Download Effect of Adrenalectomy on Miniature Inhibitory Postsynaptic

J Neurophysiol 89: 237–245, 2003;
10.1152/jn.00401.2002.
Effect of Adrenalectomy on Miniature Inhibitory Postsynaptic
Currents in the Paraventricular Nucleus of the Hypothalamus
J. M. VERKUYL AND M. JOËLS
Section of Neurobiology, Swammerdam Institute for Life Sciences, University of Amsterdam, 1098 SM Amsterdam,
The Netherlands
Submitted 30 May 2002; accepted in final form 20 September 2002
Within the rat paraventricular nucleus of the hypothalamus
(PVN) two types of neurons have been distinguished based on
morphological appearance, i.e., parvocellular and magnocellular neurons. Parvocellular neurons are key regulators of the
hypothalamus–pituitary–adrenal (HPA) activity. Thus HPA activity is driven by corticotropin-releasing hormone (CRH) and
cosecretagogues released from the parvocellular neurons. CRH
causes the release of adrenocorticotropin hormone from the
pituitary, which in turn stimulates the secretion of corticosterone from the adrenal cortex (see Whitnall 1993). Corticosterone induces peripheral effects but also feeds back to the PVN
to inhibit, via glucocorticoid receptors, CRH synthesis and
release, thus indirectly downregulating its own secretion
(Swanson and Simmons 1989). In addition to affecting the
PVN, corticosteroids also influence other brain areas, such as
the hippocampus and amygdala (De Kloet et al. 1998).
The setpoint of HPA activity is not only determined by the
humoral feedback via corticosterone but also by neuronal signals integrated in the PVN. The PVN receives excitatory inputs
from several brain areas, such as the amygdala (Feldman and
Weidenfeld 1998), the dorsomedial hypothalamus (Morin et al.
2001), and several brain stem areas (see Herman and Cullinan
1997). However, the PVN also receives a dense inhibitory
input. About 50% of the hypothalamic synapses are GABAergic (Decavel and Van den Pol 1990). Part of these involve
direct GABAergic projections to the PVN, originating, e.g., in
the suprachiasmatic nucleus (Hermes and Renaud 1993) and
arcuate nucleus (Cowley et al. 1999). Other areas, such as the
cingulate cortex (Diorio et al. 1993) and hippocampus (Herman et al. 1994)–also enriched in corticosteroid receptors–
transsynaptically inhibit the PVN via hypothalamic interneurons located among others in the bed nucleus stria terminalis
and peri-PVN regions (Boudaba et al. 1996; Roland and
Sawchenko 1993; Tasker and Dudek 1993). Nearly all CRH
parvocellular neurons express GABA receptors (Cullinan
2000), underpinning the importance of neuronal input in suppressing PVN and thus HPA activity (Herman and Cullinan
1997; Herman et al. 2002).
Since parvocellular neurons in the PVN receive humoral as
well as neuronal feedback signals, it is conceivable that these
two pathways do not work independently. This is supported by
pharmacological studies in which either the humoral or neuronal feedback signal was blocked. For instance, injection of
the GABAA receptor antagonist bicuculline close to the PVN
caused an increase of CRH, vasopressin, and c-FOS expression
in the parvocellular subregion of the PVN and increased circulating corticosterone levels (Cole and Sawchenko 2002).
Conversely, removal of the humoral feedback by adrenalec-
Address for reprint requests: J. M. Verkuyl, Section of Neurobiology, SILS,
University of Amsterdam, Kruislaan 320, 1098 SM Amsterdam, The Netherlands (E-mail: [email protected]).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked ‘‘advertisement’’
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
INTRODUCTION
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0022-3077/03 $5.00 Copyright © 2003 The American Physiological Society
237
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Verkuyl, J. M. and M. Joëls. Effect of adrenalectomy on miniature inhibitory postsynaptic currents in the paraventricular nucleus
of the hypothalamus. J Neurophysiol 89: 237–245, 2003;
10.1152/jn.00401.2002. Within the rat paraventricular nucleus of
the hypothalamus two types of neurons have been distinguished
based on morphological appearance, i.e., parvocellular and magnocellular neurons. The parvocellular neurons play a key role in regulating the activity of the hypothalamo–pituitary–adrenal axis, which is
activated, e.g., after stress exposure. These neurons receive humoral
negative feedback via the adrenal hormone corticosterone but also
neuronal inhibitory input, either directly or transsynaptically relayed
via GABAergic interneurons. In the present study we examined to
what extent the neuronal GABAergic input is influenced by the
humoral signal. To this end, miniature inhibitory postsynaptic currents
(mIPSCs) were recorded in parvo- and magnocellular neurons of
adrenalectomized rats, which lack corticosterone, and in sham-operated controls. Under visual control neurons in coronal slices containing the paraventricular nucleus were designated as putative parvocellular or magnocellular neurons: the former were located in the medial
part of the nucleus and displayed a small fusiform soma; the latter
were mostly located in the lateral part and were recognized by their
large round soma. Compared with putative magnocellular neurons,
parvocellular neurons generally exhibited a lower membrane capacitance, lower mIPSC frequency, and smaller mIPSC amplitude. Following adrenalectomy, the mIPSC frequency was significantly enhanced in parvo- but not magnocellular neurons. Other properties of
the cells were not affected. In a second series of experiments we
examined whether the increase in mIPSC frequency was due to the
absence of corticosterone or caused by other effects related to adrenalectomy. The data support the former explanation since implantation of a corticosterone releasing pellet after adrenalectomy fully
prevented the change in mIPSC frequency. We conclude that, in the
absence of humoral negative feedback, local GABAergic input of
parvocellular neurons in the paraventricular nucleus is enhanced. This
may provide a compensatory mechanism necessary for maintaining
controllable network activity.
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J. M. VERKUYL AND M. JOËLS
tomy led to increased benzodiazepine binding, as measured in
whole-hypothalamus preparations of the rat. This effect was
reversed by corticosteroid substitution (De Souza et al. 1986;
Goeders et al. 1986; Majewska et al. 1985).
We here addressed the question to what extent the local
GABAergic network in the PVN adapts if the humoral feedback signal, i.e., glucocorticoid input, is dysfunctional. To this
end rats were adrenalectomized (ADX), allowing the investigation of neuronal feedback in the absence of corticosteroids.
To monitor neuronal feedback at the synaptic level, miniature
inhibitory postsynaptic currents (mIPSCs) were recorded with
the whole-cell patch-clamp technique. Frequency, peak amplitude, and kinetic properties of mIPSCs in PVN neurons were
compared in tissue from ADX and sham-operated control rats.
Reintroduction of corticosterone in ADX rats was used to show
steroid dependence of changes after ADX.
Surgery and slice preparation
Thirty-eight male Wistar rats (Harlan CPB, Horst, The Netherlands) of
90 –190 g were group housed under standard conditions and received
food, water, and saline (ADX) ad libitum. Day/night fluctuations of
hormones of interest were standardized by a constant light/dark cycle
(08:00 –20:00/20:00 – 08:00 h). All experiments were approved by the
local Animal Experiment Committee (DEC project No. DED43). Three
days before the experiment at 09:30 h, rats were bilaterally adrenalectomized (n ⫽ 12) or sham operated (n ⫽ 14) under halothane (Sanofi Sante,
Maassluis, The Netherlands) anesthesia as described earlier (Ratka et al.
1989). In a second series of experiments, ADX rats (n ⫽ 8) received a
subcutaneous 25-mg corticosterone pellet (Innovative Research of America), which is known to result in moderately high circulating levels of
corticosterone (Ratka et al. 1989). Control ADX rats (n ⫽ 4) received a
placebo pellet.
On the day of the experiment at 09:00 h, rats were placed in a novel
environment (clean cage) for 30 min after which they were quickly
decapitated. Trunk blood was collected for determination of plasma
corticosterone by RIA. The brain was quickly removed from the skull and
placed in ice-cold carbogenated (95% O2, 5% CO2) artificial cerebrospinal fluid (ACSF) containing (in mM) 124 NaCl, 3.5 KCl, 1.25 NaH2PO4,
1.5 MgSO4, 2 CaCl2, 25 NaHCO3, and 10 glucose (all from Sigma,
Zwijndrecht, The Netherlands); pH was set at 7.4, osmolality was approximately 300 mOsm. Coronal slices (400 ␮m) at the level of the PVN
were cut on a Vibroslicer (Campden Instruments, Sileby, UK). Under a
binocular, one slice containing the PVN was selected for recording. After
an equilibration period of ⬎1 h at room temperature this slice was
transferred to the recording chamber mounted on an upright microscope,
submerged, and continuously superfused with carbogenated ACSF. To
isolate GABAA receptor-mediated synaptic currents, AMPA and NMDA
receptors were blocked with 10 ␮M CNQX (Sigma) and 10 ␮M D-AP-5
(Sigma), respectively; action potentials were blocked with 0.5 ␮M TTX
(Latoxan, Valence, France).
Recordings and analysis
An upright microscope with a 40⫻ water immersion objective and
10⫻ ocular was used to identify PVN neuron subtypes based on their
location and the shape of their cell body. Whole-cell voltage-clamp
recordings were made using an Axopatch 200B amplifier (Axon
Instruments). Patch pipettes were pulled from borosilicate glass (Science Products, Holheim, Germany) on a horizontal puller (Sutter
Instruments). The pipettes were filled with an intracellular buffer
containing (in mM) 140 CsCl, 10 HEPES, 10 EGTA, 2 MgATP, and
0.1 NaGTP (all from Sigma); pH adjusted with CsOH (Acros Organics, Geel, Belgium) to 7.2; 280 mOsm; pipette resistance 4 –7 M⍀.
J Neurophysiol • VOL
RESULTS
Identification of paraventricular neurons
Individual PVN neurons (n ⫽ 89) were identified based on
shape and location of their somata. In the in situ (live, un-
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METHODS
For later off-line visualization a limited number of cells was filled
with either Lucifer yellow (4 mg/ml; Molecular Probes, Leiden, The
Netherlands) or Alexa Hydrozin 488 (1.75 mM; Molecular Probes).
Series resistance and capacitance were monitored during the whole
recording using pCLAMP7(Axon Instruments). Recordings with an
uncompensated series resistance of less then 2.5 times the pipette
resistance were accepted for analysis.
Traces of 5 min were recorded using the gap-free acquisition mode
of pCLAMP7 at a 10-kHz sampling rate. mIPSCs were detected
off-line using CDR and WCP analysis software [J. Dempster, University of Strathclyde, Glasgow, UK, http://www.strath.ac.uk/Departments/PhysPharm/ses.htm (2002 Feb. 23)], which uses a thresholdbased event detection algorithm. Of all mIPSCs the inter-mIPSC
interval, rise time, peak amplitude, and ␶ of decay were determined.
The decay of each mIPSC was fitted with a mono- and biexponential
curve in WCP. This program uses the Levenberg–Marquardt algorithm to iteratively minimize the sum of the squared differences
between the theoretical curve and the data curve. WCP indicates the
goodness of fit with the SD of the residuals between the fitted curve
and the data points (residual SD) for each mIPSC fitted. As criterion
for the goodness of the fit the residual SD should be ⬍0.2. Fitting with
a biexponential instead of a monoexponential curve did not increase
goodness of the fit since it did not decrease the residual SD as tested
in a substantial number of cells. Also there was no significant change
in the variance of the residual of the monoexponential compared with
the variance of the residual of the biexponential, as tested with a
Student’s t-test for a random sample of individual mIPSCs of both
putative parvocellular and magnocellular neurons (n ⫽ 17). We chose
to fit with the function using the least number of parameters, i.e., the
monoexponential.
Microsoft Excel was used to select individual mIPSCs of each cell
with the following criteria: 1) peak amplitude should be larger than 10
pA; 2) rise time, taken as 10 to 90% of peak amplitude should be ⬍5
ms; 3) the ␶ of the decay time based on a monoexponential fit should
be between 4 and 50 ms. These criteria are based on earlier studies,
describing mIPSC properties in other hypothalamic nuclei or other
brain areas (Brussaard et al. 1997; Wierenga and Wadman 1999).
Based on these criteria about 14% of all initially mIPSC detected
events were discarded, equally distributed over the different treatment
groups. After this analysis, averages of the mIPSC parameters were
determined per cell. Also, mIPSC frequency was calculated by dividing the number of events by the recording time in seconds. In addition
to averaging the mIPSC parameters per cell, we also analyzed the
distribution of mIPSC interval, peak amplitude, and ␶ of decay in all
cells. Frequency distribution per cell for the inter-mIPSC interval was
fitted with a exponential curve y ⫽ A0 exp(⫺rt), with r representing
the mean of the intervals. The log of the peak amplitude (Borst et al.
1994) and ␶ of decay distributions were fitted with a Gaussian curve
y ⫽ A0 exp(⫺(t ⫺ ␮)/␴)2, where ␮ represents the mean and ␴ the SD.
The frequency distribution for the capacitance was also fitted with a
Gaussian.
Due to seasonal fluctuations in (uncontrolled) room temperature the
second series of experiments had to be corrected for temperature using
the Q10 method. The Q10 was experimentally determined by comparing the ADX groups of the two series. The Q10 for the frequency
was found to be 1.92, for peak amplitude 1.32, and for the fitted ␶ of
decay 0.86. The rise time was not temperature dependent.
Statistical analysis was performed with a two-tailed unpaired Student’s t-test. Differences in variance were tested with a F test. Differences were considered significant if P ⬍ 0.05.
EFFECT OF ADX ON
MIPSCs
IN THE PARAVENTRICULAR NUCLEUS
stained) slice preparation of the hypothalamus, subdivisions of
the PVN were clearly distinguished (Fig. 1, A and B). A medial
part could be discerned, located between the third ventricle and
a lateral cluster of large neurons. Using 400⫻ magnification,
small and usually fusiform neurons were observed within the
medial part of the PVN, with large neurons scattered in between. The latter displayed usually large round cellbodies,
similar to the cells in the lateral cluster. A limited number of
239
mIPSCs
FIG. 1. PVN neurons were characterized on basis of location within the
PVN and the shape and size of their somata. A and B: bright field photographs
of unstained slice preparations of the PVN with overlaying fluorescent micrograph. Dotted lines indicate the borders of the parvocellular (medial) and
magnocellular (lateral) subregions. *Third ventricle. A: arrow indicates a
Lucifer yellow-filled small fusiform neuron within the medial part of the PVN.
This particular neuron had a capacitance of 21.6 pF. B: arrow points to an
Alexa-filled large round neuron within the lateral part of the PVN. This neuron
had a capacitance of 33.0 pF. Scale bar, 100 ␮m. C and D are higher
magnifications of fluorescent micrographs of A and B, respectively. C: Lucifer
yellow-filled putative parvocellular neuron. D: Alexa-filled putative magnocellular neuron. Note the difference in cell soma size. Scale bar, 100 ␮m. E:
distribution of capacitance of all neurons measured in the PVN. Filled bars
represent the putative parvocellular neurons, open bars the putative magnocellular neurons. Each of the two distributions could be fitted with a single
Gaussian (r2 ⫽ 0.81 for putative parvocellular neurons and r2 ⫽ 0.89 for
putative magnocellular neurons).
J Neurophysiol • VOL
Of both groups of neurons whole-cell patch-clamp recordings at ⫺65 mV were made to study mIPSCs. Since these
recordings were made with approximately equimolar concentrations of chloride ions inside and outside, currents reversed at
0 mV (n ⫽ 3) (Fig. 2, A and B). These currents could be fully
blocked with bicuculline (n ⫽ 3), confirming that they were
indeed generated via activation of GABAA receptors (Fig. 2C).
The basal mIPSC characteristics of the two neuron groups
were studied in SHAM-operated control rats. With respect to
mIPSC characteristics the two groups of PVN neurons differed
greatly. The mIPSC frequency of putative parvocellular neurons was significantly lower than that of putative magnocellular neurons (Fig. 3, A and B and Table 1). Moreover, mIPSCs
of putative parvocellular neurons displayed a smaller peak
current than mIPSCs of putative magnocellular neurons (Fig. 3,
A and B and Table 1). The decay of the mIPSCs in putative
parvocellular neurons tended to be slower than seen in magnocellular cells, as shown for a typical example in Fig. 3C. On
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cells were stained with the intracellular dyes Lucifer yellow
(n ⫽ 5) or Alexa Hydrozin 488 (n ⫽ 7). Post hoc histological
analysis of these cells confirmed the location and shape of the
cellbody as established during the recording session (see examples in Fig. 1, A--D). Since the intracellular dyes were found
to influence the physiological properties of the cells, staining
was only performed in a limited number of cells and not
routinely applied. Only those neurons that could be identified
during the recording session as being either 1) medially located
as well as small and/or fusiform or 2) located in the lateral
cluster with a large and round cell body were included in the
present study. Based on these criteria nine cells were excluded
from further analysis. Further subdivisions as described in the
literature for stained sections (Kiss et al. 1991) could not be
made in the unstained slice preparation. In view of the location
and shape of the somata, the medially located small and fusiform neurons will be referred to as putative parvocellular
neurons; large neurons located in the lateral cluster will be
referred to as putative magnocellular neurons (Kiss et al.
1991).
These two groups of neurons differed in their basic properties. Putative parvocellular neurons had a significantly smaller
capacitance than putative magnocellular neurons (Fig. 1E and
Table 1), confirming the visual identification based on cell size.
In this analysis of the two cell groups, data from all hormonal
treatment groups (see following text) were pooled since none
of the treatments affected membrane capacitance significantly
(data not shown). Interestingly, of the 45 recorded putative
parvocellular neurons, 5 exhibited a large capacitance (Fig.
1E), although they were visually identified as a small neuron in
the medial part of the PVN. The capacitance of these putative
parvocellular neurons, i.e., 35, 40, 42, 43, and 43 pF, was
roughly two SDs removed from the mean of this group. The
capacitance of these cells was even larger than the mean
capacitance of the putative magnocellular neurons. Except for
their large capacitance, however, these cells did not differ from
the other putative parvocellular neurons with respect to their
mIPSC characteristics and the effect of adrenalectomy (see
following text); they were therefore included in the putative
parvocellular neurons group.
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TABLE
J. M. VERKUYL AND M. JOËLS
1. Capacitance and mIPSC characteristics of putative parvo- and magnocellular neurons
Capacitance (pF)
Frequency (Hz)
Peak (pA)
␶ (ms)
Parvocellular
n
Magnocellular
n
Two-tailed Student’s t-test
20.6 ⫾ 1.3
0.498 ⫾ 0.063
⫺93.1 ⫾ 16.0
15.0 ⫾ 0.8
45
8
8
8
28.9 ⫾ 1.0
2.20 ⫾ 0.27
⫺196.6 ⫾ 22.7
13.0 ⫾ 0.7
35
11
11
11
P ⬍ 0.0001
P ⬍ 0.0001
P ⬍ 0.001
P ⬍ 0.06
Data are represented as means ⫾ SE. For all visually identified PVN neurons the capacitance differed greatly between putative parvocellular and putative
magnocellular neurons. Within the SHAM-operated rats the two groups of PVN neurons also differed in mIPSC characteristics, such as frequency peak
amplitude, but not in fitted ␶ of decay. n is number of identified cells; PVN, paraventricular nucleus of the hypothalamus; mIPSC, miniature inhibitory
post-synaptic currents.
average this difference did not attain statistical significance
(Table 1).
Hormonal influences on mIPSC characteristics
J Neurophysiol • VOL
DISCUSSION
Characterization of PVN neurons
In this study we investigated to what extent absence of a
humoral inhibitory feedback signal influences the local properties of the neuronal inhibitory input to the PVN. To this end,
mIPSC characteristics of PVN neurons were compared between SHAM and ADX rats. This influence is particularly
relevant for parvocellular PVN neurons, given their key role in
the HPA axis activity. As a first step we therefore attempted to
distinguish parvocellular from magnocellular neurons, based
on the location and morphology of their somata, a criterion also
used in earlier immunohistological studies performed in the
PVN. Morphological distinction was more straightforward
than using electrophysiological criteria earlier found with sharp
electrodes (Tasker and Dudek 1991), since the presently used
whole-cell recording configuration and pipette solution precluded a meaningful comparison.
The distinction on basis of morphological characteristics
appeared to be a reliable approach since putative parvocellular and magnocellular neurons in the PVN on average
differed from each other with respect to their basic membrane capacitance and mIPSC properties, in a way that is
accordance with other findings. Thus, in general, parvocellular neurons displayed a lower membrane capacitance than
magnocellular neurons, which agrees with the difference in
their somatic surface. Interestingly, a limited number of
cells within the parvocellular cell group that were visually
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In the first series of experiments we studied the effect of
ADX on mIPSC characteristics in the PVN for both groups of
neurons. Based on the averaged numbers per cell, ADX significantly increased the mIPSC frequency by 68% in the putative parvocellular neurons (Fig. 4A and C). In addition to
averaged mIPSC frequency per cell, the distribution of mIPSC
intervals was also analyzed for all parvocellular cells in the
ADX and SHAM groups, as shown for representative examples in Fig. 4D. In all cases the interval distribution per cell
could be fitted with an exponential curve, indicating that the
mIPSCs occurred independently from each other. Moreover,
the largely increased values for the constants in the exponential
fits (see examples in Fig. 4D) confirm the considerable increase
in mIPSC frequency after ADX.
In the group of putative parvocellular neurons, no significant
effects were observed after ADX on either peak amplitude or
␶ of decay, as calculated from the averages per cell (Fig. 5A
and C). The lack of effect was confirmed when the distribution
of mIPSCs within individual cells was taken into account.
Thus, in all cells except one, the distribution of the lognormal
of the peak amplitude and ␶ of decay could be described with
a single Gaussian curve (for peak amplitude, SHAM: average
r2 ⫽ 0.80 ⫹ 0.03; ADX: 0.87 ⫹ 0.02; for ␶ of decay, SHAM:
average r2 ⫽ 0.91 ⫹ 0.03; for ADX: 0.89 ⫹ 0.03; typical
examples shown in Fig. 5, B and D). In one cell from the ADX
group, a better fit of the distribution of lognormal of the peak
amplitude was obtained with a double Gaussian. Importantly,
after ADX there was no change in the mean as well as the
variances of the distributions (as tested with an F test), indicating that the distributions of the peak amplitude and the fitted
␶ of decay were in all respects comparable for the ADX and
SHAM groups.
Within the putative magnocellular neurons, ADX resulted in
a small but nonsignificant increase of the mIPSC frequency,
based on the averaged numbers per cell (P ⫽ 0.27; Fig. 4, B
and D). Similar to what was seen in the putative parvocellular
neurons, ADX did not affect peak amplitude or ␶ of decay in
putative magnocellular neurons (Fig. 5, A and C). Also, the
frequency distribution of mIPSC interval in putative magnocellular neurons (representative examples in Fig. 4F) as well as
the distribution of the lognormal of peak amplitude and ␶ of
decay (Fig. 5, B and D) were fully comparable for the ADX
and SHAM groups.
In the second series of experiments we investigated whether
the effects as seen in putative parvocellular neurons after ADX
were caused by the absence of corticosteroids. If so, restoring
corticosterone level to that of the SHAM-operated controls
should normalize the mIPSC characteristics. To this end, eight
ADX rats received a subcutaneous corticosterone pellet (25
mg). Pellet implantation indeed resulted in comparable corticosterone levels (9.63 ⫾ 0.39 ␮g/dl; n ⫽ 8) as observed in
SHAM-operated controls (9.95 ⫾ 2.50 ␮g/dl; n ⫽ 14). In this
second experimental series, control ADX rats received a placebo pellet. As predicted, the mIPSC frequency of putative
parvocellular neurons in the corticosterone-replaced ADX rats
was indeed decreased compared with the frequency in rats
receiving a placebo pellet (P ⬍ 0.002; Fig. 6). The temperature
corrected mIPSC frequency of ADX rats receiving corticosterone replacement was comparable to that of SHAM rats. Compared with the placebo-treated ADX group, corticosterone replacement also changed the averaged ␶ of decay (20%, P ⬍
0.05) and peak amplitude (42%, not significant) but these
changes were substantially less pronounced than the ⬎150%
change in mIPSC frequency.
EFFECT OF ADX ON
MIPSCs
IN THE PARAVENTRICULAR NUCLEUS
241
FIG. 2. Original traces recorded from typical parvocellular neurons showing that mIPSCs are carried by chloride ions and generated by GABAA
receptors. A: recordings were made with equimolar concentrations of chloride
ions inside and outside the cell. Therefore the currents reverse at 0 mV and are
inward at negative holding potentials. B: IV plot of traces shown in A. C:
currents were completely blocked by 10 ␮M bicuculline (BIC).
identified as having a small cell soma and were located in
the medial part of the PVN had a very large capacitance.
Except for their large capacitance, however, these cells do
not differ from the other putative parvocellular neurons in
their mIPSC characteristics or the effect of adrenalectomy.
The large capacitance but small cell soma could indicate
that the dendrites, particularly large diameter first order
branches, also contribute to the capacitance measurement.
We can presently not exclude that this small group of
J Neurophysiol • VOL
FIG. 3. Visually identified PVN neurons differed in their mIPSC characteristics. A: original trace recorded from PVN neurons. Left traces: visually
identified putative parvocellular neurons. Right traces: putative magnocellular
neurons. Note the difference in peak amplitude and frequency. For more
detailed comparison, B shows a blowup of the original trace of a putative
parvocellular (left) and magnocellular neuron (right). C: averaged mIPSCs of
typical putative parvocellular (solid black line) and putative magnocellular
neuron (dashed line). For comparison of the ␶ of decay, the peak IPSC
amplitude of the putative parvocellular neuron was scaled to that of the
magnocellular neuron (gray line).
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neurons represents a subset of parvocellular neurons that is
morphologically different from the majority of cells.
Magno- and parvocellular neurons also differed from each
other with respect to the mIPSC characteristics. While mIPSCs
in magnocellular neurons displayed a high-frequency, large
peak amplitude and fast decay, parvocellular neuron mIPSCs
were low in frequency, had a small peak amplitude, and were
more slowly decaying. The mIPSC characteristics for magnocellular neurons as found in this study closely resemble those
described for magnocellular neurons in the SON (Brussaard et
al. 1997). The higher frequency of mIPSCs in magnocellular
neurons may be related to the fact that the percentage of
inhibitory synapses making contact with the soma is higher in
the magnocellular than the parvocellular region, as established
with electronmicroscopy (Decavel and Van den Pol 1990).
Since the present recordings mostly reflect somatic currents,
the high mIPSC frequency of the magnocellular neurons may
be caused by the high number of somatic GABAergic synapses
on these cells. The difference in peak amplitude or quantal
amplitude could point to a difference in the number of postsyn-
242
J. M. VERKUYL AND M. JOËLS
aptic GABAA receptors (Nusser et al. 1997). Indications for
higher levels of GABAergic receptors in magnocellular than
parvocellular neurons come from in situ hybridization studies,
showing that the magnocellular section of the PVN consisJ Neurophysiol • VOL
tently exhibits a higher expression of GABAA receptor subunits than the parvocellular section (Cullinan and Wolfe 2000).
The small difference in mIPSC ␶ of decay between parvo- and
magnocellular neurons may represent differences in synaptic
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FIG. 4. Effect of ADX on PVN
neurons. A: original traces of putative parvocellular neurons. Left traces: SHAM rats. Right traces: ADX
rats. Note the increases in mIPSC
frequency. B: original traces of putative magnocellular neurons. Left
traces: SHAM rats. Right traces:
ADX rats. C: histogram showing increased mIPSC frequency in putative
parvocellular neurons from ADX
rats. Black bar represents the averaged (⫹SE) mIPSC frequency of putative parvocellular neurons from
SHAM rats; the gray bar the averaged mIPSC frequency of putative
parvocellular neurons from ADX
rats. *Statistically significant at P ⬍
0.01 (unpaired two-tailed Student’s
t-test). D: typical examples of distribution of the mIPSC interval (bin
size 1 ms) from a putative parvocellular neuron of a SHAM rat (left) and
an ADX rat (right). Distributions
were fitted with an exponential curve
(solid line). Note higher constants for
the cell of the ADX rat. E: adrenalectomy did not affect (P ⫽ 0.27)
mIPSC frequency of magnocellular
neurons. Open bar: averaged (⫹SE)
mIPSC frequency of putative magnocellular neurons from SHAM rats.
Light gray bar: averaged mIPSC frequency of magnocellular parvocellular neurons from ADX rats. F: typical examples of distribution of the
mIPSC interval (note smaller bin size
of 0.25 ms) from a putative magnocellular neuron of a SHAM rat (left)
and an ADX rat (right). Both curves
were fitted with an exponential
curve. Note comparable constants for
both groups.
EFFECT OF ADX ON
MIPSCs
IN THE PARAVENTRICULAR NUCLEUS
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FIG. 5. Adrenalectomy did not affect peak amplitude (A and B) or ␶ of decay (C and D) for both the putative parvocellular
neurons and magnocellular neurons. A: histogram showing average (⫹SE) peak amplitude of putative parvocellular neurons in
SHAM (black bar) and ADX rats (gray bar) and putative magnocellular neurons in SHAM (white bar) and ADX (light gray) bar.
B: typical example of distribution of the mIPSC peak amplitude for a single putative parvocellular neuron in a SHAM rat (top left)
and ADX rat (top right). Representative examples for putative magnocellular are given below. C: histogram showing average
(⫹SE) ␶ of decay of putative parvocellular cell in SHAM (black bar) and ADX rats (gray bar) and of putative magnocellular
neurons in SHAM (white bar) and ADX rats (light gray bar). D: typical example of distribution of the mIPSC ␶ of decay for a single
putative parvocellular neurons in a SHAM (top left) and an ADX rat (top right). Similarly, examples of putative magnocellular
neurons are given below.
J Neurophysiol • VOL
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244
J. M. VERKUYL AND M. JOËLS
parameters such as subunit composition, transmitter uptake, or
diffusion of GABA in the synaptic cleft (Cherubini and Conti
2001).
Effect of ADX
In the HPA system corticosteroids feed back primarily on
the PVN, to downregulate HPA activity. This is done in concert with direct or transsynaptic inhibitory inputs to the PVN
from higher brain areas and local hypothalamic areas. To
investigate to what extent absence of a humoral inhibitory
feedback signal in the PVN influences the local properties of
the neuronal inhibitory input to the PVN, the ADX model was
selected.
The data show that corticosteroids and the GABAergic innervation indeed do not work independently. Reducing corticosteroids levels by ADX increased the mIPSC frequency of
parvocellular neurons; mIPSC frequency of magnocellular
neurons–which are not directly involved in the HPA system–
was not altered, indicating a specific effect on the GABAergic
system involved in stress. Restoring corticosteroid levels in
ADX rats reduced mIPSC frequency of parvocellular neurons
to SHAM level, emphasizing that the effect of ADX is indeed
due to the absence of corticosterone. Tau of decay was also
slightly but significantly changed when comparing ADX rats
receiving corticosterone to ADX rats receiving a placebo.
Perhaps this difference can be explained by the fluctuating
corticosterone levels seen in SHAM rats versus the rather
constant and moderately high levels of corticosterone in ADX
rats receiving corticosterone via a pellet.
J Neurophysiol • VOL
The increase in local GABAergic transmission after ADX is
supported by earlier pharmalogical studies. In whole hypothalamus preparations several groups showed increased agonist
binding to the benzodiazepine receptor complex after ADX.
This effect could be reversed by corticosteroid substitution (De
Souza et al. 1986; Goeders et al. 1986; Majewska et al. 1985).
Recently, Miklos and Kovacs (2002) found that 7 days after
ADX the number of GABAergic terminals specifically on
CRH-positive neurons was significantly increased, as established with electron microscopy. The latter observation indicates that the increased mIPSC frequency seen in our study
most likely reflects an increase in the number of GABAergic
terminals, rather than an increase in release probability. This
change in synaptic innervation after ADX could take place in
several ways. Thus corticosterone could affect synaptic contacts directly in the PVN. Corticosterone may also act at the
level of the limbic structures projecting to the PVN, thus
indirectly affecting synaptic contacts in the hypothalamus.
What could be the functional meaning of the ADX-induced
increase in GABAergic transmission locally in the PVN? In the
ADX model, the corticosteroid feedback signal–which normally downregulates HPA activity–is no longer present. It was
shown that the lack of feedback leads to increased levels of
ACTH secretagogues, in particular CRH and vasopressin (de
Goeij et al. 1993; Sawchenko 1987). In brain slices, Kasai and
Yamashita (1988) found that the spontaneous firing rate of
neurons in the parvocellular region from ADX rats was higher
than that of intact rats. In that study, synaptic inputs from other
areas were near absent. Apparently, the intrinsic firing rate of
parvocellular neurons is increased after ADX. We here show
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FIG. 6. Subcutaneous placement of a
corticosterone-releasing pellet in ADX
rats, thus restoring the corticosterone
level to that in SHAM operated controls,
decreased the mIPSC frequency of putative parvocellular neurons compared with
values seen in ADX rats receiving a placebo pellet. A: original traces of putative
parvocellular neurons from ADX rats receiving a 25-mg corticosterone pellet. B:
original traces of putative parvocellular
neurons of ADX rats receiving a placebo
pellet. C: histogram showing the averaged mIPSC frequency of putative parvocellular neurons from ADX rats receiving
a 25-mg corticosterone pellet (white
striped bar) and ADX rats receiving a
placebo pellet (gray striped bar). *Statistically significant at P ⫽ 0.002 with an
unpaired two-tailed Student’s t-test.
EFFECT OF ADX ON
MIPSCs
IN THE PARAVENTRICULAR NUCLEUS
that the local synaptic inhibition, however, is increased. Generally, GABAergic innervations are thought of as being important for synchronizing neuronal activity. The increased mIPSC
frequency might therefore provide a compensatory mechanism
necessary for maintaining controllable network activity.
We thank Drs. J. A. Groot and W. van Raamsdonk for the use of equipment,
Dr. H. Karst for the adrenalectomy operations and Drs. R. Lingeman and A. B.
Brussaard for helpful discussions.
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