstration of innervation of this region by nerve endings containing these substances, the mapping of the relevant neural pathways and receptors, and the demonstration of physiologically meaningful e¡ects caused by activation of those pathways. The existence of noradrenergic innervation of the auditory brainstem has been known for some time and a recent report describes innervation of the VNTB/RPO in particular (Wynne and Robertson, 1996). One report also exists of an extensive serotoninergic innervation of the SOC (Harvey et al., 1993). So far, however, there is no de¢nitive evidence as to the source of either the noradrenergic or serotoninergic innervation of the VNTB/RPO, although likely candidates are the A5 and A7 cell groups (Wynne and Robertson, 1996) and the locus coeruleus (Klepper and Herbert, 1991). As far as the peptides are concerned, there is similar lack of de¢nitive information regarding possible pathways (Adams, 1993). mRNAs for CCK and SOM precursors are present in cells of the inferior colliculus (mainly the central nucleus and external cortex) with
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CCK being particularly prominent. SOM precursor is
ing auditory pathways, the action of these substances in
also manufactured in the ventral CN and in low con-
the intact animal could have profound e¡ects on the
centrations in cells of the superior paraolivary nucleus
processing of ascending information and on the centri-
(SPN). Within the main nuclei of the SOC itself, immu-
fugal signals that exit the brainstem to in£uence the
nocytochemical (Ho¡man et al., 1993 ; Ryan et al.,
functioning of the peripheral receptor organ.
1991) and in-situ hybridisation experiments (Wynne et al., 1995) provide evidence that VNTB/RPO cells in the rat manufacture ENK and it has been suggested that
Acknowledgments
some or all of these cells may belong to the group of medial olivocochlear neurones that project from the
Supported by grants from the National Health and
VNTB to the inner ear (Ho¡man et al., 1993 ; Ryan
Medical Research Council, The Australian Research
et al., 1991). Thus the present demonstration of inhib-
Council and The University of Western Australia. The
itory e¡ects of ENK on VNTB cells may re£ect intrinsic
authors are indebted to D. Bornow for animal care and
connections of enkephalinergic cells within this region.
solution preparation.
Previous slice experiments and double labelling anatomical studies have demonstration that many cells in the VNTB/RPO possess local axon collaterals with terminal arbors, which is certainly consistent with this notion (Robertson, 1996 ; Warr and Beck, 1996). As far as SP is concerned, previous reports indicate
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that the SOC itself is devoid of neuronal somata con-
Juiz, D.A. Godfrey, and E. Mugnaini (Eds.), The Mammalian
taining this substance (Wynne et al., 1995). However, it
Cochlear Nuclei : Organization and Function. Plenum, pp. 133^
has been reported that cells in the dorsomedial zones and external cortex of the IC express mRNA for preprotachykinin, a precursor peptide for SP (Wynne et
141. Adams, J.C. and Mugnaini, E. (1985) Patterns of immunostaining with antisera to peptides in the auditory brainstem of cat. Soc. Neurosci. Abstr. 11, 32.
al., 1995). SP immunoreactivity has also been directly
Banks, M.I. and Smith, P.H. (1992). Intracellular recordings from
demonstrated in cells of the IC (Adams, 1993) and in
neurobiotin-labeled cells in brain slices of the rat medial nucleus
terminals in the ventromedial zones of the cat SOC (Adams and Mugnaini, 1985). It is therefore possible that there may be a SP-utilising descending pathway
of the trapezoid body. J. Neurosci. 12, 2819^2837. Brown, M.C. (1993) Fiber pathways and branching patterns of biocytin labeled olivocochlear neurones in the mouse brainstem. J. Comp. Neurol. 337, 600^613.
from the IC to the VNTB/RPO in the rat. Of particular
Caspary, D.M., Rybak, L.P. and Faingold, C.L. (1985) The e¡ects of
interest in this regard is the clear association of SP
inhibitory and excitatory amino-acid neurotransmitters on the re-
sensitivity
with
the
AHP2
type
of
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sponse
properties
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Drescher (Ed.), Auditory Biochemistry. CC Thomas, Spring¢eld, IL.
cumstantial evidence for the notion that cells with the
Davies, W.E. and Owen, C. (1985) The nature of neurotransmitters in
AHP2 spike shape may belong to the medial olivo-
the mammalian lower auditory system. In : D.G. Drescher (Ed.),
cochlear neurone population originating in the VNTB/ RPO and terminating on the receptor cells of the inner ear. There is also direct anatomical evidence for an IC
Auditory Biochemistry. CC Thomas, Spring¢eld, IL. Epelbaum J. (1986) Somatostatin in the central nervous system : physiology and pathological modi¢cations. Prog. Neurobiol 27, 63^ 100.
projection to medial olivocochlear neurones (Vetter et
Grothe, B. and Sanes, D.H. (1993) Bilateral inhibition by glycinergic
al., 1993). The available evidence therefore suggests that
a¡erents in the medial superior olive. J. Neurophysiol. 69, 1192^
these cells may be the selective targets of a descending system originating in the IC, that releases SP from its terminals. The strong excitatory e¡ect of SP seen in the
1196. Harvey, J.A., McMaster, S.E. and Romano, A.G. (1993) Methylenedioxyamphetamnine : neurotoxic e¡ects on serotoninergic projections to brainstem nuclei in the rat. Brain Res. 619, 1^14.
present study would suggest that this pathway might
Helfert, R.H., Snead, C.R. and Altschuler, R.A. (1991) The ascending
serve to raise the level of activity in the olivocochlear
auditory pathways. In : Altschuler et al. (Eds.), Neurobiology of
projection.
Hearing : The Central Auditory System, Raven Press, New York,
More work is needed to establish a clear functional role for the changes in excitability described here. In addition, the list of neurotransmitters/neuromodulators
pp. 1^25. Ho¡man, D.W., Hochreiter, J.S., Landry, D.R., Brimijoin, M.R., Treadwell, M.D., Gardner, P.D. and Altschuler, R.A. (1993) Localization of preproenkephalin mRNA-expression cells in rat audi-
that has been investigated here is by no means exhaus-
tory brainstem with in situ hybridization. Hear. Res. 69, 1^9.
tive (Adams, 1993). Nonetheless, the present results
Hille, B. (1992) Ionic Channels of Excitable Membranes, 2nd edn.,
provide clear evidence that neurones in the VNTB/ RPO are potentially subject to a wide variety of modulatory chemical in£uences. Given the emerging picture of a role for the VNTB in both ascending and descend-
Sinauer, pp. 182^193. Irvine, D.R.F. (1986) The Auditory Brainstem. In : D. Ottoson (Ed.), Progess in Sensory Physiology, Vol. 7, Springer, Berlin, 1986, pp. 79^121. Klepper, A. and Herbert, H. (1991) Distribution and origin of nor-
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adrenergic and serotinergic ¢bres in the cochlear nucleus and inferior colliculus of the rat. Brain Res. 557, 190^201.
Thompson, A.M. and Thompson, G.C. (1993) Relationship of descending inferior colliculus projections to olivocochlear neurons.
Morley, B.J., Farley, G.R. and Javel, E. (1985) Putative neurotranmitter receptors in the central auditory system. In : D.G. Drescher (Ed.), Auditory Biochemistry. CC Thomas, Spring¢eld, IL. Noble, F., Derrien, M. and Roques, B.P. (1993) Modulation of opioid
J. Comp. Neurol. 335, 402^412. Vater, M. (1995) Ultrastructural and immunocytochemical observations on the superior olivary complex of the mustached bat. J. Comp. Neurol. 358, 155^180.
antinociception by CCK at the supraspinal level : evidence of reg-
Vetter, D.E., Saldana, E. and Mugnaini, E. (1993) Inputs from the
ulatory mechanisms between CCK and enkephalin systems in the
inferior colliculus to medial olivocochlear neurons in the rat : a
control of pain. Br. J. Pharmacol. 109, 1064^1070.
double label study with PHA-L and cholera toxin. Hear. Res.
Osen, K.K., Mugnaini, E., Dahl, A.L. and Christiansen, A.H. (1984)
70, 173^186.
Histochemical localization of acetylcholine esterase in the cochlear
Warr, W.B. (1992) Organization of olivocochlear e¡erent systems in
and superior olivary nuclei : a reappraisal with emphasis on the
mammals. In : D.B. Webster et al. (Eds.), Mammalian Auditory
cochlear granule cell system. Arch. Ital. Biol. 122, 169^212.
Pathway : Neuroanatomy, Springer Handbook of Auditory Re-
Potashner, S.J., Morst, D.K., Oliver, D.L. and Jones, D.R. (1985)
search, pp. 410^448.
Identi¢cation of glutamatergic and asparatergic pathways in the
Warr, W.B. and Beck, J.E. (1996) Multiple projections from the ven-
auditory sysem. In : D.G. Drescher (Ed.), Auditory Biochemistry.
tral nucleus of the trapezoid body in the rat. Hear. Res. 93, 83^
CC Thomas, Spring¢eld, IL.
101.
Robertson, D. (1996) Physiology and morphology of cells in the ven-
Winter, I.M., Robertson, D. and Cole, K.S. (1989) Descending pro-
tral nucleus of the trapezoid body and rostral periolivary regions
jections from auditory brainstem nuclei to the cochlea and coch-
of the rat superior olivary complex studied in slices. Aud. Neurosci. 2, 12^31.
lear nucleus of the guinea pig. J. Comp. Neurol. 280, 143^157. Wu, S.H. and Kelly, J.B. (1991) Physiological properties of neurons in
Ross, C.D., Godfrey, D.A. and Parli, J.A. (1995) Amino acid concentrations and seleceted enzyme activities in rat auditory, olfactory and visual systems. Neurochem. Res. 20, 1483^1490.
the mouse superior olive : membrane characteristics and post-synaptic responses studied in vitro. J. Neurophysiol. 65, 230^246. Wu, S.H. and Kelly, J.B. (1995) Inhibition in the superior olivary
Ryan, A.F., Simmons, D.M., Watts, A.G. and Swanson, L.W. (1991) Enkephalin mRNA production by cochlear and vestibular e¡erent neurons in the gerbil brainstem. Exp. Brain Res. 87, 259^267. Schwartz, I.R. (1985) Autoradiographic studies of amino acid labeling of neural elements in the auditory brainstem. In : D.G. Drescher (Ed.), Auditory Biochemistry. CC Thomas, Spring¢eld, IL. Shepherd, B., Neurobiology, 3rd edn., Oxford, New York, 1994, pp. 170^187.
complex :
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Wynne, B. and Robertson, D. (1996) Localization of dopamine- hydroxylase-loke immunoreactivity in the superior olivary complex of the rat. Audiol. Neurootol. 1, 54^64. Wynne, B., Harvey, A.R., Robertson, D. and Sirinathsinghji, D.J.S. (1995) Neurotransmitter and neuromodulator systems of the rat inferior colliculus and auditory brainstem studied by in situ hy-
Sheri¡, F.E. and Henderson, Z. (1994) Cholinergic neurons in the ventral trapezoid nucleus project to the cochlear nuclei in the rat. Neuroscience 58, 627^633.
bridization. J. Chem. Neuroanat. 9, 289^300. Zook, J.M. and Kuwabara, N. (1993) Superior olivary cells with descending projections to the cochlear nucleus. In : M.A. Merchan,
Spangler, K.M., Cant, M.B., Henkel, C.K., Farley, G.R. and Warr, W.B. (1987) Descending projections from the superior olivary
J.M. Juiz, D.A. Godfrey, and E. Mugnaini (Eds.), The Mammalian Cochlear Nuclei. Plenum, pp. 143^151.
complex to the cochlear nucleus of the cat. J. Comp. Neurol. 259, 452^465.
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E¡ects of bioamines and peptides on neurones in the ventral nucleus of trapezoid body and rostral periolivary regions of the rat superior olivary complex: an in vitro investigation Xueyong Wang, Donald Robertson * The Auditory Laboratory, Department of Physiology, The University of Western Australia, Nedlands, WA 6907, Australia
Received 25 June 1996; revised 12 November 1996; accepted 22 November 1996
Abstract
Intracellular microelectrode recordings were made from single neurones of the ventral nucleus of trapezoid body and rostral periolivary regions in the rat auditory brainstem, using in vitro slice techniques. Bath application was used to examine the effects of putative neurotransmitters and neuromodulators on cell responses to constant depolarizing current pulsse. Noradrenaline exerted excitatory effects (increased firing rate) that were probably mediated by K-receptors, whereas inhibitory effects (decreased firing rate) were probably mediated by L-receptors. Serotonin also produced either excitatory or inhibitory effects in different cells. Of the neuroactive peptides, substance P and enkephalin were especially potent. Substance P was found to be exclusively excitatory and enkephalin was exclusively inhibitory. Choleycystokinin exerted either inhibitory or excitatory effects in a small percentage of cells. Somatostatin had only very weak or non-existent effects. These effects were able to be elicited under conditions of synaptic blockade, indicating they they were mediated by direct action on the cells in question. Most effects on firing rate were accompanied by either depolarization or hyperpolarization of the resting membrane potential although in many cases this change in membrane potential was small. Changes in cell access resistance were also relatively difficult to detect, but in the case of both noradrenaline and substance P, clear increases in cell access resistance were recorded in a number of cells. These could be obtained in the presence of tetrodotoxin, again indicating a direct action of these substances rather than an indirect action mediated via synaptic connections. Although the exact mechanisms of action remain to be investigated in each case, it is clear that neurones in this region of the auditory brainstem are potentially subject to a wide variety of modulatory influences that could be important in auditory processing. Keywords :
Peptide
Auditory brainstem; Superior olivary complex; Rat; Brain slice; Neurotransmitter; Neuromodulator; Bioamine;
1. Introduction
* Corresponding author. Fax: +61 (9) 380 1025; e-mail: [email protected] AHP, afterhyperpolarisation; AVCN, anteroventral cochlear nucleus; cAMP, cyclicAMP; CCK, cholecystokinin; CN, cochlear nucleus; DAG, diacylglycerol; ENK, enkephalin; 5-HT, serotonin; IC, inferior colliculus; IP3, inositol trisphosphate; LSO, lateral superior olivary nucleus; MSO, medial superior olivary nucleus; MNTB, medial nucleus of trapezoid body; NE, noradrenaline; PVCN, posteroventral cochlear nucleus ; RPO, rostral periolivary zone; SOM, somatostatin; SP, substance P; SPN, superior paraolivary nucleus; SOC, superior olivary complex; TTX, tetrodotoxin; VNLL, ventral nucleus of the lateral lemniscus; VNTB, ventral nucleus of trapezoid body Abbreviations :
The mammalian superior olivary complex (SOC) is an important centre for the integration of ascending auditory information, receiving inputs from the anteroventral (AVCN) and posteroventral (PVCN) cochlear nuclei and projecting to the lateral lemniscal nuclei and the inferior colliculus (IC) (see Irvine, 1986; Helfert et al., 1991 for reviews). This region is also the target of descending pathways emanating from the IC and possibly elsewhere (Vetter et al., 1993 ; Thompson and Thompson, 1993 ; Klepper and Herbert, 1991; Harvey et al., 1993) and is itself the source of descending e¡erent systems that terminate in the cochlear nucleus and
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auditory pathway (Warr and Beck, 1996), the VNTB and RPO are targets of descending pathways from the IC (Thompson and Thompson, 1993; Vetter et al., 1993) and are a major source of the descending pathways to the cochlear nucleus (CN) and cochlea (Warr, 1992). Thus any physiological action of the substances tested could have far-reaching implications for both the central processing of auditory information and the functioning of the peripheral receptor organ. 2. Methods
Fig. 1. Schematic representation of the anatomy of rat brainstem at the level of the SOC. Upper section is more caudal than lower section. The VNTB and RPO from which recordings were made are shown as stippled areas.
on the peripheral receptor cells and primary sensory neurones of the cochlea (Warr, 1992; Winter et al., 1989; Sheri¡ and Henderson, 1994; Spangler et al., 1987; Brown, 1993; Zook and Kuwabara, 1993). There is emerging evidence for the functional role of classical excitatory and inhibitory amino acid neurotransmitters such as acetylcholine, glutamate, glycine and GABA in the SOC (Robertson, 1996; Grothe and Sanes, 1993; Wu and Kelly, 1991, 1995; Caspary et al., 1985; Davies and Owen, 1985; Morley et al., 1985; Potashner et al., 1985; Schwartz, 1985; Vater, 1995; Ross et al., 1995). However, immunocytochemical and in-situ hybridization studies indicate a wide variety of other neurotransmitters and neuromodulators are present, either within intrinsic neurones of the region or as putative transmitters/modulators used by its ascending and descending inputs (Ho¡man et al., 1993; Ryan et al., 1991; Harvey et al., 1993; Wynne et al., 1995; Wynne and Robertson, 1996). It was, therefore, the purpose of the present study to investigate the action of some of these non-amino acid transmitters and neuromodulators on cells in the SOC. To investigate the e¡ects of these substances, we used a slice preparation of the rat auditory brainstem. We investigated the action of two bioamines; noradrenaline (NE) and serotonin (5-HT) and a number of neuroactive peptides in a region of the rat SOC that has so far received little attention: the ventral nucleus of the trapezoid body (VNTB) and the associated rostral periolivary zone (RPO) (cf., Osen et al., 1984). The VNTB is a thin sheet of cells lying ventral to the main SOC. The RPO is more or less continuous with the VNTB and is a zone of sparsely distributed neurones lying sandwiched between the rostral edge of the LSO and ventro-caudal limit of the ventral nucleus of the lateral lemniscus (VNLL). Some authors (Vetter et al., 1993) consider the RPO and VNTB as one continuous structure. As well as containing neurones of the ascending
Slices were prepared from 99 PVG/c and 7 Wistar rats of either sex, ranging in age from 3.5 weeks to about 8 weeks postnatal, using methods described in detail previously (Robertson, 1996). All procedures conformed to the Code of Practice of the National Health and Medical Research Council of Australia and were approved by an institutional Animal Ethics Experimentation Committee. The composition of the ¢nal bathing £uid was 130 mM NaCl, 3 mM KCl, 1.2 mM KH2PO4 , 20 mM NaHCO3 , 2.4 mM CaCl2 , 1.3 mM MgSO4, 10 mM. The solution was continuously bubbled with 95% O2 and 5% CO2 and the ¢nal pH was 7.4. Selected slices containing the VNTB and RPO were mounted in the recording chamber and were continuously perfused on both sides at £ow rates of 4^6 ml/min. For the present studies, the temperature of the superfusate was constant at 25³C, in order to eliminate perturbations in temperature during solution changes. Cell viability and recording stability did not appear to be compromised by this procedure and general cell properties were in conformity with those reported in an earlier study (Robertson, 1996). The gross morphology of the brainstem slices is illustrated schematically in Fig. 1. Single neurones in VNTB and RPO were impaled using glass microelectrodes made from borosilicate ¢lament glass 1.2 mm OD (Clark Electromedical) using a Brown Flaming P-87 puller. Electrodes were ¢lled either with 2 M potassium acetate, or 2 M potassium acetate with 3^4% biocytin Table 1 Summary of e¡ects of bioamines and peptides on single cells in VNTB/RPO Drug tested Concentration (WM) Number of cells (+) (3) (0) NE 0.1^20 83 7 25 5-HT 5 ^20 22 3 15 SP 0.1^10 27 0 16 ENK 1 ^30 0 12 2 CCK 0.1^0.5 3 4 18 SOM 0.1^0.5 1 0 14 Excitatory e¡ects designated as (+), inhibitory e¡ects as (3) and no clear e¡ect (0).
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hydrochloride (Sigma). Electrode DC resistances measured in situ ranged from 90 to 150 M6. The microelectrode was advanced through the slice using a Narashige WR-90 micromanipulator, and the cell membrane potentials were recorded with an Axoclamp 2B ampli¢er, Digidata interface and pClamp (ver. 6.0) software. The pClamp software was also used to specify the timing and amplitude of current pulses delivered through the recording microelectrode. Estimates of cell access resistance were obtained by applying 0.1 nA hyperpolarizing current pulses (50 ms duration) and carefully adjusting the capacity compensation and bridge balance to remove, as far as possible, the contribution of electrode resistance. The change in membrane potential was measured at the end of the hyperpolarizing pulse. For drug applications during single cell recording, known concentrations of the drugs were added to the superfusate, without interruption of the £ow through the chamber. Drug solutions were freshly prepared for each experiment and were continuously bubbled with 95% O2 and 5% CO2 before use. The solutions were held in a series of reservoirs that could be switched to connect them to the inlet to the perfusion chamber. In the typical experimental protocol, the DC value of membrane potential, the cell access resistance, the rate of spontaneous action potentials, and the number of action potentials in response to a standard depolarizing pulse were all recorded before, during and after the test solution was introduced into the recording chamber. The depolarizing current pulse used was 250 ms in duration repeated every 10 s. Drugs were applied for periods ranging from 10 to 40 s before switching back to control solution. The chemicals used were: noradrenaline (NE: Sigma), serotonin (5-HT: Sigma), substance P
Fig. 2. Typical baseline data from a single neurone in VNTB. A,B: reconstruction of biocytin-¢lled cell showing location within VNTB. A: Low-power reconstruction; bottom: at higher power. Scale bar=1 mm in (A) and 100 Wm in (B). C: Responses of same cell in (A) and (B) to a current injection series. Note repetitive discharge and spike shape typical of AHP2 category (Robertson, 1996). Vertical scale bar in (C): 40 mV; horizontal scale bar: 20 ms. Resting potential of cell was 374 mV. See Abbreviation List.
Fig. 3. Results from two di¡erent cells illustrating methods of measurement and showing typical excitatory and inhibitory e¡ects of noradrenaline. Upper two panels in each half of ¢gure show samples of access resistance and resting membrane potential. Note signi¢cant changes in membrane potential. Middle panels show plots of number of number of action potentials evoked by standard depolarizing pulses of 0.05 nA. Arrows indicate time of switching of bathing solution. Records below are digitized traces of original recordings of membrane potential showing samples taken at the times indicated by the numbers. Note depolarization and increased ¢ring rate in one case and hyperpolarization and cessation of ¢ring in other case. Fluctuations in action potential height are artifacts caused by lowered digital sampling rate that was used to store long records. Real action potential amplitude was stable throughout experiment.
(SP: synthetic acetate salt, Sigma), cholecystokinin (CCK: fragment 26^33 amide, CCK-8 sulphated, Sigma), enkephalin (ENK: synthetic acetate salt of leucine enkephalin, Sigma) and somatostatin (SOM: synthetic, Sigma). Non-NMDA receptors were blocked by CNQX (Tocris Neuramin) and action potentials were blocked by tetrodotoxin (TTX: Sigma). For noradrenergic receptors, the selective blockers propranolol and phentolamine and the agonist phenylephrine were used. For SP receptors, the selective agonists used were Senkitide (succinyl^Asp6 MePhe8)^SP for NK3 receptors and (Cys3 6 Tyr8Pro9)^SP for NK1 receptors (Auspep). In a number of experiments, synaptic transmission was ;
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lier description (Robertson, 1996) in which it was reported that there was a strong tendancy for AHP1 cells to have ¢ne rapidly tapering aspinous dendrites and axonal arborization apparently limited to the SOC, whereas AHP2 cells possessed thick spiny dendrites and had at least one axonal branch that ascended dorsally to exit from the SOC. The one aberrant cell in the biocytin-¢lled sample was located within the nearby MNTB. This cell showed the single onset spike and marked recti¢cation in response to current injection that is characteristic of the principal cells of this nucleus (Banks and Smith, 1992). Fig. 4. Illustration of e¡ects of three di¡erent drugs on one cell. Record is interrupted between each drug application as indicated by broken bars on time axis. Upper two panels: Cell access resistance and resting membrane potential. Lower panel: Number of action potentials evoked by standard depolarising current pulse. Note signi¢cant excitatory e¡ects of NE, SP and inhibitory e¡ect of ENK.
blocked by omitting CaCl2 from the bathing medium and in some cases also adding 4 mM MgCl2. For morphological identi¢cation, cells were injected with biocytin via the recording microelectrode using 100 ms depolarizing pulses at a rate of 5/s. Histological processing and cell reconstruction were carried out using standard techniques described elsewhere (Robertson, 1996). 3. Results
3.1. General cell properties
Fig. 2 shows typical recordings obtained from a cell that was injected with biocytin. Also shown in the ¢gure is the histological reconstruction showing the cell's location and morphology. The cell was clearly located within the VNTB/RPO. Its spike shape was typical of the type that has been classi¢ed previously as AHP2 (Robertson, 1996) and it showed morphological features consistent with previous descriptions for this physiological category, viz. numerous thick, slowly tapering spinous dendrites and an axon that ascended dorsally out of the SOC. Of the total sample of 233 cells, 40 were injected with biocytin and all except one of the cells that were retrieved in subsequent histological processing were found to be located within the VNTB/RPO. On the basis of their action potential shape, 50% of these ¢lled cells fell into the category de¢ned as AHP1 (Robertson, 1996; and see Fig. 7), 43% were classi¢ed as AHP2 and 7% were AHP3. A detailed description of the detailed morphology of the biocytin-¢lled cells is beyond the scope of the present paper. However, the present sample con¢rmed an ear-
3.2. Responses to neurotransmitters/modulators
When applied to the bath in micromolar or submicromolar concentrations, all but one of the putative neurotransmitters and neuromodulators tested showed e¡ects on cell excitability. The e¡ects were recorded as either an increase or decrease in the action potential rate evoked by a constant depolarizing current pulse. Accompanying the ¢ring rate change, there was usually a detectable change in resting membrane potential, either a depolarization or hyperpolarization depending on whether the e¡ect was excitatory or inhibitory. All e¡ects on excitability were reversible after washing out the drug (Fig. 3). Some substances were found to be multipotent, able to exert either excitatory or inhibitory e¡ects in di¡erent cells, whereas other substances were found to be exclusively excitatory or inhibitory in all cells in which they produced any detectable e¡ect. Noradrenaline was investigated most intensively. It produced either inhibitory or excitatory e¡ects in di¡erent cells (Fig. 3). Concentrations ranging from 1 to 10 WM were tested and the type of response (excitatory or inhibitory) in any one cell did not appear to be dependent on concentration over this range. The e¡ects of NE were tested in 115 cells, with 83 showing clear excitatory e¡ects, 7 being clearly inhibited and 25 failing to exhibit either a clear increase or decrease in excitability. The excitatory e¡ects typically consisted of a small depolarization of the resting membrane potential (mean change: 3.3 þ 0.7 mV in 1 WM NE), and a clear increase in the number of action potentials evoked by a test depolarizing pulse delivered through the recording microelectrode. The rate of action potentials evoked inTable 2 Summary of results testing cell responses to more than one drug Drugs tested Cells tested Cells responding to all NE and CCK 20 3 NE and SP 21 15 NE and 5-HT 28 18 NE and ENK 9 6 ENK, NE and SP 7 4 5-HT, NE and CCK 10 1
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creased by 5 times or more in some instances, with an average increase of 482 þ86% in 10 WM NE. In some cells, the depolarization evoked by NE application was large enough to also cause spontaneous ¢ring of the cells in addition to an increase in the action potential rate during the test current pulse. Inhibitory e¡ects of NE consisted of a hyperpolarization and a decrease in the number of action potentials produced by a standard depolarizing current pulse. The average hyperpolarization produced was 4.3 þ 0.9 mV, and the action potential ¢ring rate during the test pulse usually fell to zero during this period. Strong excitatory or inhibitory e¡ects were also observed in response to 5-HT application. Forty cells in the VNTB/RPO were tested with this substance, of which 22 showed excitation, 3 showed inhibition and 15 showed equivocal or absent changes. As for NE, inhibitory e¡ects were accompanied by depolarizations or hyperpolarization of the resting membrane potential. For the excitatory e¡ects, the mean depolarization was 3.7 þ 0.6 mV and the mean increase in action potential ¢ring rate above control was 362 þ 45% in 10 WM 5-HT. All but one of the neuroactive peptides tested was found to be active in VNTB and RPO neurones. ENK had only inhibitory e¡ects which were apparent in 12 of the 14 cell tested. No clear e¡ect was seen in the remaining cells. The decrease in excitability observed with enkephalin was accompanied by a small hyperpolarization of the resting membrane potential (mean: change
Fig. 5. Results from one cell showing e¡ects of L-blocker propranolol and illustrating persistent e¡ect of noradrenaline in low calcium solution. Upper two panels: Cell access resistance and resting membrane potential. Lower panel: Number of action potentials evoked by standard depolarising current pulse. Time axis interrupted as shown by broken bars. Open bar above time axis: Period of application of 2.5 WM propranolol. Note reversible elimination of inhibitory e¡ect of 1 WM noradrenaline (NE1). Cross-hatched bar: Period of application of 0 Ca2 solution. Note increased excitability caused by low calcium solution, but persistent inhibitory e¡ect 5 WM noradrenaline (NE5).
Fig. 6. Illustration in one cell of excitatory e¡ects of 1^5 WM noradrenaline (NE1,5,2.5) and 1 WM K-agonist phenylephrine (PL1), and blockage of excitatory e¡ect of 2.5 WM NE by K-blocker phentolamine at 2.5 WM. Open bar above time axis: Period of application of phentolamine. Time axis interrupted as shown by broken bars.
1.5 þ 0.2 mV). Forty-two cells were tested for e¡ects of SP and in 27 of these there was a strong excitatory e¡ect with a clear depolarization of the resting membrane potential (mean change: 2.1 þ 0.5 mV for 1 WM SP) being evident during the period of increased action potential ¢ring to the test depolarizing current pulse. The mean increase in ¢ring rate observed was 377 þ73%. The other cells showed no obvious change in change in excitability. CCK was e¡ective on only a relatively small proportion of the cells tested, but was active at concentrations as low as 0.2 WM. When tested in 25 cells CCK was found to increase excitability in 3 and decrease it in 4. Excitatory e¡ects of CCK were accompanied by a small depolarizing change in resting potential (mean change: 2.7 þ 1.3 mV), whereas inhibitory e¡ects were associated with a clear hyperpolarization (4.9 þ 0.4 mV). The least e¡ective substance tested was SOM. Of 15 cells tested only one cell showed any change of excitability (a weak excitatory e¡ect). Table 1 summarizes the results obtained for all substances. In a total of 95 cells the responses to more than one substance were tested. It was a common observation that one cell could be responsive to more than one of the neuroactive substances tested. Fig. 4 shows a typical example, and the results for all cells tested are summarized in Table 2. 3.3. Blockage of synaptic transmission
The e¡ects described above could be elicited under conditions designed to block chemical synaptic trans-
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Fig. 7. Illustration of spike shape varieties found in four di¡erent cells. The ¢rst example on the left is of a cell classi¢ed as AHP1, with a single, slow deep afterhyperpolarisation. The remaining examples are of cells classi¢ed as AHP2, possessing a rapid early component to the afterhyperpolarisation that varied in prominence.
mission. In 14 cells the e¡ects of NE, SP and ENK were investigated before, during and after calcium was omitted from the bathing medium. The interpretation of these experiments was somewhat complicated by the fact that the low calcium solution itself usually caused a signi¢cant increase in ¢ring of the cell. Nevertheless, clear e¡ects of applied NE (9 cells), SP (3 cells) and ENK (1 cell) could be seen during the period of perfusion of the chamber with low calcium solution. Inhibitory e¡ects were relatively easy to demonstrate in such cases as illustrated in Fig. 5. In the case of excitatory actions, clear additional increases in ¢ring that seemed to be due to the applied drug were seen, but reversibility was poor until the normal levels of calcium were restored. Other experiments were carried out in which TTX was included in the bathing medium, resulting in the complete blockade of all action potentials. Under these conditions, NE (2 cells), and SP (1 cell) were observed to cause a signi¢cant reversible depolarizations of the cells as was the case without TTX present. In addition, signi¢cant reversible increases in cell access resistance were also measured for both NE and SP in the absence of action potentials. The e¡ects of SP were also tested in two cells in the presence of 5 WM CNQX, a selective blocker of nonNMDA type glutamate receptors. In both cases the e¡ect of SP persisted despite this selective blockade of synaptic transmission. 3.4. Selective antagonists and agonists
In the case of NE and SP, various antagonists and agonists were also investigated. The excitatory e¡ects of NE were blocked in 6 out of 7 cells by the K-receptor blocker phentolamine at concentrations ranging from 1^20 WM. Fig. 6 shows an example of this result in which the excitatory e¡ect of 2.5 WM NE was blocked in the presence of 2.5 WM phentolamine. In 12 out of a further 13 cells, it was consistently found that the Kreceptor agonist phenylephrine, at concentrations from
25
1^5 WM elicited similar excitatory e¡ects to NE (Fig. 6). Inhibitory e¡ects of NE were totally blocked by the Lreceptor blocker propranolol (1^20 WM) in all three cells tested (Fig. 5). In one cell, 1 WM phenylephrine was applied to a cell in which action potential generation was blocked by including TTX in the bathing medium. The phenylephrine produced a depolarisation that was similar in magnitude to that elicited by 1 WM NE, although the time course of the depolarisation with phenylephrine was longer. In cells in which SP elicited excitatory responses the e¡ect of two selective SP receptor agonists were also tested. All three cells tested with 0.1^1.0 WM of the NK1 agonist showed excitatory e¡ects that were comparable to those seen with SP. All three cells tested with the NK3 agonist failed to show any change in excitability for the same range of concentrations. 3.5. Relationship to spike shape
Cells in VNTB/RPO have been classi¢ed according to their spike shape and it has been suggested that such spike shape categories may correspond to di¡erent functional classes (Robertson, 1996). It was therefore of interest to know if these di¡erent spike shape categories showed a di¡erential sensitivity to di¡erent drugs, since this might indicate that di¡erent functional classes are subject to synaptic in£uence from di¡erent neurotransmitter systems. Fig. 7 illustrates typical examples of spike shapes found in the present study. Essentially, the scheme of Robertson (1996) was con¢rmed, in that at least two basic spike categories, dubbed AHP1, AHP2 could be identi¢ed. AHP1 cells showed a single slow, deep, bowl-shaped afterhyperpolarisation, whereas AHP2 cells showed an early rapid component to their afterhyperpolarisation, with a second, slower component showing variation in amplitude, shape and time course in di¡erent cells. A third and more rare category (AHP3) was identi¢ed in an earlier study (AHP3) in which the second component was more prominent and clearly separable from the ¢rst rapid component. Such cells were also seen in the present study but their numbers were too small for any meaningful analysis of drug e¡ects. In the present sample, 118 cells were classi¢ed as AHP1 and 89 as AHP2. Although not all cells in each category were responsive, examples were found in both groups that responded to at least one of the basic ¢ve test substances listed in Table 1. However, some di¡erences were apparent in the susceptibility of the di¡erent cell types to di¡erent transmitters. It was found for example, that AHP1 and AHP2 cells were equally likely to be responsive to NE (82% and 77% respectively; M2 test, P s 0.9), whereas AHP1 cells tended to be more likely to show excitatory responses to 5-HT than were AHP2 cells (82% and 50% respec-
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X. Wang, D. Robertson / Hearing Research 106 (1997) 20^28
tively; M2 test, P 6 0.1). The most striking di¡erence was found in the case of SP which was e¡ective in all 22 AHP2 cells tested, whereas only 5 out of 17 (29%) of AHP1 cells tested showed any change in excitability to this transmitter (M2 test, P 6 0.001). 4. Discussion
The present study shows that the bioamines NE and 5-HT, and a number of peptides found to be neuroactive in a variety of other systems, have signi¢cant e¡ects on excitability of neurones in the VNTB/RPO of the auditory brainstem. The possibility therefore exists that these substances may have a direct neurotransmitter role in these regions. Altenatively, or in addition, these substances may exert a modulatory in£uence on the actions of other transmitters utilised by the regions' inputs and/or intrinsic neuronal circuitry. The only substance we tested that did not show substantial e¡ects on neuronal excitability in VNTB/RPO was SOM, although it has been reported to have e¡ects elsewhere in the nervous system (for review see Epelbaum, 1986). These e¡ects were found using intracellular microelectrode recording from single cells in slices, and they consisted of membrane potential changes and clear increases or decreases in the number of action potentials evoked by the cells in response to ¢xed amplitude depolarizing current pulses delivered through the recording microelectrode. The e¡ects found were not caused by some non-speci¢c artifact related to the physical act of changing the bathing solution because (1) not all drug solutions produced the same e¡ect in the one cell, (2) changing over to an identical control solution caused no e¡ect, (3) the e¡ects were related to the concentration of drug applied, (4) e¡ects were reversible on washing out the test solutions, and (5) speci¢c antagonists blocked the e¡ects of various drugs. In terms of the cellular site of action, we believe that the results support the view that the e¡ects are mediated, in large part, by a direct action of the neuroactive substances on the cells from which recording were being made. However, another possibility that cannot be totally ruled out is that the drugs were acting pre-synaptically to evoke transmitter release and that this could indirectly modulate excitability. Although we carried out a number of experiments which indicated that synaptic transmission is not required for the e¡ects observed, these experiments su¡ered from a number of limitations. The increase in cell excitability caused by low calcium solutions tended to mask excitatory drug e¡ects and prolonged the recovery. Similarly, the results with CNQX indicated that glutamergic non-NMDA receptor pathways do not need to be activated for the e¡ects to be observed, but they provide no information about the possible involvement of presynaptic pathways
using other transmitters. The experiments with TTX also are consistent with a direct action of NE and SP, but they too do not rule out the possibility of a modulation of pre-synaptic transmitter release that is independent of voltage-gated sodium channels. In other neuronal systems, it is known that NE, 5HT, ENK, SP and CCK exert e¡ects at synapses by acting on receptors that are linked to a variety of second-messenger intracellular pathways. Examples have been reported of the involvement of the inositol trisphosphate (IP3), diacylglycerol (DAG) cyclicAMP and Ca2 -regulated pathways. These second messenger pathways act to modulate the state of a variety of ion channels and so produce a wide variety of post-synaptic changes in membrane conductance, excitability and ¢ring patterns (see Hille, 1992; Shepherd, 1994 for reviews). Further levels of complexity arise from the fact that many of these substances can also act presynaptically to modulate transmitter release and that there is frequent co-existence of and possible interactions between the di¡erent transmitter/modulator systems (see, for example Noble et al., 1993). Thus, the failure to ¢nd a direct action of SOM in the present study does not rule out other possible roles for this peptide that we have not investigated. Further work will be required before it can be stated whether the precise mechanisms of action of all these substances in the VNTB/RPO are similar to those which have been reported elsewhere. The existence of neuronal sensitivity to these substances does not, of itself, provide evidence that they have a role in regulating cell excitability in the VNTB/ RPO under in vivo conditions. Further evidence would consist of the anatomical demonstration of innervation of this region by nerve endings containing these substances, the mapping of the relevant neural pathways and receptors, and the demonstration of physiologically meaningful e¡ects caused by activation of those pathways. The existence of noradrenergic innervation of the auditory brainstem has been known for some time and a recent report describes innervation of the VNTB/RPO in particular (Wynne and Robertson, 1996). One report also exists of an extensive serotoninergic innervation of the SOC (Harvey et al., 1993). So far, however, there is no de¢nitive evidence as to the source of either the noradrenergic or serotoninergic innervation of the VNTB/RPO, although likely candidates are the A5 and A7 cell groups (Wynne and Robertson, 1996) and the locus coeruleus (Klepper and Herbert, 1991). As far as the peptides are concerned, there is similar lack of de¢nitive information regarding possible pathways (Adams, 1993). mRNAs for CCK and SOM precursors are present in cells of the inferior colliculus (mainly the central nucleus and external cortex) with
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27
CCK being particularly prominent. SOM precursor is
ing auditory pathways, the action of these substances in
also manufactured in the ventral CN and in low con-
the intact animal could have profound e¡ects on the
centrations in cells of the superior paraolivary nucleus
processing of ascending information and on the centri-
(SPN). Within the main nuclei of the SOC itself, immu-
fugal signals that exit the brainstem to in£uence the
nocytochemical (Ho¡man et al., 1993 ; Ryan et al.,
functioning of the peripheral receptor organ.
1991) and in-situ hybridisation experiments (Wynne et al., 1995) provide evidence that VNTB/RPO cells in the rat manufacture ENK and it has been suggested that
Acknowledgments
some or all of these cells may belong to the group of medial olivocochlear neurones that project from the
Supported by grants from the National Health and
VNTB to the inner ear (Ho¡man et al., 1993 ; Ryan
Medical Research Council, The Australian Research
et al., 1991). Thus the present demonstration of inhib-
Council and The University of Western Australia. The
itory e¡ects of ENK on VNTB cells may re£ect intrinsic
authors are indebted to D. Bornow for animal care and
connections of enkephalinergic cells within this region.
solution preparation.
Previous slice experiments and double labelling anatomical studies have demonstration that many cells in the VNTB/RPO possess local axon collaterals with terminal arbors, which is certainly consistent with this notion (Robertson, 1996 ; Warr and Beck, 1996). As far as SP is concerned, previous reports indicate
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