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Endocrinology 146(3):1048 –1052
Copyright © 2005 by The Endocrine Society
doi: 10.1210/en.2004-1415
Electrotonic Coupling in the Anterior Pituitary of a
Teleost Fish
Berta Levavi-Sivan, Corinne L. Bloch, Michael J. Gutnick, and Ilya A. Fleidervish
Department of Animal Sciences (B.L.-S., C.L.B.) and Koret School of Veterinary Medicine, (C.L.B., M.J.G., I.A.F.) Faculty of
Agricultural, Food, and Environmental Quality Sciences, The Hebrew University, Rehovot 76100, Israel
The anterior pituitary of teleost fish contains a variety of
endocrine cells, which, under control from the hypothalamus,
release trophic hormones and thereby play a major role in
reproduction, social behavior, and growth. In fish, hypothalamic fibers directly innervate the pituitary. The hypothalamic hormones released from these fibers bind to membrane
receptors on pituitary cells, triggering action potentials, a rise
in cytosolic calcium, and exocytosis. It is unclear whether
these activities are confined to the stimulated cell or propagate to adjacent cells. We addressed this issue using whole cell
and perforated patch-clamp techniques in a novel, hypothalamo-pituitary slice preparation from the tilapia fish
(Oreochromis niloticus). Pituitary cells at rest generated occasional spontaneous spikes and sharp depolarizations of
lower amplitude. The latter probably represented spikes in
T
HE ANTERIOR PITUITARY of the teleost fish contains
a variety of endocrine cells, which, under direct control
from the hypothalamus, release trophic hormones and
thereby play a major role in fish reproduction, social behavior, and growth. The binding of hypothalamic hormones to
membrane receptors on a pituitary cell triggers action potentials, a rise in cytosolic calcium, and exocytosis (for review, see Ref. 1). It is not clear whether these activities remain
spatially confined to the stimulated cell or propagate to adjacent cells.
In the pituitary of mammals, endocrine cells with the same
and different hormonal contents as well as nonendocrine
folliculostellate cells (FS) communicate via gap junctions (GJ)
(2– 6). GJ in the anterior pituitary of the rat were first observed on endocrine cells by Fletcher et al. (2) and were later
also found on FS cells (3). At the GJ, pre- and postjunctional
hemichannels comprised of connexin protein subunits bind
and form nonspecific channels that allow the direct cell to cell
passage of ions and small molecules. The functional characteristics of the GJ, such as the efficiency and symmetry of
the electrical coupling, depend on the different connexins
that form the channels (for review, see Ref. 7). Cells in the
anterior pituitary of mammals express the connexins Cx43,
Cx26, and Cx36 (8, 9). Morand et al. (4) showed the existence
of homologous cell to cell communication between FS cells
and heterologous communication between prolactin cells
First Published Online December 16, 2004
Abbreviations: FS, Folliculostellate cell; GJ, gap junction; IR-DIC,
infrared differential interference contrast; LY, Lucifer Yellow.
Endocrinology is published monthly by The Endocrine Society (http://
www.endo-society.org), the foremost professional society serving the
endocrine community.
neighboring, electrotonically coupled cells. The presence of
electrotonic communication, probably mediated by gap junctions, was also supported by the finding that Lucifer Yellow
diffuses between cells. To quantify this connectivity, we performed simultaneous recording from pairs of adjacent cells.
Thirty-three percent of the cells exhibited strong reciprocal
coupling. Coupling coefficients ranged between 0.18 and 0.31,
and coupling resistances ranged between 16 and 39 GOhm.
The electrical junctions were effective low pass filters, attenuating action potentials much more than low frequency waveforms. We conclude that electrical activities of anterior pituitary cells in teleost fish are synchronized by coupling
through gap junctions. Regulation of this coupling may play
a critical role in determining complex patterns of pituitary
hormone secretion. (Endocrinology 146: 1048 –1052, 2005)
and FS cells in culture. Guerineau et al. (5) reported spontaneous increases in intracellular Ca2⫹, occurring synchronously in small groups of excitable cells in slices of guinea pig
pituitary. This rapid coactivation involves cell to cell communication via GJ, mostly (but not exclusively) between
endocrine cells. The functional significance of these GJ is not
yet clear. Other than short-range communication within
small groups of synchronous secreting cells, there is evidence
of a long-range mechanism synchronizing different groups
of secreting cells across the pituitary through GJ with FS cells,
which extend throughout the pituitary (5, 6).
Abraham et al. (10) observed GJ in the pituitary of teleosts
using a freeze-etching technique. The functional correlates of
this finding have not yet been explored. The aim of the
present study was to seek physiological evidence of electrical
coupling in the anterior pituitary of tilapia and to characterize it. First, we looked for dye coupling between endocrine
cells in hypothalamo-pituitary slices and observed small
clusters of coupled cells. Then we quantified the electrical
connectivity by performing simultaneous recording from
pairs of adjacent cells. Here, we present the first functional
characterization of coupling in the pituitary of a teleost fish.
Materials and Methods
Preparation of hypothalamo-pituitary slices
Young (6 –10 wk old) tilapia fishes of both sexes were anesthetized
with 2-phenoxyethanol (1 ml/liter; Sigma-Aldrich Corp., St. Louis, MO)
and decapitated. Brains were removed and cooled to 2– 4 C in gassed (5%
CO2-95% O2) Ringer’s saline containing 124 mm NaCl, 3 mm KCl, 2 mm
CaCl2, 2 mm MgSO4, 1.25 mm NaH2PO4, 26 mm NaHCO3, and 10 mm
glucose (pH 7.35). The top of the brain was trimmed, and the remaining
block was glued to the stage of a Vibratome (series 1000, Vibratome Co.,
St. Louis, MO) with the trimmed surface down, so that the plane of the
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Levavi-Sivan et al. • Brief Communications
Endocrinology, March 2005, 146(3):1048 –1052
knife was tangential with respect to the infundibular stalk. A single
300-␮m slice that contained the neuro- and adenohypophysis, hypothalamus, and infundibular stalk was cut and transferred to a holding
chamber until used for electrophysiological recording. Only cells from
the anterior pituitary were recorded. All experimental procedures were
in compliance with the animal care and use guidelines at Hebrew University and were approved by the local administrative panel on laboratory animal care committee.
V2 V1
ⴱ
ⴱ关CC1 ⴱ CC2ⴱ共1 ⫺ CC1兲 ⫺ 共1 ⫺ CC1兲兴
I2 I1
Rc1 ⫽
V2
V1
CC1 ⴱ
ⴱ CC2 ⫺
I2
I1
(2)
V1
ⴱ Rc1 ⴱ 共1 ⫺ CC2)
I1
Rc2 ⫽
V1
Rc1 ⴱ CC2 ⫺
ⴱ CC2 ⴱ 共1 ⫺ CC1兲
I1
(3)
冉
Electrophysiology
For recording, slices were transferred to a chamber attached to the
stage of an upright microscope (Axioskop FS, Zeiss, Oberkochen, Germany) continuously superfused with Ringer’s saline at room temperature. Endocrine cells were viewed with a ⫻60, 0.9 numerical aperture,
water immersion objective lens (Olympus, Munich, Germany). Single
and paired whole cell recordings were made under infrared differential
interference contrast (IR-DIC) microscopic control using the patchclamp technique (11). Patch pipettes, pulled from borosilicate glass
capillaries (Hilgenberg, Maisfield, Germany) on a Narishige PP83 puller,
had resistances of 2.5– 4.5 m⍀. Membrane currents were recorded using
either the conventional, whole cell, patch-clamp technique or the nystatin-perforated, patch-clamp technique. The standard patch pipette
solution contained 135 mm potassium gluconate, 2 mm MgCl2, 1 mm
CaCl2, 11 mm EGTA, 3 mm ATP (magnesium salt), and 10 mm HEPES
(potassium salt), pH 7.25. For the patch-pipette solution for nystatinperforated patch recordings, a stock solution containing 10 mg/ml nystatin (Sigma-Aldrich Corp.) in acidified methanol was prepared and
added to the pipette solution to a final concentration of 400 ␮g/ml. An
Axoclamp-2A amplifier (Axon Instruments, Union City, CA) in Bridge
mode and an Axopatch-200B amplifier in fast current clamp mode were
used to record membrane voltages. Care was taken to maintain membrane access resistance as low as possible (usually 5–7 m⍀ and always
⬍10 m⍀). Command current protocols were generated, and data were
acquired on-line with a Digidata 1320A interface (Axon Instruments).
Data were low pass filtered at 10 kHz (⫺3 dB, four-pole Bessel or
one-pole Butterworth built-in filter) and sampled at a digitalization
frequency of 20 kHz. Voltages were not corrected for liquid junction
potential. Data were analyzed using PClamp 9 (Axon Instruments) and
Microcal Origin 6.0 software.
Intracellular Lucifer Yellow (LY)
The fluorescent dye LY (1 mm) was introduced into pituitary cells
using patch pipettes. Dye transfer between coupled cells was visualized
during the experiment using Axioskop FS epifluorescent equipment
(filter set 05, BP395– 440/LP 470, Zeiss). Images at selected time points
after breakthrough were acquired using an Axiocam HR color CCD
camera and AxioVision software (Zeiss).
1049
冊
where Rc1 is the resistance of the coupling of cell 1 to cell 2, and Rc2 is
the resistance of the coupling of cell 2 to cell 1. Rc1 and Rc2 can differ if
the coupling is asymmetrical.
Statistics
Numerical data in the text are presented as the mean ⫾ sem. Differences were assessed by t test.
Results
Adenopituitary cells were recorded from a hypothalamopituitary slice preparation. Whole cell and perforated patchclamp recordings in current clamp mode revealed a resting
potential of ⫺62 ⫾ 2 mV (n ⫽ 9), a very high apparent input
resistance of 4.9 ⫾ 0.3 G⍀ (n ⫽ 6). When depolarized above
the voltage threshold of ⫺35 ⫾ 1 mV (n ⫽ 6), the cells
generated action potentials that were either nonovershooting
or just overshooting. In whole cell recordings, the spike parameters were stable during the first 3–5 min after break-in.
However, as the recording progressed, spike amplitudes and
velocities of the rising phase decreased, whereas spike
threshold increased. These changes were probably due to
run-down of Na⫹ and Ca2⫹ conductances. Indeed, in perforated patch recordings, satisfactory access resistance was
usually achieved within minutes after seal formation, and
passive and active membrane properties were stable for
more than 40 min.
Cells recorded using the perforated patch-clamp technique occasionally exhibited spontaneous spikes and spikelets at rest (Fig. 1A, top). These lower amplitude sharp potentials could represent postsynaptic potentials, or they
might reflect action potentials generated in neighboring cells
in a coupled cluster and transferred to the recorded cell
Analysis
The strength of electrotonic coupling between adjacent cells was
evaluated as described by Devor and Yarom (12). The coupling coefficient (CC) was calculated as follows:
CC ⫽
Vpost
Rpost
⫽
Vpre Rpost⫹Rc
(1)
where Vpost and Vpre are steady state voltage responses of the post- and
prejunctional cells, respectively, to the prolonged negative and positive
current pulses to the prejunctional cell, Rc represents the resistance of the
coupling, and Rpost is the input resistance of the postjunctional cell when
coupled to cells other than the prejunctional cell.
Equation 1 can only be used to calculate Rc if Rpost is very much
smaller than Rc, because only then can Rpost be calculated from the slope
of a linear portion of the V-I relationship. However, because the input
resistance of the pituitary cell is so high (on the order of G⍀s), it is very
unlikely that this assumption is valid. Therefore, the strength and symmetry of the coupling between two adjacent cells were evaluated by
solving equations using the following four experimentally measured
parameters (12): V1/I1, V2/I2, CC1, and CC2:
FIG. 1. Spikelets in adenopituitary cells. A, Top, Adenopituitary cells
at rest exhibit spontaneous firing, and spikelets (marked with asterisks) were observed between action potentials. Bottom, When the cell
was hyperpolarized to ⫺70 mV, both forms of spontaneous activity
stopped. B, Left, An average spikelet (n ⫽ 100). Middle, An average
spike (n ⫽ 100). Right, An overlay of average spike and spikelet.
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Endocrinology, March 2005, 146(3):1048 –1052
through GJ. Because there is no evidence of chemical synapses in the adenopituitary cells, the latter possibility is more
likely. This conclusion is strengthened by the fact that hyperpolarizing the cell to a potential of ⫺70 mV by current
injection completely eliminated both the action potentials
and the spikelets (Fig. 1A, bottom). This indicates that the
cluster of coupled cells is relatively small, such that injection
of hyperpolarizing current into one cell is enough to silence
all of the others. Figure 1B presents an overall average of 100
spikelets and spikes, aligned at half-amplitude of the rising
phase, recorded from the same cell. Spikelets had amplitudes
of 3–7 mV, and their rates of rise and decay were significantly
slower than those of full action potentials.
To clarify whether the spikelets indeed represent action
potentials in neighboring cells that were transferred through
GJ, we carried out dye transfer experiments with the low
molecular mass (457 Da) fluorescent dye LY. In a live
hypothalamo-pituitary slice preparation, the neuro- and
adenohypophyses, hypothalamus, and infundibular stalk
were clearly visualized at low magnification (Fig. 2A, left).
Higher magnification IR-DIC video microscopy of the anterior pituitary (Fig. 2A, right) revealed individual, closely
packed endocrine cells, each approximately 6 ␮m in diameter. Figure 2B, left, illustrates an example of a live pituitary
cell (yellow arrow) dialyzed with LY-containing solution via
the patch pipette. A few seconds after break-in, one of the
neighboring cells (green arrow) also became stained, indicating LY diffusion from the dialyzed cell to its coupled partner.
By 3 min after break-in (Fig. 2B, right), the intensity of staining of the coupled partner was increased, and two additional,
more distant cells had also been stained. No LY diffusion was
observed in other adjacent cells. In similar experiments, dye
FIG. 2. Intercellular communications in fish anterior pituitary: fluorescent dye coupling. A, Left, Live slice preparation containing the
hypothalamus and pituitary gland as seen in the recording chamber.
Note that the hypothalamus (Hypothal), infundibular stalk (Stalk),
and neuro- and adenohypophysis (Neuro, Adeno) are readily identifiable. Right, Whole cell recording using an LY-containing pipette
from a visually identified endocrine cell (yellow arrow) under higher
magnification, IR-DIC microscopic control. B, Left, LY introduced into
this cell (yellow arrow) almost instantly labeled it. One of the adjacent
cells (green arrow) was also stained, but more weakly. Right, Three
minutes after the break-in, the staining of this cell intensified, and at
least two other neighboring cells (green arrows) became stained as
well.
Levavi-Sivan et al. • Brief Communications
coupling to at least one of the neighboring cells was observed
in about 45% of cells stained with LY (five of 11 cells tested).
In no case did the extent of dye coupling exceed five cells.
The presence of electrotonic coupling was tested in simultaneous current clamp recordings from nine adjacent cell
pairs by injecting hyperpolarizing current pulses into one of
the recorded cells and measuring voltage responses in both.
Three pairs, including the one shown in Fig. 3, A and C, were
found to be reciprocally coupled, i.e. current injected into
either one of the cells elicited a measurable voltage deflection
in the other. The six remaining equidistant pairs (an example
is shown in Fig. 3D) were defined as not coupled, because
current injection in one of them produced no voltage change
in the other.
The strength and symmetry of the coupling in the near-DC
frequency range were evaluated by measuring the voltage
response in pre- and postjunctional cells at the end of 0.5-sec
hyperpolarizing current pulses (V1 and V2, in response to
either I1 or I2; Fig. 3B; see Materials and Methods). In the
coupled pair shown in Fig. 3C, a V1/I1 of 4.5 G⍀ and a CC1
of 0.18 were calculated upon current injection into cell 1, and
a V2/I2 of 6.2 G⍀ and a CC2 of 0.24 were calculated upon
current injection into cell 2. Accordingly, the coupling resistance Rc1 was 39 G⍀, and Rc2 was 16 G⍀. In the three coupled
pairs, CC varied between 0.18 and 0.31, and Rc varied between 16 and 39 G⍀. In all pairs, a clear difference (18 –33%)
between CC1 and CC2 was evident, raising the possibility of
coupling asymmetry.
Because of membrane capacitance, electrical coupling is
much less efficient during rapid voltage changes compared
with near-DC frequency events. Examination of the voltage
waveforms in two coupled cells in response to an injection
of depolarizing current into one of them (Fig. 3E) showed
that a full-blown action potential in one cell only produces
a small voltage deflection in its coupled partner, whereas a
slowly rising passive depolarization is subject to significantly
less attenuation. In the example shown in Fig. 3F, the coupling coefficients during the rising phase of the action potential, CC1 (0.008) and CC2 (0.032), were calculated as the
ratio between the post- and the prejunctional voltages, measured at the time of threshold and at the peak of the action
potential in the prejunctional cell. In the three coupled pairs
analyzed, the coupling coefficient during the rising phase of
the action potential fell to only 4 –12% of the coupling coefficient measured for an unchanging voltage. In the example
in Fig. 3F, the coupling was asymmetrical. This may have
resulted in part from a difference in upstroke velocity of
action potentials in the two cells (dV/dtmax ⫽ 81 V/sec in cell
1 and 11 V/sec in cell 2).
Discussion
In this study we provide the first direct physiological evidence of electrical coupling in the anterior pituitary of the
teleost fish. Spikelets were recorded from some of these cells
using the perforated patch-clamp technique. Dye coupling of
small clusters of cells was observed in 45% of the stained
cells, with a maximal LY spread of five cells. The results of
our dye-coupling experiments are similar to those reported
for guinea pig anterior pituitary, where dye diffusion to
Levavi-Sivan et al. • Brief Communications
Endocrinology, March 2005, 146(3):1048 –1052
1051
FIG. 3. Intercellular communications in fish anterior pituitary: electrotonic coupling. A, Simultaneous, IR-DIC-guided, whole cell recording
from a pair of adjacent endocrine cells in the anterior pituitary. B, A model of two electrotonically coupled cells (adapted from Ref. 12). R1 and
C1 represent membrane resistance and capacitance of cell 1, respectively, and R2 and C2 represent membrane resistance and capacitance of
cell 2, respectively. Rc signifies a coupling resistance connecting two cells. Current was injected into either cell 1 (I1) or cell 2 (I2). C, 0.5-s, 8-pA
negative current pulses injected into cell 1 (left) and cell 2 (right) elicited voltage responses in both cells. The postjunctional responses (V2 on
left and V1 on right) to the current pulses from either direction were similarly attenuated and exhibited a slower time course than the
prejunctional response. Note that pre- and postjunctional responses are plotted on different voltage scales. D, Under a similar stimulation
protocol, no postjunctional response was observed in a pair of adjacent cells that are not electrotonically coupled. E, 0.5-s, 8-pA depolarizing
current pulses injected into cell 1 (left) and cell 2 (right) elicited an action potential in the prejunctional cell and a very small voltage deflection
in the postjunctional cell. Note that the postjunctional responses to a prejunctional action potential were attenuated more significantly than
those to the passive waveforms. F, Measurement of electrotonic coupling during an action potential. Traces are spike-triggered averages of 30
pre- and postjunctional voltage sweeps from the same pair as in E. Dashed lines indicate times of the threshold and of the peak action potential
in the prejunctional cell; arrows show the voltage values at these times which were used for CC calculations. Note that during the falling phase
of the prejunctional action potential, the postjunctional cell continues to depolarize even more effectively than during the upstroke due to a
high transjunctional voltage difference and lower frequency.
neighboring cells was seen in 47% of the clusters tested, and
the extent of the coupling did not exceed six cells (5). Simultaneous double-recording experiments from nine pairs of
adjacent cells also supported the presence of functional GJ,
showing reciprocal coupling in three of the tested pairs. The
postjunctional response was delayed, slowed, and attenuated relative to that of the prejunctional cell. Thus, the electrical junctions act as very effective, low pass filters, and
action potentials are strongly attenuated, whereas low frequency voltage changes are not. In all of the coupled pairs
recorded, CC1 was different from CC2, indicating coupling
asymmetry. The cells in the anterior pituitary of mammals
express several types of connexins (8, 9). Directional asymmetry of electrical coupling could be the result of heterotypic
GJ, which are formed by hemichannels comprised of different connexins (13). Connexins are expressed in different tissues of teleosts (14, 15), but to date their expression has not
been studied in the pituitary. Based on the limited sample of
coupled pairs, we cannot yet conclude whether this asymmetry is due to rectification of the coupling conductance or
to a difference in the apparent input resistance of two cells.
When discussing the above findings, several technical
points should be kept in mind. First, although dye coupling
indicates the presence of coupling, it does not necessarily
capture the entire cluster of electrically coupled cells. Several
studies have suggested that some connexin combinations are
less permeable to LY molecules than others (16, 17), and LY
diffusion may be limited (18). Thus, although dye coupling
with LY does demonstrate the existence of coupling, it is not
sufficient for quantification, and it is likely that the coupled
clusters are larger than the extent of dye coupling might
imply. In contrast, the fact that moderate hyperpolarization
of the recorded cell was always enough to completely silence
the spike activity of an entire cluster supports the hypothesis
that the number of cells coupled to each other is quite limited.
Other methods used to indicate the presence of GJ include
demonstrating the expression of connexins. Connexin expression is a necessary, but not sufficient, condition for the
existence of GJ communication between neighboring cells.
Connexins may, for example, create hemichannels on the cell
membrane that do not allow cell to cell passage of molecules
(for review, see Ref. 19). We used a direct, electrophysiological method to show the presence of electrical coupling in the
teleost pituitary. Ephaptic transmission or electrical field interactions as alternative explanations for the coupling (for
review, see Ref. 20) are unlikely, because in 66% of the recorded pairs, injection of hyperpolarizing or depolarizing
current into one of the cells produced no change in voltage
in the neighboring cell. This indicates that the observed interactions were indeed the result of a specific coupling
pattern.
GJ are found on most cell types in vertebrates (for review,
see Ref. 19) and have been observed in all endocrine glands
investigated to date (for review, see Refs. 21 and 22). The
physiological significance of the electrical coupling in the
teleost pituitary is not yet clear. One functional consequence
of the presence of these junctions is to reduce the spontaneous firing of these tiny, high resistance pituitary cells. Indeed,
with the observed high input resistance of about 5 G⍀, a
depolarizing current of only 8 pA (the equivalent of simultaneously opening four Na⫹ channels) at spike threshold
would be capable of producing a full-blown action potential.
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Endocrinology, March 2005, 146(3):1048 –1052
Without being coupled to other cells, the input resistances
would be even higher, and the sporadic opening of Na⫹
channels could lead to haphazard spike generation. The presence of electrical coupling thus may be helpful in preventing
firing without a hypothalamic signal.
Another likely role for the coupling is to synchronize the
evoked release from pituitary cells. Because these junctions
operate as low pass filters, this is not a spike to spike synchronization, but one that operates on a slower scale. It has
been shown (23) that in the mammalian pituitary, gonadotropes respond to GnRH application with a slow (⬍1 Hz)
membrane potential oscillation reflecting sequential activation of factors controlling intracellular Ca2⫹ and, hence, release. The filtering characteristics of the connections we report here would be optimal for disseminating such an
oscillation among a population of pituitary cells without
passing individual spikes that ride on their crest. This could
be an important mechanism for synchronizing hormone release on a functionally relevant time scale.
In the present experiments we did not attempt to identify
the hormonal content of the recorded cells, and specific patterns of coupling have yet to be determined. Unlike mammals, in which the different types of cells are scattered
throughout the pituitary, the cells of the anterior pituitary of
teleosts are segregated into distinct regions according to the
characteristic hormone they secrete (24). In addition, GnRH
has broader effects in fish, inducing the secretion of several
pituitary hormones (24, 25). In light of these unique organizational features of the tilapia pituitary, questions of coupling specificity and cluster size are especially intriguing.
Intrapituitary communication between different types of
cells may contribute to complex patterns of pituitary hormone secretion.
Acknowledgments
Received October 27, 2004. Accepted December 8, 2004.
Address all correspondence and requests for reprints to: Dr. Berta
Levavi-Sivan, Department of Animal Sciences, Faculty of Agricultural,
Food and Environmental Quality Sciences, The Hebrew University, P.O.
Box 12, Rehovot 76100, Israel. E-mail: [email protected].
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Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the
endocrine community.