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The Journal of Neuroscience, February 15, 1998, 18(4):1399-1407
Outward Currents in Drosophila Larval Neurons:
dunce Lacks a Maintained Outward Current Component
Downregulated by cAMP
Ricardo
Delgado2,
Ronald
Davis3,
Maria Rosa
Bono1,
Ramon
Latorre1, 2, and
Pedro
Labarca1, 2
1 Departamento de Biologia, Facultad de Ciencias,
Universidad de Chile, 2 Centro de Estudios Cientificos de
Santiago, Casilla 16443, Santiago 9, Chile, and 3 Division
of Neuroscience, Department of Cell Biology, Baylor College of
Medicine, Houston, Texas 77030
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ABSTRACT |
Outward current modulation by cAMP was investigated in wild type
(wt) and dunce (dnc)
Drosophila larval neurons. dnc is
deficient in a cAMP phosphodiesterase and has altered memory. Outward
current modulation by cAMP was investigated by acute or chronic
exposure to cAMP analogs. The analysis included a scrutiny of outward
current modulation by cAMP in neurons from the mushroom bodies (mrb). In Drosophila, the mrb are the centers of olfactory
acquisition and retention. Based on outward current patterns, neurons
were classified into four types. Downmodulation of outward currents induced by acute application of cAMP analogs was reversible and found
only in type I and type IV neurons. In the general wt neuron population, approximately half of neurons exhibited cAMP-modulated, 4-aminopyridine (4-AP)-sensitive currents. On the other hand, a
significantly larger fraction of mrb neurons in wt (70%) was endowed
with cAMP-modulated, 4-AP-sensitive currents. Only 30% of the
dnc neurons displayed outward currents modulated by
cAMP. The deficit of cAMP-modulated outward currents was most severe in
neurons derived from the mrb of dnc individuals. Only
4% of the mrb neurons of dnc were cAMP-modulated. The
dnc defect can be induced by chronic exposure of wt
neurons to cAMP analogs. These results document for the first time a
well defined electrophysiological neuron phenotype in correlation with
the dnc defect. Moreover, this study demonstrates that
in dnc mutants such a deficiency affects most severely
neurons in brain centers of acquisition and retention.
Key words:
Drosophila; neurons; outward currents; cAMP; downmodulation; mushroom bodies; dunce mutants
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INTRODUCTION |
In Drosophila, there is
solid evidence that the cAMP cascade, involving a Ca- and
calmodulin-responsive adenylyl cyclase, protein kinase A, and a cAMP
phosphodiesterase, is relevant for the events leading to the retention
of olfactory information (Tully et al., 1994 ; Davis, 1996). Mutants
altering the cAMP cascade, such as dunce (dnc)
and rutabaga (rut), are deficient in short-term memory (Tully et al., 1994). The evidence indicates also that DCO, a gene encoding protein kinase A, plays a role in
retention and acquisition (Drain et al., 1991; Skoulakis et al., 1993). As in Drosophila, behavioral plasticity associated with cAMP
is found in vertebrate organisms (Kandel and Schwartz, 1982; Frey et
al., 1993; Huang et al., 1994; Weisskopf et al., 1994). The dnc, rut, and DCO genes express
preferentially in the mushroom bodies (mrb) (Nighorn et al., 1991; Han
et al., 1992; Skoulakis et al., 1993), brain structures crucial to
acquisition and retention in Drosophila (Heisenberg et al.,
1985; Debelle and Heisenberg, 1994; Davis, 1996). The memory mutants of
the fruit fly, with an altered cAMP cascade, such as dnc and
rut, exhibit defective plasticity in peripheral synapses
(Corfas and Dudai, 1989; Zhong and Wu, 1991; Delgado et al., 1992), and
cAMP analogs can abolish plasticity in the normal larval neuromuscular
synapse (Delgado et al., 1992). One target of cAMP modulation is ion
channels (Levitan, 1988; Moreno et al., 1995). Modulation of ion
channels, via cAMP-associated mechanisms, has been implicated in
changes in synaptic efficacy (Kandel and Schwartz, 1982). In
Drosophila, cAMP modulation of K+
conductances has been described (Delgado et al., 1991; Zhong and Wu,
1993; Wright and Zhong, 1995), and a hyperpolarized resting potential
was reported in dnc larval muscle (Delgado et al., 1991). Moreover, in dnc, synaptic plasticity in motor end plates
could be restored by K+ channel blockers (Delgado et
al., 1992), suggesting that the effects of cAMP are mediated by
K+ channel conductance. In support of this notion,
Zhao and Wu (1997) reported recently that cytochalasin arrested
embryonic neuroblasts ("giant neurons") from dnc and
rut exhibit abnormal spontaneous spikes and altered firing
patterns in correlation with altered K+ conductance.
Despite this, the fundamental question of whether mrb neurons from
Drosophila mutants deficient in cAMP cascade exhibit altered
K+ conductances remains open. We have taken
advantage of the availability of wild-type (wt) and dnc
enhancer detector lines to address the following questions: (1) do mrb
neurons represent a particular set of neurons distinguishable
electrophysiologically and pharmacologically from the general
population of neurons; and (2) does the dnc defect gives
rise to specific electrophysiological deficiencies in mrb neurons?
A preliminary account of this work was communicated previously (Delgado
and Labarca, 1997).
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MATERIALS AND METHODS |
Fly stocks. In most experiments neurons from enhancer
detector fly line 221 (derived from Canton-S), taken as wild type, and line dnc1,221, derived from dnc1,
were used. Both enhancer detector lines express the lacZ
gene preferentially in the mrb (Fig.
1A).
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Figure 1.
A, Staining of mushroom bodies in
larval brain of enhancer detector lines. Left, Enhancer
detector line 221 (wt); right, enhancer detector line
221,dnc1. Staining was achieved following the
method of Han et al. (1992). Scale bar, 250 µm. B.
FDG+ mrb neuron from enhancer detector line. Left,
Neurons in culture under Hoffman optics; right, same
under fluorescence microscope. FDG staining of mushroom body neurons was performed as described in Materials and Methods. Scale bar, 50 µm.
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Tissue culture. Cell culture was achieved by following the
method of Wu et al. (1983) with slight modifications. Fifteen to 30 larval brains were incubated for 15 min in 1 ml of PBS endowed with
trypsin, (0.0125%, type III; Sigma, St. Louis, MO) at 37°C. The
tissue was pelleted, washed twice in 1 ml of culture media made of
Drosophila Schneider (Life Technologies, Gaithersburg, MD),
supplemented with 5% fetal bovine serum (Life Technologies) and
gentamicin (50 µg/ml, Lab Astorga, Santiago, Chile), and resuspended in 1 ml of media. The suspension was gently passed repeatedly through a
fire-polished Pasteur pipette until tissue dissociation was achieved.
Cells were plated (25010; Corning, Corning, NY) to a final volume of
1.7 ml/plate and kept at room temperature (20°C).
Electrophysiology. Whole-cell recording was performed in 2- to 5-d-old cell cultures using an Axopatch 1-C amplifier (Axon Instruments Inc., Foster City, CA). The patch-clamp station included a
Nikon Diaphot TMD microscope endowed with fluorescence and Hoffman optics. Pulse protocols were generated using pClamp 5.5 software (Axon
Instruments). Before recording, the culture media was replaced with 2 ml of external solution made of (in mM) 140 NaCl, 2 KCl, 4 MgCl2, 2 CaCl2, and 5 HEPES, pH
7.2. Unless specified, 3-4 M patch pipettes (Kimax-51; Fisher
Scientific, Pittsburgh, PA) were filled with standard internal solution
made of (in mM) 70 KF, 70 KCl, 2 MgCl2,
1 CaCl2, 11 EGTA, and 10 HEPES, pH 7.2. Cell capacitance averaged 1.9 ± 0.1 pF (n = 24) in wt
and 1.8 ± 0.1 pF (n = 42) in dnc
neurons. Assuming a spherical shape, the average cell radius amounts to
3.9 µm, with an average surface area of 191 µm2/cell. A large fraction of dissociated neurons
(50%) lacked or displayed very small outward currents (<10 pA) 2-5 d
after plating. Such neurons were not considered in the analysis. Flow
cytometry indicated that some 30% of freshly dissociated neurons were
permeable to propidium iodide. Thus, it can be assumed that neurons
void of outward currents in 2- to 5-d-old primary cultures represented damaged cells.
Voltage-gated currents, monitored 1 min after establishing whole-cell
conditions, displayed steady amplitudes, and, in all cases, the effect
of pharmacological agents on outward currents was tested only after
showing that currents were of constant amplitude during three
consecutive voltage jumps from the holding voltage ( 120 mV) to 0 mV.
The effects of acute application of cAMP analogs (100 µM
8-bromo-cAMP or dibutyryl-cAMP; Sigma) and K+
channel blockers were tested by release of the appropriate compound for
20-30 sec from a pipette located 10-20 µm from the cell surface by
means of a picospitzer. Similar results were obtained with both cAMP
analogs, and they were used indistinctly. For simplicity, in the text
and figures cAMP analogs are referred to as X-cAMP. Control studies
demonstrated that picospitzer application of external solution, lacking
cAMP analogs or K+-channel blockers, from a pipette
located 10-20 µm from the cell surface had no effect on outward
current amplitude. Under such experimental conditions, high-resistance
patches lasted, on average, 4.8 ± 0.7 min (n = 12).
Identification of mrb neurons. Neurons derived from larval
mrb of enhancer detector fly lines were identified under the
fluorescence microscope using fluorescein
di-( -D-galactopyranoside) (FDG; Molecular Probes,
Eugene, OR), a substrate of -galactosidase (Fig.
1B). Cleavage of FDG releases fluorescein, which
excites at 490 nm and emits at 514 nm. Loading of neurons with FDG was achieved by means of a 60 sec hyposmotic shock with diluted external solution enriched with 1 mM FDG. Analysis by flow cytometry
revealed that ~2.5% of freshly dissociated neurons were FDG+. This
estimate was in good agreement with that obtained by an independent
count of FDG+ neurons in 2- to 5-d-old culture plates, which yielded 1.2 ± 0.5% (n = 3) FDG+ cells, and with a
previous estimate by Wright and Zhong (1995).
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RESULTS |
Outward currents in wt neurons
Outward currents properties were scrutinized in wt neurons
(n = 502) from wt enhancer detector line 221, as
described in Materials and Methods. This allowed us to define four cell
types on the basis of outward current inactivation patterns (Fig.
2A, Table 1). Identical neuron types were observed
in a sample (n = 60) from Canton-S larvae. Neuron
classification was followed by a study of outward current sensitivity
to cAMP and to K+ channel blockers.
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Figure 2.
Classification of neurons in wt
Drosophila larvae. Whole-cell currents were recorded as
described in Materials and Methods. A, Neuron types in
wt larvae. Neurons were classified based on time constants of
inactivation and relative amplitude of exponential components, at
V = 0 mV, as explained in Results. Records show various examples
of outward current patterns in different neurons types. Outward
currents were elicited by 20 mV depolarizing steps, from 60 to 20 mV,
in 20 mV intervals. Holding potential was 120 mV. B.
Analysis of inactivation times in neurons with outward currents
inactivating along a single exponential (n = 128).
Left, Single exponential outward current inactivation
patterns. Currents were triggered by depolarizing excursions from a
120 mV holding to 0 mV. Lines on top of
current records are best fit of single exponential function to data.
Right, Distribution of inactivation times. The
solid line is best fit to data of two Gaussian curves, with peaks at 92 and 158 msec. The relative contribution of each Gaussian component is 0.58 and 0.42, respectively;
n = 128. r2 = 0.96.
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A fraction of wt neurons (25%) displayed slowly inactivating outward
currents ( i > 50 msec) with an inactivation time course (at V = 0 mV) that could be accounted for with a single-component exponential fit (Fig. 2B). In these neurons, analysis
of the distribution of outward current inactivation times, at V = 0 mV, yielded two Gaussian curves, with peaks at 92 and 158 msec,
respectively (see histogram in Fig. 2B). In turn,
acute exposure to cAMP analogs, applied via a picospitzer, to neurons
exhibiting outward inactivation along a single exponential showed that
a fraction of them responded to the X-cAMP with a decrement in outward
current amplitude. In such cells, inactivation times at V = 0 mV
averaged 162 ± 8 msec (n = 12) (Table 1). This is
in coincidence with the peak at 158 msec in the histogram shown in
Figure 2B. Thus, the Gaussian component with a
maximum at 158 msec correlates with neurons exhibiting outward currents
downregulated by cAMP. Such neurons were classified as type I (Fig.
2A). In 6 of 12 type I neurons tested, the outward current component affected by X-cAMP was a maintained one (Fig. 3A, top records).
In six other type I neurons, cAMP analogs affected a slowly
inactivating outward current component (Fig. 3A, bottom records). Diminishment in outward current amplitude reverted 1-2 min after acute application of cAMP analogs ceased (Fig. 3A,
inset). In type I neurons, 4-AP (100 µM) blocked
outward currents with properties similar to cAMP-sensitive ones (Fig.
3B). Furthermore, when stimulation with X-cAMP was preceded
by exposure to 4-AP, no effect of cAMP analogs on outward current
amplitude was observed in type I neurons. In type I neurons outward
currents insensitive to 4-AP inactivated with i = 22 ± 4 msec.
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Figure 3.
Properties of outward currents in type I neurons.
In all experiments shown hereunder, currents were elicited by jumping
the voltage from a 120 mV holding potential to 0 mV. Currents shown in records labeled 3 were obtained by subtraction of
currents recorded after exposure to X-cAMP or 100 µM 4-AP
(2) from control outward currents
(1). A, top record,
Maintained outward current abolished by cAMP analogs. Bottom
record, Slowly inactivating cAMP-sensitive outward currents.
Inset, Reversibility of effect of X-cAMP on outward
currents in type I neurons. Reversibility was monitored 1-2 min after
exposure to X-cAMP. B, Blockade by 100 µM
4-AP of cAMP-sensitive currents in type I neurons.
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Neurons displaying outward currents that inactivated along a single
exponential at V = 0 mV and that did not respond to cAMP analogs
(Fig. 4A) had
inactivation times averaging 89 ± 3.6 msec, in correspondence
with the major peak at 92 msec in the histogram in Figure
2B. These neurons were classified as type II neurons (Table 1). In such neurons tetraethylammonium (TEA, 1 mM)
but not 4-AP (100 µM) was effective in blocking outward
currents, which, at V = 0 mV, inactivated with i = 120 ± 13 msec (n = 6; Fig. 4B).
Currents that remained after exposing type II neurons to 1 mM TEA inactivated with i = 12 ± 2 msec.
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Figure 4.
Properties of outward currents in type II and type
III neurons. Currents shown in records labeled 3 were
obtained by subtraction of control outward currents in records labeled
1 from currents recorded after exposure to X-cAMP, 1 mM TEA, or 100 µM 4-AP in records labeled
2. A. Lack of effect of cAMP analog on
outward currents in type II neurons. B, top
record, Blockade by 1 mM TEA of outward currents in
type II neurons. Bottom record, Lack of effect of 100 mM 4-AP on outward currents in type II neurons. C. Lack of effect of cAMP analogs on outward currents in
type III neurons. D. Insensitivity of outward currents
to TEA (1 mM, top record) and 100 4-AP (100 µM, bottom record) in type III
neurons.
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Type III neurons, which composed 4.6% of the sample, exhibited outward
currents dominated by rapidly inactivating outward currents (Fig.
2A ,Table 1). In all type III neurons tested
(n = 10) outward currents were refractory to X-cAMP
(Fig. 4C) and poorly sensitive to TEA (1 mM) and
4-AP (100 µM) (Fig. 4D).
Type IV neurons represented the largest subset of neurons (69.9%). At
V = 0 mV they displayed outward currents with fast and slow
inactivating components of about similar amplitudes (Table 1). From a
sample of 46 type IV neurons, 28 exhibited currents that were
diminished by exposure to X-cAMP. In all cases, cAMP analogs abolished
a slowly inactivating outward current, blocked by 4-AP (100 µM; Fig. 5A).
The effect of X-cAMP on outward currents reverted within 1-2 min after
acute application ceased (Fig. 5B). The extent of
inactivation of cAMP-modulated outward currents in wt neurons was
investigated further during a long 400 msec depolarizing pulse. The
analysis indicated the presence of two cAMP-sensitive currents,
distinguishable by the extent of their inactivation at the end of a
prolonged depolarization, as documented in Figure 5B, inset.
The major peak in the histogram corresponds to cAMP-sensitive currents
found in type IV and type I neurons, in which inactivation was almost
complete at the end of the 400 msec pulse. The minor peak corresponds
to cAMP-sensitive currents exhibiting little inactivation at the end of
the 400 msec depolarizing pulse, found in type I neurons and in neurons
from the mrb. In the fraction of type IV neurons that were sensitive to
cAMP, outward currents that remained after exposure to cAMP analogs
inactivated with i = 8 ± 1 msec. In type IV
neurons void of cAMP-sensitive currents, TEA (1 mM) blocked
an outward component, which, at V = 0 mV, inactivated with
i = 86 ± 10 msec (Fig. 5C). Because in
wt, all type I neurons and 60% type IV neurons displayed outward currents modulated by cAMP, it is expected that ~50% of wt neurons exhibit such currents.
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Figure 5.
Properties of outward currents in type IV neurons.
Currents shown in records labeled 3 were obtained by
subtraction of control outward currents in records labeled
1 from currents recorded after exposure to X-cAMP, 1 mM TEA, or 100 µM 4-AP in records labeled 2. A, Block of X-cAMP-sensitive outward
currents by 100 µM 4-AP. Top record,
cAMP-dependent outward currents in a type IV neuron. Bottom
record, In the same neuron as in top record
after exposure to X-cAMP, outward currents were allowed to recover for
2 min. Then, 100 µM 4-AP was applied via the picospitzer.
B, Further evidence for reversibility of X-cAMP
modulation of outward currents in type IV neuron. Inset,
Extent of inactivation of cAMP-sensitive currents at the end of a 400 msec depolarization to 0 mV in wt neurons. The histogram was built with
data from 60 neurons. I, Amplitude of cAMP-sensitive
currents at the end of the depolarizing pulse; I peak,
peak amplitude of cAMP-sensitive currents at the beginning of the
depolarization. The solid line represents the best
fitting to two Gaussian components, with peaks at 0.7 and 0.18. C, Outward current block by 1 mM TEA in type
IV neuron lacking cAMP-sensitive currents. Top record,
Lack of effect of X-cAMP in the type IV neuron. Bottom
record, 1 mM TEA blocks outward currents in same
type IV neuron as in top record.
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Outward currents in wt mrb neurons
A sample of 40 mrb neurons was tested to investigate cAMP
modulation of outward currents. In 27 of these neurons X-cAMP was effective in downregulating outward currents. Current subtraction revealed that cAMP-sensitive outward currents were either maintained or
slowly inactivating (Fig.
6A) and were blocked by
4-AP (Fig. 6B). These observations indicate that
~70% of mrb neurons display outward currents downregulated by cAMP.
To a p  0.05 level of confidence, mrb neurons differ
from the whole wt neuron population in that they contained a larger
fraction of units exhibiting cAMP-sensitive outward currents. The
presence of a small outward current component, displaying fast
inactivation and blocked by 4-AP (100 µM; Fig. 6B, bottom record), was also apparent in half of mrb
neurons tested for 4-AP sensitivity. Both type II and type III neurons
were detected in those mrb neurons that did not display outward
currents sensitive to cAMP. Thus, the same neuron types seen in the
general neuron population seemed to be present in the mrb.
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Figure 6.
Properties of cAMP-modulated outward currents in
mushroom body neurons. Mushroom body neurons were identified under the
fluorescence microscope as described in Materials and Methods. Currents
shown in records labeled 3 were obtained by subtraction
from control outward currents in records labeled 1 and
outward currents recorded after exposure to X-cAMP or 100 µM 4-AP in records labeled 2. A, cAMP-modulated outward currents in mushroom body
neuron. Top record, Maintained, cAMP-sensitive outward
current component. Bottom record, Slowly inactivating,
cAMP-sensitive outward current in mushroom body neuron.
B, Blockade by 100 µM 4-AP of maintained and slowly inactivating outward currents in mushroom body neurons. Top record, 4-AP-sensitive, maintained outward current.
Bottom record, 4-AP-sensitive, slowly inactivating
outward current. Notice in record labeled 3 the presence
of a fast-inactivating, 4-AP-sensitive outward current component.
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Outward currents in dnc neurons
Outward currents were investigated also in a sample of neurons
(n = 267) derived from larval brains of the
221,dnc line. It was found that, as a population,
dnc neurons differed from wt. First, type I neurons were
almost absent in dnc (Table 1, Fig. 7A, inset). In fact, only
three such neurons could be found in the sample. The effects of cAMP on
outward currents could be tested in one of these neurons. X-cAMP caused
decrements in outward current amplitude, owing to inhibition of a
slowly inactivating current. In another type I neuron, 4-AP (100 µM) was effective in blocking a slowly inactivating
outward current component, similar to that inhibited by X-cAMP (Fig.
7A). In the dnc sample, type III neurons were
fourfold more abundant than in wt. Similar results were obtained in a
set of 60 neurons from dncM14 larvae (Table
2). Otherwise, the electrophysiological
and pharmacological properties of outward currents in dnc
neurons were similar to those found in wt (Table 1).
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Figure 7.
cAMP-sensitive outward currents in
dnc neurons and wt neurons exposed chronically to
X-cAMP. cAMP-sensitive currents in records labeled 3
were obtained by subtraction from control record in 1
and record obtained after exposure to X-cAMP in 2.
A, Top record, Abolishment of slowly
inactivating outward current by X-cAMP in type I neuron from
dnc. Bottom record, blockade of outward
currents by 100 µM 4-AP in type I dnc
neuron. Inset, Distribution of inactivation times in
dnc neurons exhibiting single exponential inactivation. The distribution displays a single peak and is well fitted to a single
Gaussian component and peaks at 91.6 msec (n = 38;
r2 = 0.97). B, Properties of
cAMP-modulated currents in dnc neurons. Top
record, Abolishment of slowly inactivating outward current by
X-cAMP in type IV neurons from dnc. Bottom
record, Blockade by 100 µM 4-AP of slowly
inactivating outward current in type IV neuron from dnc.
C, 4-AP-sensitive outward currents in wt neuron after chronic
exposure to X-cAMP. D, cAMP-sensitive, slowly
inactivating outward current component in dnc mrb
neuron.
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From 27 type IV dnc neurons tested, 12 displayed slowly
inactivating cAMP-sensitive outward currents, blocked by 4-AP (Fig. 7B). Thus, as a population, ~30% of dnc
neurons display cAMP-sensitive outward currents. This percentage, to a
p 0.05 level of confidence, is significantly lower
than the fraction of neurons exhibiting cAMP-sensitive currents
observed in the wt population. The extent of inactivation of
cAMP-sensitive currents at the end of a prolonged, 400 msec
depolarization in dnc averaged 84 ± 6%
(n = 30), relative to peak currents measured at the
beginning of the depolarizing pulse. This percentage of inactivation at
the end of a 400 msec depolarization is similar to that of
cAMP-sensitive currents in type IV in wt individuals, as documented in
Figure 5, inset.
Effect of chronic exposure of wt neurons to cAMP
Further studies were performed to determine whether a chronic
increase in intracellular levels of cAMP suffices to account for the
deficit of dnc neurons exhibiting cAMP-sensitive outward currents. wt neurons were exposed chronically (12 hr) to a 100 µM concentration of either cAMP analog before recording.
As shown in Table 2, in a sample of 60 such neurons, the presence of
type I cells could not be documented. Given that type I neurons made 11% of the wt population, one would predict in this sample, to a
p = 0.05 level of confidence, the occurrence of at
least two type I neurons. Thus, chronic exposure to X-cAMP causes a
significant drop in the number of type I neurons in the wt population.
Thirty wt neurons chronically exposed to cAMP analogs were tested also for 4-AP sensitivity, to asses the fraction of wt neurons exhibiting cAMP-sensitive outward currents. Outward currents blocked by 4-AP could
be documented in seven of these neurons (Fig. 7C). This number is significantly less than that expected for wt neurons not
exposed chronically to cAMP analogs but is similar to that of
dnc (p = 0.05 level of significance).
Moreover, the amplitude of 4-AP-sensitive currents in wt neurons
exposed chronically to cAMP analogs (37 ± 3 pA; n = 7) was significantly smaller than in type IV neurons not exposed
chronically to the cAMP analog (150 ± 17; n = 46)
(Table 1). In addition to causing a significant decrement of type I
neurons, chronic exposure of wt neurons to cAMP analogs produced an
increase in the fraction of type III neurons to levels similar to those
found in the dnc population (Table 2).
The reversibility of the decrement in amplitude of cAMP-modulated
current seen in wt neurons after chronic incubation in X-cAMP was also
investigated. To this purpose, outward currents were recorded from
these neurons for a 3 min period after whole-cell recording conditions
had been established. During this period, in which they were exposed to
cAMP-free solution applied via the picospitzer, no increase in outward
current amplitude was observed. In a second protocol, designed to
investigate reversibility of the effects of chronic application of cAMP
analogs on outward currents, whole-cell recording was performed in 60 wt neurons that had been exposed for 12 hr to X-cAMP after they were
incubated for 1 hr in a cAMP-free solution. In this sample, no type I
neuron was detected, and the fraction of type III neurons was still
significantly larger than that expected for wt neurons not incubated
chronically with cAMP analogs. The amplitude of slowly inactivating,
cAMP-modulated currents in these neurons (62 ± 12 pA;
n = 6) was larger (p 0.1 level of significance) than the amplitude of cAMP-sensitive currents
recorded from wt neurons exposed chronically to cAMP but that were not
subjected to incubation for 1 hr in a medium free of cAMP analogs
before recording (37 ± 3 pA; n = 7). This result
suggests a slow reversal of the effect of chronic exposure to cAMP on
the slowly inactivating cAMP-sensitive current.
mrb neurons from dnc
From a sample of 37 dnc mrb neurons tested, only two
(5%) were found to exhibit outward currents downmodulated by cAMP. In both cases, exposure to cAMP analogs abolished a slowly inactivating outward current component (Fig. 7D). Thus, in dnc
there is a significant drop in the fraction of mrb neurons displaying
cAMP-sensitive currents, compared with the dnc neuron
population as a whole. This difference is still more dramatic when mrb
neurons from dnc are compared with mrb body neurons from wt
larvae. Such a comparison reveals that in dnc, the fraction
of mrb neurons exhibiting outward currents down modulated by cAMP is
14-fold smaller than in mrb neurons from normal individuals.
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DISCUSSION |
There is convincing evidence that acquisition and retention of
olfactory clues in the fruit fly depend critically on the mrb. In
Drosophila, these bilateral integrative brain centers are
made of a few thousand neurons that receive inputs from the antennal and optical lobes, as well as from other sensory centers. Their axons
establish connections with other structures in the brain (Nassel, 1987;
Davis, 1996). Altered mrb structure, or chemical ablation of the mrb,
causes loss of acquisition and retention (Heisenberg et al., 1985;
Debelle and Heisenberg, 1994). Moreover, genes engaged in acquisition
and retention, such as dnc, are expressed preferentially in
the mrb. Thus, mrb neurons are a logical locus to search for
electrophysiological correlates to altered acquisition and retention in
neurological mutants of the fruit fly. The establishment of such
correlates in the brain of Drosophila should provide
important clues on the functional aspects of acquisition and retention
in the nervous system. From the present study it can be concluded that,
in Drosophila larval neurons, outward currents downmodulated by cAMP segregate to specific neurons, namely type I and type IV
neurons. Therefore, downregulation by cAMP of K+
conductances is not restricted to neurons from the mrb. Indeed, the
fraction of wt neurons exhibiting outward current modulation by the
cyclic nucleotide in the whole neuron sample (~50%) is well above
the fraction of neurons expected to be contributed by the mrb (1-5%).
Our results indicate also that mrb neurons can display a
4-AP-sensitive, fast-inactivating outward current component refractory
to cAMP. This outward current differs from fast-inactivating outward
currents found in type III neurons, in which significant blockade by
4-AP requires concentrations well above 1 mM. As a whole,
our results point to a differential functional expression of
K+ channels in larval neurons. Differential
expression of K+ channels has been reported
previously by various groups in the brain of Drosophila. For
example, Shaker, which yields 4-AP-sensitive A-type currents
(Covarrubias et al., 1991), distributes nonuniformly, expressing
preferentially in the mrb (Schwarz et al., 1990). In turn, Baker and
Salkoff (1990) found that only a fraction of neurons derived from the
thoracic ganglia of late-stage Drosophila pupae exhibited
Shaker currents. More recently, Zhao and Wu (1997) reported a differential expression of K+ currents in
"giant" neurons derived from cytokinesis-arrested Drosophila embryonic neuroblasts.
cAMP-modulated K+ conductances in mrb neurons
exhibited properties similar to those monitored in type I and type IV
neurons. However, a significantly larger fraction of wt mrb neurons
possessed cAMP-sensitive currents. On the other hand, the fraction of
mrb neurons exhibiting cAMP-modulated currents in dnc was
14-fold smaller than in normal individuals. These observations provide the first direct clue that deficiencies in a cAMP phosphodiesterase, expressing preferentially in the mrb, correlates with altered electrophysiology in neurons derived from these brain structures attributable to a significant decrement of cAMP-modulated
K+ conductances.
The comparison of neuron populations evidenced significant differences
between wt and dnc. First, in the mutant, type I neurons were almost absent, whereas type III neurons were four times more abundant. Chronic exposure of wt neurons to cAMP analogs (12 hr) caused
a loss of type I neurons and an increase in the number of type III
neurons, which mimics the properties of the dnc neuronal population. This result, to a first approximation, reveals that in wt,
chronic increases in cAMP suffice to yield the dnc neuronal population. cAMP-modulated outward currents in larval neurons were of
two types: maintained or slowly inactivating. The experimental evidence
suggests that slowly inactivating and maintained cAMP-sensitive currents are two functionally different outward components. In wt,
maintained currents were found exclusively in type I neurons and in
neurons from the mrb. Only the maintained cAMP-sensitive component was
absent in dnc. On the other hand, both cAMP-sensitive currents were blocked by 100 µ 4-AP and had similar
activation properties. Recent studies in our laboratory indicate that
cAMP-sensitive outward currents such as those monitored in larval
neurons are present in adult neurons (P. Labarca and R. Delgado,
unpublished results). Chronic exposure to cAMP analogs of wt larval
neurons abolished the maintained component and caused a 6-fold
diminishment in amplitude of the slowly inactivating cAMP-sensitive
current. Thus, the two currents might differ in their susceptibility to downmodulation by cAMP. Perhaps these two currents correspond to
splicing variants, or mRNA editions, of a same K+
channel. Alternatively, it could be that the different inactivation properties of cAMP-sensitive currents result from regulatory units that
control the extent of channel inactivation during long depolarizing episodes.
The effects of transient, acute increases in cAMP differ from those
caused by chronic exposure to the cyclic nucleotide. Thus, although the
effects on cAMP-sensitive outward currents induced by acute application
of cAMP analogs reversed within 1-2 min, full reversal of the effects
of chronic exposure could not be documented for up to 1 hr. Perhaps
chronic increases in cAMP, lasting many hours, hinders expression of
cAMP-sensitive maintained currents, for example, by interfering with
the channel-forming protein synthesis or by blocking protein
maturation. According to the results obtained here, reversal of the
effects of chronic increases in cAMP in Drosophila neurons
would require a longer time span than the maximal experimental time
span tested.
In neurons from Drosophila embryos, outward currents seemed
to be accounted for mainly by three K+ channel
genes, namely Shal, Shaw, and Shab
(Tsunoda and Salkoff, 1995). Shal is, by and large, the
major constituent of A-type currents in such neurons. Shaw
would associate with noninactivating, weakly voltage-dependent outward
currents, and Shab would build delayed rectifier
K+ currents. Further studies would be necessary to
establish to what extent outward current patterns in larval and adult
neurons coincide with those found in neurons from Drosophila
embryos and in "giant" neurons, derived from cytokinesis-arrested
embryonic neuroblasts (Zhao and Wu, 1997). Based on available evidence, it would be expected that A-type currents present in type III larval
neurons associate mostly with Shal and not Shaker
(Solc et al., 1987; Baker and Salkoff, 1990; Tsunoda and Salkoff,
1995). Fast-inactivating outward currents, insensitive to cAMP and
4-AP, such as those recorded in type III neurons, were apparent in type I and type IV neurons. Similarly, type II neurons were found to posses
a TEA-insensitive outward current with fast inactivation times.
Therefore, A currents, insensitive to 4-AP (0.1 µM) and TEA (1 mM), would be present in all neuron types. Lacking
their pharmacological profiles, it seems imprudent at this moment to attempt to associate Shaw and Shab
K+ currents reported in embryonic neurons (Tsunoda
and Salkoff, 1995) with K+ conductances found in
larval and adult neurons, in particular with 4-AP sensitive currents
downmodulated by cAMP. It is worth pointing out, however, that when
expressed in Xenopus oocytes, Shab
channels are insensitive to 4-AP (Covarrubias et al., 1991). Thus, a
suitable candidate to account for the maintained, 4-AP-sensitive outward currents downmodulated by cAMP is Shaw. Shaw is
sensitive to 4-AP but not to TEA (Wei et al., 1990). Kv3.1, a
Shaw homolog expressing preferentially in fast-spiking
neurons from mice (Messengill et al., 1997), is sensitive also to 4-AP
(Yokoyama et al., 1989), and another Shaw homolog, Kv3, is
amenable to downmodulation by cAMP through cAMP-dependent protein
kinase (Moreno et al., 1995).
The mechanisms by which cAMP downmodulates outward currents in
Drosophila neurons remain to be established. Downmodulation of K+ currents by the cyclic nucleotide has been
reported to operate indirectly through protein kinase A (Levitan, 1988,
Drain et al., 1994; Moreno et al., 1995). Probably a similar mechanism
accounts for downmodulation of K+ currents in
Drosophila. Up to now, direct modulation of
K+ channels by cyclic nucleotides, including
modulation of K+ channels in Drosophila
(Delgado et al., 1991; Gomez and Nassi, 1995; Jorquera et al., 1995;
Labarca et al., 1996), has been reported to give place to increases in
channel open probability.
| |
FOOTNOTES |
Received Aug. 28, 1997; revised Dec. 4, 1997; accepted Dec 5, 1997.
This work was supported by Fondo Nacional de Investigación Grant
1950457 and a Presidential Chair in Science to P.L. R.L. holds a
Presidential Chair in Science. R.D. held a C.A.I. fellowship. Institutional support of a group of Chilean companies (Empresas CMPC,
CGE, Codelco, COPEC, Minera Escondida, Novagas, Business Design
Associates, and Xerox Chile) is recognized. Part of this work was
performed during the tenure of a John Simon Guggenheim Fellowship to
P.L. P.L. is an International Scholar of the Howard Hughes Medical
Institute.
Correspondence should be addressed to Pedro Labarca, Centro de Estudios
Cientificos de Santiago, Casilla 16443, Santiago 9, Chile.
| |
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Top
Abstract
Introduction
