Electrical activity of caudal neurosecretory neurons in seawater- and freshwater-adapted flounder: responses to cholinergic agonists
School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK
* Author for correspondence (e-mail: Cathy.McCrohan{at}man.ac.uk)
Accepted 30 July 2003
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Summary |
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Key words: flounder, Platichthys flesus, neurosecretory system, Dahlgren cell, acetylcholine, nicotine, oxotremorine, electrophysiology, osmoregulation
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Introduction |
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Using intracellular recording from an isolated in vitro CNSS
preparation, Hubbard et al.
(1996a) identified two types
of Dahlgren cell in the flounder, based on electrophysiological criteria. Type
1 cells were spontaneously active, whereas type 2 cells were less numerous,
normally quiescent and relatively inexcitable. The two cell types have similar
morphology (Hubbard et al.,
1996a
) and apparently both colocalise UI and UII (A. Ashworth,
unpublished observation; Yamada et al.,
1986
; Larson et al.,
1987
; Ichikawa et al.,
1988
). This raised the possibility that type 1 and type 2 cells
represent different functional states of a common neuron type. Around 60% of
type 1 cells exhibit characteristic bursting activity
(Brierley et al., 2001
),
reminiscent of that described for other magnocellular neuroendocrine cells,
such as mammalian vasopressin neurons
(Armstrong et al., 1994
;
Stern and Armstrong, 1995
;
Leng et al., 1999
). Bursting
activity has been reported to facilitate release of neuropeptide
(Cazalis et al., 1985
),
suggesting that the pattern of electrical activity in these cells may be at
least as important as overall firing frequency in regulating release of
neurohormone into the circulation.
Using a homologous radioimmunoassay for flounder UII, Winter et al.
(1999) reported elevated
levels of the peptide in plasma taken from fully seawater-adapted (SWA)
compared to freshwater-adapted (FWA) fish. This suggests that the activity
patterns of Dahlgren cells may differ between the two states of adaptation,
leading to raised UII secretion in seawater conditions. Such differences have
not yet been investigated; previous electrophysiological studies in the
flounder were confined to CNSS from SWA fish.
Our electrophysiological studies of flounder CNSS in vitro have
identified neuromodulators of Dahlgren cell activity, which probably arise
in vivo from descending pathways. For example, superfusion with
adrenergic agonists led to hyperpolarisation of type 1 and type 2 cells
(Hubbard et al., 1996b); type
1, but not type 2, cells were similarly inhibited by serotonin
(Hubbard et al., 1997
). A
further potential modulator of CNSS activity is acetylcholine (ACh). Pandey
(1981
) reported the presence
of acetylcholinesterase in CNSS from a number of freshwater teleost species
and, in a study using H3+ choline uptake, Conlon and Balment
(1996
) confirmed ACh synthesis
within isolated trout CNSS, together with marked ACh release in response to a
depolarising (high K+) stimulus. A preliminary electrophysiological
study (Brierley et al., 2000
)
demonstrated cholinergic excitation of a sub-population of flounder type 1
Dahlgren cells; indeed, ACh is the only neurotransmitter so far shown to
depolarise these neurons. This raises the possibility that ACh may play a role
in regulating Dahlgren cell activity during the transition between freshwater
and seawater.
In this study we first aimed to compare electrophysiological properties and
spontaneous activity patterns of Dahlgren cells in CNSS taken from fully SWA
and FWA flounders. We then tested the hypothesis that ACh modulates Dahlgren
cell activity and that its modulatory effects are related to the adaptive
state of the system. Both nicotinic and muscarinic effects were investigated,
since mammalian vasopressin neurons are modulated by both these pathways
(Michels et al., 1991;
Renaud and Borque, 1991
;
Mori et al., 1994
;
Zaninetti et al., 2002
). We
used both intracellular and extracellular (multi-unit) recordings from the
isolated CNSS preparation.
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Materials and methods |
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Intracellular recordings from Dahlgren cells
Intracellular recordings were made from individual Dahlgren cells using
glass microelectrodes (40-80 M when filled with 3 mol l-1
potassium acetate). Electrodes were fabricated using a one-stage pull on a
Flaming-Brown type P-97 micropipette puller (Sutter Instrument Co., CA, USA)
and thin-walled, filamented, borosilicate glass (GC150F-10, Harvard Apparatus,
Kent, UK). The electrode was connected to an Axoclamp 2A amplifier (Axon
Instruments, CA, USA), data captured (sampling rate
8 kHz) via a
CED 1401 interface (Cambridge Electronic Design, UK) and stored and analysed
(off-line) using CED Spike2 (v4.01) software. Once a neuron had been
penetrated, the electrode was bridge-balanced (using 500 ms, -0.3 nA pulses at
0.3 Hz) and the cell's spontaneous electrical activity and resting membrane
potential were monitored for at least 30 min. Cells with membrane potentials
more negative than -50 mV and that generated the action potential waveform
characteristic of Dahlgren cells (Hubbard
et al., 1996a
) were considered viable. Stable recordings of up to
3 h were possible. Measurement protocols for membrane parameters followed
Hubbard et al. (1996a
). For
measurement of action potential duration (measured at half maximal height)
only action potentials which were not preceded by another spike for >10 s
were sampled, as Dahlgren cells exhibit frequency-dependent spike broadening
(Brierley et al., 2001
).
Extracellular recordings from the CNSS
Extracellular recordings were made from pre-terminal segments 2-4 using a
large (300-600 µm tip diameter) glass suction electrode, filled with
Ringer. The electrode was placed directly on the surface of the cord to record
activity in nearby neuronal somata and axonal tracts. Signals were recorded
differentially and amplified (x10K) using a Neurolog AC NL104 amplifier
(Digitimer, UK) and filtered (AC NL125; 5 Hz and 1.2 kHz cut-off frequencies
plus a 50 Hz notch filter). A CED converter and Spike2 (v4.01) software were
used for data storage (sampling rate 8-12 kHz) and analysis. Stable recordings
of up to 8 h were possible. Recordings were analysed off-line using CED Spike2
software to separate out activity of up to eight individual units. Briefly,
the software generates an action potential template for each unit and scans
the recording for similar waveforms (60-90% fit required). Activity from
Dahlgren cells was positively identified by their long duration (4-8 ms),
triphasic action potential waveform. Units were selected randomly but only
included in the final analysis when >90% (estimated empirically) of
activity was successfully extracted from the original recording. Action
potentials from separated units are illustrated as event marks using the
Spike2 software option. Estimation of overall activity during extracellular
recordings was achieved using the channel process option, in which waveforms
are rectified (all negative values replaced by positive values) and then
integrated via the smooth function (time constant=1 s).
Pharmacological experiments
Drugs (ACh, Sigma-Aldrich, UK; nicotine, RBI, UK; oxotremorine M, Tocris
Cookson, UK) (100 µmol l-1 in appropriate Ringer) were cooled to
8-12°C prior to bath superfusion over the CNSS. Drugs were superfused for
10 min, followed by a return to normal Ringer. There was a minimum of 20 min
between successive drug applications.
Data analysis
Data are presented as mean ± S.E.M., except for burst
data (Table 2), which are
presented as ± S.D. to allow assessment of the variability
in durations of different phases of activity. Statistical analysis (student's
two tailed t-test) was carried out using Graphpad Prism (v3.0).
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Results |
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Type 1 cells from SWA CNSS exhibit a `sag' potential in response to a
hyperpolarising injected current pulse, which is more pronounced at
depolarised membrane potentials (Brierley
et al., 2001). In this study, we found similar sag potentials in
type 1 cells from FWA CNSS (Fig.
1). Furthermore, as for SWA cells
(Brierley et al., 2001
), the
sag potential in FWA cells was voltage-dependent. This is illustrated in
Fig. 1B, which shows responses
to an injected hyperpolarising current pulse (-1.2 nA) in a FWA type 1
Dahlgren cell, measured at three levels of membrane potential (resting
membrane potential, RMP=-60 mV, and RMP±10 mV). The degree of sag
(measured as the difference in membrane potential between the initial peak
deflection and that at pulse termination) for RMP+10 mV (6.0±2.1 mV)
was significantly greater (P<0.05, N=10 cells) than that
measured at RMP (1.7±0.4 mV) and RMP-10 mV (0.4±0.2 mV). There
was no significant difference between sag potentials in FWA compared to SWA
cells at RMP or at RMP±10 mV.
The sag potential is followed by a depolarising after-potential (DAP)
immediately following termination of the current pulse in both SWA
(Fig. 1A;
Brierley et al., 2001) and FWA
(Fig. 1) cells. Like the sag
potential, this DAP is voltage-dependent. At RMP and more negative potentials,
DAP amplitude was <2 mV and not significantly different between SWA
(N=10) and FWA (N=6) cells (P>0.5). However, at
the depolarised membrane potential (RMP+10 mV) DAP amplitude was significantly
greater (P<0.005) than at more negative potentials, sometimes
causing the cell to overshoot threshold and fire
(Fig. 1B). In addition, DAPs
evoked in depolarised SWA type 1 cells were significantly larger than in
depolarised FWA type 1 cells (SWA=7.0±0.7 mV, N=10;
FWA=3.6±0.5 mV, N=6; P=0.005).
Recordings from type 2 cells (SWA 8 cells; FWA 3 cells; not shown) revealed the presence of both sag potentials and DAP, suggesting similar membrane properties in spontaneously active type 1 and in the relatively inexcitable type 2 Dahlgren cells. Sag potentials (ca. 1 mV at RMP) and DAPs (ca. 2.5 mV) were not significantly different between SWA and FWA type 2 cells.
Since type 2 cells are not spontaneously active, and therefore not recorded extracellularly, the remaining analyses described in this paper were confined to type 1 Dahlgren cells.
Extracellular recording from CNSS
In some preparations (N=8) a type 1 Dahlgren cell was recorded
simultaneously both intracellularly, from the more rostral soma, and
extracellularly from the descending axon tract. This allowed measurement of
axonal conduction velocity (0.73±0.16 ms-1), which
indicates, as suggested by previous histochemical evidence
(Pandey, 1981), that the
Dahlgren cell axons are unmyelinated. Extracellular recording revealed typical
long-duration action potentials (4-8 ms when recorded extracellularly)
characteristic of Dahlgren cells (Fig.
2A). It also revealed the presence of electrical activity from
another, unidentified, neuron type with much shorter duration action
potentials (<1 ms), indicating the presence of non-Dahlgren cells (here
termed
neurons; Fig.
2A) within the CNSS. Spontaneous
neuron activity was more
commonly recorded from FWA (ca. 50% of recordings) than from SWA (ca. 20% of
recordings) preparations but was always triggered following muscarinic
activation (see below).
|
Suction electrode recording allowed us to monitor the activity of several Dahlgren cells in the same preparation. Fig. 2B shows an example of a SWA recording analysed using Spike2 to separate out activity of individual units. Typically between 2 and 6 units were analysed in this way in each preparation. Cells from SWA (N=40 from 8 fish) and FWA (N=40 from 12 fish) preparations were recorded for 60 min. Activity was classified as: `bursting', comprising discrete action potential bursts of >20 s separated by periods of >20 s inactivity; `phasic', consisting of irregular activity including periods of higher frequency (typically 1-3 Hz), lower frequency (<1 Hz) and no firing; `tonic' with continuous activity of ca. 1 Hz and few or no silent periods; `silent', cells whose presence only became apparent when the cell became spontaneously active during the recording period. Similar activity patterns were also distinguished from intracellular recordings (Fig. 2C). Table 2 summarises the firing activity of bursting, phasic and tonically active Dahlgren cells from SWA and FWA CNSS (silent cells could not be included in this analysis). The proportions of cells displaying different classes of activity pattern did not differ between SWA and FWA preparations. Furthermore, there was no significant difference in firing frequencies (mean or maximum), or of burst duration or cycle period in bursting cells. However, FWA bursting cells were more variable in terms of burst duration and cycle period compared to SWA, as shown by the larger S.D. for these parameters for FWA cells. This greater variation was mainly accounted for by a longer deceleration phase for FWA bursts. The minimum burst duration from FWA and SWA cells was similar (34 s and 42 s respectively), as was the mean acceleration phase duration (i.e. time from the onset of a burst to peak spike frequency). However, although the mean deceleration phase duration was not significantly different (P>0.5), the coefficients of variation for this parameter were very different at 49% for SWA and 91% for FWA, suggesting a difference in the mechanisms underlying SWA and FWA burst termination.
Transitions between firing patterns in individual Dahlgren cells
During the 60 min extracellular recording period, some units underwent a
spontaneous transition between one type of firing pattern and another
(Fig. 3), supporting our view
that different classes of activity do not represent different classes of
Dahlgren cell, but rather that the same cell type may fire in different
ways.
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Spontaneous transitions occurred in 7/40 (17.5%) SWA and 11/40 (27.5%) FWA Dahlgren cells. All of the transitions in SWA Dahlgren cells involved tonic activity. The majority were between tonic activity and silence (5/7 cells: 71%; Fig. 3A) and two (29%) involved tonic-phasic transitions. There were no transitions to or from bursting activity in SWA Dahlgren cells. FWA cells underwent a greater variety of transitions (Fig. 3B). Tonically active FWA cells underwent transitions to all other firing patterns (one became bursting, one phasic and two silent) and two silent cells became active (one tonic, one bursting). In contrast to the SWA observations, 36% of the transitions involved a change to bursting activity (Fig. 3B) and there was a single observation of a regularly bursting neuron becoming phasic.
Responses to acetylcholine and ACh receptor agonists
In a preliminary study, Brierley et al.
(2000) observed both
depolarising and hyperpolarising responses to acetylcholine in Dahlgren cells
and, in a subset of cells, no response at all. In this study, superfusion with
ACh (100 µmol l-1) elicited similar inconsistent responses in
cells recorded extracellularly, from SWA (N=3 preparations)
(Fig. 4) and FWA (N=3)
CNSS. In the example shown in Fig.
4 cells were excited (Fig.
4v), inhibited (Fig.
4iii) and/or altered their firing pattern
(Fig. 4ii, bursting to phasic).
This range of responses suggested differential ACh receptor (AChR) expression
in individual neurons. The contribution of ionotropic and metabotropic AChR
was assessed using the agonists nicotine and oxotremorine, respectively.
|
In both SWA and FWA preparations, superfusion of the broad-spectrum
muscarinic agonist, oxotremorine (100 µmol l-1, 600 s) led to an
increase in overall activity in the CNSS. However, this was largely due to
excitation of neurons, seen as a marked increase in frequency of short
duration (<1 ms) action potentials (Fig.
5A). The effects of oxotremorine on
neurons occurred
within 30 s of onset of superfusion and typically washed out within 200 s.
Extracellular recording sites varied in their access to Dahlgren cell and
neurons. Some recordings had little or no Dahlgren cell activity
(Fig. 5A), whereas others
contained activity from >4 Dahlgren cells
(Fig. 5B,C). In the latter,
neuron activity often became apparent only after further analysis
(e.g. Fig. 5B) as the
small-amplitude
neuron signal (<20 µV) was obscured. However,
rectification and integration (RIT) of the raw data signal to enable
quantification of total activity, highlighted underlying excitation by
oxotremorine (Fig. 5B).
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The effect of oxotremorine on Dahlgren cells was largely inhibitory, leading to cessation of activity followed by recovery after 10-20 min washout (Figs 5B,C, 6A,B). All FWA type 1 cells (18 units from 7 fish) and 93% of SWA cells (26/28 cells from 9 fish) ceased firing following the onset of superfusion (Fig. 6A,B). Following washout with normal Ringer, 68% of SWA and 33% of FWA cells resumed their previous activity pattern. The remaining cells generated a new pattern of activity or remained silent.
|
The inhibitory effect of oxotremorine was examined further using intracellular recordings. In FWA type 1 Dahlgren cells, oxotremorine induced hyperpolarisation in 4/6 cells (three tonically and one phasically active) (Fig. 6C) leading to cessation of activity for up to 1200 s. Peak hyperpolarisation (20.5±5.2 mV) was reached within 200 s, after which, even in the continued presence of the agonist, membrane potential began to repolarise towards RMP (Fig. 6C). The two non-inhibited neurons (one silent, one bursting) were both transiently and weakly depolarized, by around 5 mV, during the superfusion period only. Three SWA type 1 Dahlgren cells, recorded intracellularly (two tonically active and one bursting) were superfused with oxotremorine. One of the tonically active neurons showed no response, the second was depolarized (Fig. 6D), eliciting an increase (from 1 to 3 Hz) in firing rate. The bursting neuron was transiently hyperpolarized, by about 20 mV, followed by membrane repolarisation as described above for FWA cells.
Superfusion with nicotine (100 µmol l-1, 600 s) induced bursting activity in previously non-bursting SWA Dahlgren cells (N=8/18 (44%) non-bursting cells) recorded intra- (Fig. 7A) or extracellularly (not shown). However, nicotine had no effect on SWA neurons showing ongoing burst patterns (N=11 spontaneously bursting cells; Fig. 7B). A much smaller proportion of non-bursting FWA Dahlgren cells responded to nicotine in this way, with only 3/21 cells (14%) cells generating burst patterns. For both SWA and FWA cells, the transition to bursting typically took >300 s to occur after the initial onset of nicotine superfusion.
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No SWA Dahlgren cells were inhibited by nicotine, but 57% (13/23 cells) of
FWA cells were. This is illustrated in Fig.
7C, in which three FWA bursting cells were inhibited during or
following superfusion with nicotine. Nicotine did not evoke neuron
activity (not shown) in either SWA or FWA CNSS (data from 10 fish).
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Discussion |
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The sag potential and depolarising after-potential (DAP) have been
suggested as a mechanism to facilitate repetitive firing (bursting) in
Dahlgren cells (Brierley et al.,
2001). DAPs occur following the action potential
afterhyperpolarisation (AHP), taking the membrane potential towards threshold
for the next spike. Although the ion channel activation underlying this
response has yet to be characterised in these cells, it is reminiscent of the
excitatory hyperpolarisation-activated cation current (IH)
described for other rhythmic neuronal and cardiac pacemaker cells
(Irisawa, 1987
;
Erickson et al., 1993
;
Vasilyev and Barish, 2002
).
Voltage-dependent sag potentials during hyperpolarising current injection were
observed in FWA as well as SWA cells, especially when held at depolarised
membrane potentials. However, the DAP was significantly larger in SWA compared
to FWA cells, which might be expected to lead to more effective maintenance of
ongoing firing activity in the former. However, this was not reflected in a
difference in burst parameters between SWA and FWA cells. Although type 2
cells are usually relatively inexcitable, they do appear to generate a sag
potential response, reinforcing the hypothesis that type 2 and type 1 Dahlgren
cells may represent different activity states within a single neuronal
population.
Not only were similar patterns of activity recorded from SWA and FWA
Dahlgren cells, but also the proportions of cells showing these patterns of
activity were not different. This was unexpected bearing in mind the known
involvement of the CNSS and the urotensins in osmoregulatory adaptation (for a
review, see Winter et al.,
2000). There are several possible explanations for this. The first
is the absence of extrinsic input to the CNSS in the in vitro,
isolated preparation used here. Studies using a range of species have
identified descending neuromodulatory input to the CNSS, including
monoaminergic pathways (Audet and
Chevalier, 1981
; Miller and
Kriebel, 1986
; McKeon et al.,
1988
; Yulis et al.,
1990
; Oka et al.,
1997
; Hubbard et al.,
1996b
,
1997
). It is possible that
differences in Dahlgren cell activity following SW-FW adaptation depend on
ongoing descending input and are thus not retained in vitro.
Secondly, any link between electrical activity of the neuroendocrine cells and
neuropeptide release from their terminals is unlikely to be straightforward.
The amount of peptide released from the storage organ (urophysis) in response
to depolarisation may depend more on the level of peptide available for
release; we previously showed that the amount of stored peptide in the
urophysis is significantly greater in fully SWA compared to FWA flounder
(Winter et al., 1999
). Local
control of release at the level of the terminals is also a possibility; this
would not necessarily be reflected by changes in Dahlgren cell activity.
However, this raises the question of the function of patterned, and especially
bursting, activity in these cells.
Since Dahlgren cells were often seen to change their activity pattern from
one type to another (`transitions'), it is proposed that they represent a
fairly homogeneous population in which the activity of individuals can change
over time. It was assumed that extracellular recordings included only type 1
neurons, since type 2 are quiescent, relatively inexcitable and show
pronounced spike frequency accommodation when induced to fire
(Hubbard et al., 1996a).
However, type 1 neurons too may often be silent (16% of SWA cells;
Brierley et al., 2001
). It was
not possible using extracellular recording to quantify the proportion of
silent type 1 cells in SWA and FWA preparations so there remains the
possibility that more cells are quiescent in FWA fish, leading to reduced
peptide release.
Transitions between activity patterns showed some differences between SWA
and FWA preparations. Specifically, spontaneous transitions in SWA Dahlgren
cells never involved bursting activity, whereas transitions to or from
bursting activity were common in FWA cells. This suggests that bursting
activity may be more stable in SW than in FW conditions. Further evidence for
this is the greater variability of burst cycle period and duration observed in
FWA compared to SWA cells. Differences in the robustness of bursting activity
could result from the greater variability in osmoregulatory state of
individual fish in FW. Although plasma osmolality is tightly maintained by SWA
fish (ca. 324 mOsmol kg-1 H2O;
Bond et al., 2002) osmolality
in FWA fish is much more variable, as indicated by significantly greater
S.D. (H. Bond, personal communication), suggesting that individual
fish cope with low salinity media with differing degrees of precision.
The effects of ACh and agonists reported here support the hypothesis that
ACh plays a role in regulating CNSS activity. Central cholinergic pathways
have profound effects on rhythmic neuronal activity in a number of systems.
For example, in the mammalian hippocampus
(Cobb et al., 1999), supraoptic
nucleus (Zaninetti et al.,
1999
), somatosensory cortex
(Buhl et al., 1998
) and
entorhinal cortex (Klink and Alonso,
1997
), ACh can initiate burst-mode activity and promote synchrony
within neuronal populations. Furthermore, mammalian osmoregulatory vasopressin
neurons, which have comparable activity patterns to bursting Dahlgren cells
(Armstrong et al., 1994
), are
stimulated via both nicotinic and muscarinic pathways. The main
source of ACh input to the flounder CNSS in vivo is unclear. Conlon
and Balment (1996
)
demonstrated ACh synthesis and release in the isolated CNSS. However,
descending spinal pathways involving ACh may also be present, as reported for
several freshwater teleosts by Pandey
(1989
).
Muscarinic actions of ACh on both SWA and FWA Dahlgren cells were largely
inhibitory. Indeed some cells showed 20 mV hyperpolarisation in response to
oxotremorine, which is consistent with the presence of muscarinic receptors on
the Dahlgren cells themselves. In addition, oxotremorine caused marked
activation of neurons. The identity of these neurons is unknown. They
are unlikely to be motoneurons, as these are not thought to be present in the
terminal segments of spinal cord.
neurons showed more spontaneous
activity in FWA than in SWA CNSS, suggesting that they may have a role related
to the osmoregulatory function of the system. Cohen et al.
(1990
) described a dense
plexus of serotonin immunoreactive fibres in the CNSS of the molly
(Poecilia latipinna). It is possible that
neurons correspond
to these fibres and might therefore mediate at least some of the reported
inhibition of Dahlgren cells via serotonin
(Hubbard et al., 1997
).
Acetylcholine appeared to promote bursting activity in Dahlgren cells, via nicotinic receptors. However, this effect was more pronounced in SWA than in FWA CNSS. This would be expected to enhance release of urotensins. In FWA CNSS, more than half of cells recorded were inhibited by nicotine. Thus responses of FWA Dahlgren cells, both muscarinic and nicotinic, were largely inhibitory.
Cholinergic effects on Dahlgren cells appear to involve a balance between reducing overall activity and promoting secretion-efficient bursting. In FWA CNSS, responses tend towards inhibition, whereas in SWA fish, bursting activity is promoted. These differential effects of AChR ligands on SWA and FWA preparations, though subtle, may contribute, together with those of other modulators, to differential activity patterns in vivo. Our results suggest that differences in functional expression of ACh receptors may occur during SW-FW adaptation, thus altering the Dahlgren cells' response to descending inputs.
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Acknowledgments |
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References |
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