Bursting properties of caudal neurosecretory cells in the flounder Platichthys flesus, in vitro
School of Biological Sciences, University of Manchester, Manchester, UK
*Author for correspondence (e-mail: Cathy.McCrohan{at}man.ac.uk)
Accepted May 24, 2001
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Summary |
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Key words: neurosecretion, bursting, electrophysiology, flounder, Platichthys flesus, osmoregulation.
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Introduction |
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The CNSS receives descending input, largely from the hindbrain (Cohen and Kriebel, 1989). Immunohistochemical and biochemical studies indicate adrenergic, serotonergic, cholinergic and peptidergic (gonadotropin-releasing hormone and neuropeptide Y) inputs to the CNSS (Audet and Chevalier, 1981; McKeon et al., 1988; Miller and Kriebel, 1986; Yulis et al., 1990; Oka et al., 1997). The origin and physiological role of these inputs is unknown. We have begun to characterise the actions of specific neurotransmitters/modulators (monoamines, acetylcholine) on Dahlgren cells in an in vitro CNSS preparation from the euryhaline flounder (Hubbard et al., 1996a; Hubbard et al., 1997; Brierley et al., 2000). Descending input to the Dahlgren cells is likely to influence the electrical output of the CNSS by modulating both intrinsic cellular and local network properties and, hence, to affect patterns of peptide secretion. However, the spontaneous firing patterns of type 1 Dahlgren cells have not yet been described in detail.
Two subtypes of Dahlgren cell (type 1 and type 2) have been identified in seawater-adapted flounder using electrophysiological criteria (Hubbard et al., 1996b). It is unlikely that these represent separate populations secreting either UI or UII, since immunocytochemical studies indicate that the two peptides are colocalised in >90% of all Dahlgren cells (Larson et al., 1987; A. Ashworth, unpublished). Type 2 cells are electrically silent in the in vitro system and can only be induced to fire single action potentials, during depolarising current injection, owing to spike frequency accommodation (Hubbard et al., 1996b). In contrast, the more numerous type 1 cells are usually spontaneously active, often generating characteristic bursting activity. The ability to generate prolonged high frequency bursts of spikes is a common feature of secretory cells in both vertebrates and invertebrates (e.g. mammalian hypothalamic neurons) (Lincoln and Wakerley, 1974) (pancreatic ß-cells) (Bertram and Sherman, 2000) (Aplysia brasiliana R15 neuron) (Dudek et al., 1979). Each burst of electrical activity facilitates the release of a bolus of neuropeptide in sufficient quantity transiently to increase circulating levels. Bursting properties depend on intrinsic neuronal properties or on local network interactions, or may reflect a combination of the two. Furthermore, the modulation of the intrinsic burst properties of the Dahlgren cells is likely to be reflected in changes in neuropeptide secretion via the urophysis.
The European flounder is of marine origin and is one of the few fish species that can fully adapt its osmoregulatory physiology to both sea and fresh water. In this study, we have exploited the highly accessible in vitro preparation of the CNSS of seawater-adapted flounder, in order to characterise bursting parameters of type 1 Dahlgren cells in the absence of external inputs. The aims were, firstly, to define intrinsic cellular parameters of Dahlgren cell bursting activity that are likely to be modulated by descending input to the CNSS during the process of physiological adaptation, thus leading to altered peptide secretion in vivo. This was achieved using intracellular recordings from individual type 1 Dahlgren cells. Secondly, extracellular recordings were carried out to determine the relative firing patterns of groups of Dahlgren (presumably type 1) cells and, in particular, whether their bursting activity is synchronised or coordinated. Thirdly, experiments were carried out to determine whether a known neuromodulator substance could influence ongoing bursting activity in Dahlgren cells. 5-Hydroxytryptamine (5-HT) was chosen for these experiments since this neuromodulator has been shown to be present in the CNSS (Cohen et al., 1990) and to hyperpolarise Dahlgren cells directly (Hubbard et al., 1997).
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Materials and methods |
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Intracellular recordings of Dahlgren cell activity
Intracellular recordings were made from individual Dahlgren cells using glass microelectrodes filled with 3moll-1 potassium acetate with resistances of 50100M. The electrode was connected to an Axoclamp 2A amplifier (Axon Instruments, CA, USA), data captured via a CED 1401 converter (Cambridge Electronic Design, UK) and stored and analysed using CED SIGNAL version 1.72 and Spike2 version 2.01 software. Once a cell was penetrated, the bridge-balance facility in the amplifier was adjusted whilst passing 500ms, -0.3nA pulses at 0.3Hz. Only cells that maintained a steady resting membrane potential of at least -50mV, and that could generate overshooting action potentials, were considered viable. Type 1 cells were impaled and held for up to 120min, and their spontaneous activity was recorded continuously. Responses to hyperpolarising current pulses (501000ms, -0.1 to -1.5nA) were recorded. In addition, depolarised and hyperpolarised membrane potentials could be imposed by continuous current injection in order to examine the response to current pulses at different holding potentials.
Extracellular recordings from the CNSS
Extracellular recordings were made under bath conditions similar to those described above, using either two fine silver wire hook electrodes (0.5mm diameter wire, flattened to approx. 1mm to increase the area of contact, 1mm apart) placed under the spinal cord, or a suction electrode with pipette diameter approx. 0.5mm, at pre-terminal segments 2 or 3. Stable recordings could be maintained for more than 6h. Signals were recorded differentially and amplified using a Neurolog AC NL104 amplifier (Digitimer, UK) and filtered (AC NL125; 5Hz and 1.2kHz cut-off frequencies plus a 50Hz notch filter). A CED converter was used to digitise the signals and CED Spike2 software for storage and analysis.
Bath and focal application of 5-HT
Both focal (during intracellular recordings) and bath (for extracellular recordings) drug applications were achieved using simple gravity-fed systems with a 10ml and 50ml reservoir, respectively. Focal applications (210s) of 5-HT (100µmoll-1) via large diameter glass electrodes (placed 100500µm upstream of the recording site) restricted 5-HT applications to the spinal cord segment from which recordings were being made. The perfusion of the spinal cord in normal Ringer was maintained throughout. In contrast, during extracellular recording, the whole CNSS was superfused with 5-HT (100µmoll-1). The preparation was superfused with normal Ringer for 2h prior to any 5-HT application. The CNSS was then superfused with 5-HT and allowed to equilibrate in this medium for 30min, after which it was returned to normal Ringer for up to a further 3.5h.
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Results |
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Bursting properties of Dahlgren cells
Bursts of action potentials lasted approx. 120s, and were separated by periods of inactivity lasting between 70s and 600s (Fig.1A), yielding a mean cycle period of 382±49.4s. Burst duration and total number of spikes per burst were remarkably consistent both within and between cells in vitro (Table1), suggesting at least some intrinsic or local component to burst generation. Fig.1B shows a typical burst together with a plot of instantaneous spike frequency. There is an initial acceleration phase (to a maximum frequency of 4.5±0.4Hz after 28.7±3.5s), followed by slower deceleration and burst termination. The time taken and the number of spikes generated to reach maximum spike frequency were again consistent within and between cells, as were the parameters relating to the deceleration phase (Table1).
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Spontaneous and evoked bursts
During generation of spontaneous bursting, bursts appeared to be triggered by a compound excitatory postsynaptic potential (EPSP), which led to depolarisation of the membrane by 7.0±0.7mV (N=14 cells) until action potential threshold was reached (Fig.2). The compound EPSP consisted of a slow wave of depolarisation, upon which were superimposed fast, possibly unitary, EPSPs of 24mV amplitude and 30ms duration. The origin of these inputs in this in vitro preparation is unknown.
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Extracellular recording from Dahlgren cells
Extracellular recording from the spinal cord (N=17 preparations) enabled the simultaneous monitoring of activity of a number of Dahlgren cells, which could often be separated by differences in apparent spike amplitude (Fig.5A). Where activity was discernible from a single Dahlgren cell it was possible to distinguish bursting activity with burst durations (approx. 120s) comparable to those displayed by single cells recorded intracellularly (Fig. 5B). The most notable observation was that Dahlgren cells did not burst synchronously. Instead, the combined activity of a small population of Dahlgren cells often contributed to much longer superbursts, whose duration varied from 500 to 900s (Fig.5C).
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Discussion |
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The DAP provides a potential means for maintaining repetitive firing since each action potential and associated AHP is followed by a rebound depolarisation, which may reach threshold. In the Dahlgren cells, the sag potential took around 200ms to develop; this is similar to the duration of the AHP and could account in part for the maximum spike frequency of 5Hz seen during a burst. Another limit to firing rate within a burst was action potential shape. At maximum firing frequency, spike duration increased and AHP amplitude reduced. The latter would lead to decreased activation of the inward current underlying DAP and thus delay the subsequent action potential, leading to a slowing of firing rate in the latter part of the burst. The occurrence of DAP was dependent on membrane potential; it appeared only in relatively depolarised cells. This suggests that bursting is, at least partially, dependent on background excitation, either from descending pathways or local interneuronal networks. Even in our isolated, in vitro preparation, bursts appeared to be triggered by excitatory synaptic inputs, which must have been generated locally. A population of local serotonergic interneurons was identified within the CNSS of the molly Poecilia latipinna (Cohen et al., 1990), which may form part of a rhythm generating network, providing phasic input to Dahlgren cells. However, since 5-HT is inhibitory to these cells, excitatory neurons, possibly cholinergic, must also be involved. Acetylcholine is present in large amounts in the CNSS of teleosts (Conlon and Balment, 1996) and has been shown in preliminary experiments to excite flounder Dahlgren cells (Brierley et al., 2000). The mechanism underlying burst termination is unknown since spike duration and AHP returned to their original level as firing rate declined.
Simultaneous extracellular recording from a group of Dahlgren cells showed no evidence for synchronised bursting. In contrast, close coupling of magnocellular oxytocin neurons leads to synchronised population bursts which have been linked to pulsatile release of high concentrations of peptide for phasic milk ejection (Poulain and Wakerley, 1982). These bursts have high frequency and short duration compared to those recorded in Dahlgren cells, typically comprising 7080 spikes within 24s. The asynchronous bursting activity of the Dahlgren cells resembles more that of vasopressin hypothalamic neurons, with longer and less intense bursts. During spontaneous phasic activity, the latter show burst durations of up to 100s and intraburst firing rates up to 15 spikess-1, with no synchronisation of bursting activity between cells (Poulain and Wakerley, 1982). Thus, for vasopressin neurons, and probably Dahlgren cells, the bursts provide an efficient means of secreting hormone, whilst asynchrony of bursts ensures continuous rather than pulsatile output. The lack of apparent coupling between the activity of Dahlgren cells does not, however, rule out the possibility of local interactions at the level of the neurosecretory axon terminals. Cioni and De Vito (Cioni and De Vito, 2000) reported colocalisation of UI and UII with nitric oxide synthase in axon terminals in the CNSS of the teleost Oreochromis niloticus. This raises the possibility that nitric oxide produced in response to neuronal activity could act locally to modulate further release of urotensins. The occurrence of the superbursts recorded extracellularly in Dahlgren cells may have functional significance or may represent an artefact owing to the relatively small number of cells being sampled.
Pharmacological concentrations of 5-HT terminated individual bursts and inhibited bursting in the Dahlgren cell population, showing that experimental manipulation of the activity of the system using known neuromodulators is achievable. As suggested by Poulain and Wakerley (Poulain and Wakerley, 1982), it is likely that neurosecretory cell activity would be modulated in two ways by input pathways. Subtle modifications of peptide secretion are probably achieved by alterations in burst frequency and duration, via modification of intrinsic parameters that facilitate bursts, such as membrane potential, AHP and DAP. However, the amount of peptide secreted might also depend on recruitment of silent or tonically active cells. Like Dahlgren cells, vasopressin neurons show three different activity patterns (slow irregular, fast continuous and phasic), which probably represent different activity states of the same neuron type (Poulain and Wakerley, 1982).
Bursting activity in Dahlgren cells is relatively homogeneous in the in vitro system, and will enable future characterisation of factors that influence CNSS activity and urotensin secretion. Considerable variation in the bursting pattern must occur in vivo, in response to changes, for example, in osmotic status. Both intra- and extracellular recording from the CNSS of flounder in vivo are feasible, and will allow us to investigate the functional role of this discrete neurosecretory system at the cellular level within its physiological context.
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Acknowledgments |
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References |
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