30 E
of which are blocked by the opioid receptor antagonist 2.2. Drugs
naloxone [50]. Thus, the hyperthermia evoked by these factors is the result of a decreased capacity to dissipate
All drugs were initially dissolved in Milli-Q H 0 unless
2
heat, and an enhanced ability to produce and retain heat. otherwise stated. TTX Sigma Chemical Co., St. Louis,
The POA also contains gonadotropin hormone-releasing MO, USA was dissolved in Milli-Q H O and diluted to
2
hormone GnRH neurons that project to the median the appropriate volume with 0.1 acetic acid final
eminence, and the neuropeptide released from these neu- concentration 1 mM; pH 4–5. Cesium chloride CsCl;
rons acts as the primary secretagogue in the release of J.T. Baker Inc., Phillipsburg, NJ, USA was prepared as a
gonadotropins i.e., follicle stimulating hormone, luteiniz- stock solution of 0.2 M. N-ethyl-N-phenylamino-1,2-di-
ing hormone LH from the anterior pituitary [15,42]. methyl-6-methylamino pyrimidinium chloride ZD 7288;
POA neurons receive inhibitory GABAergic input [28], as Tocris Cookson Inc., Ballwin, MO, USA was prepared as
well as synaptic input from b-endorphin neurons emanat- a stock solution of 10 mM. Nickel chloride hexahydrate
ing from the arcuate nucleus [9,18]. Activation of GABA NiCl ; Sigma was prepared as a stock solution of 0.1 M.
B 2
receptors inhibits naloxone-stimulated LH release in a 4-Aminopyridine 4-AP; Aldrich Chemical Co., Mil-
variety of experimental models [7,11,30], thereby indicat- waukee, WI, USA was prepared as a stock solution of 0.1
ing an indirect opioid modulation of LH secretion through M. Tetraethylammonium TEA; Sigma was prepared as a
an interaction with GABAergic neurons. There is, how- stock solution of 10 mM. DAMGO Peninsula Laborator-
ever, ample evidence to suggest that opioid peptides and ies Inc., Belmont, CA, USA and met-enkephalin Penin-
GABAergic ligands affect changes in reproductive status sula were prepared as stock solutions of 1 and 5 mM,
by acting at the level of the POA to directly inhibit GnRH respectively. 6-Baclofen 6-b-aminomethyl-4-chlo-
and thereby LH secretion [25,28,45]. robenzenepropanoic acid; Sigma was dissolved in 0.1 N
The purpose of the present study was to characterize the HCl to a stock concentration of 40 mM. Barium chloride
prominent intrinsic and inhibitory, agonist-activated con- dihydrate BaCl ; Sigma was prepared as a stock solution
2
ductances in POA neurons of the ovariectomized female of 10 mM. Aliquots of the various stock solutions were
guinea pig. To this end, whole-cell and intracellular stored at 2808C TTX, DAMGO, met-enkephalin, ba-
recordings were made from coronal hypothalamic slices. clofen, 2208C ZD 7288or 48C CsCl , NiCl , 4-AP,
2 2
2 4
Methionine met-enkephalin and [
D
-Ala , N-Me-Phe , TEA, BaCl until used for experimentation.
2 5
Gly-ol ]-enkephalin DAMGO, opioid peptides selective for the m-receptor [17], were used to assess the effects of
2.3. Hypothalamic slice preparation m-opioid receptor activation. Likewise, the selective
GABA receptor agonist baclofen [6] was used to evaluate On the day of experimentation, the animal was decapi-
B
the effects of GABA receptor activation. The results are
tated, and its brain rapidly removed from the skull. The
B
discussed in a context pertinent to the important role of the brain was rinsed with ice-cold artificial cerebrospinal fluid
POA in physiological processes such as thermoregulation aCSF; NaCl, 124; KCl, 5; NaH PO , 2.6; dextrose, 10;
2 4
and reproduction. HEPES, 10; MgSO , 2; CaCl , 2; in mM and the
4 2
hypothalamus immediately dissected. Four coronal slices 450 mM through the rostro-caudal extent of the POA
were cut with the aid of a vibratome. The slices ranged
2. Materials and methods from the organum vasculosum laminae terminalis at the
rostral end slice one to the anterior hypothalamic border 2.1. Animals
at the caudal end slice four. We then transferred the slices to a multi-well auxiliary chamber containing oxygenated
Female Topeka guinea pigs 440–670 g were obtained 95 O , 5 CO aCSF, where they were kept until
2 2
from our institutional breeding facility, and maintained electrophysiological recording.
under conditions of constant temperature 72.460.18F and light lights on between 06:30 and 20:30 h. Animals were
2.4. Electrophysiology housed individually, with food and water provided ad
libitum. They were ovariectomized while under ketamine During recording, slices were maintained in a chamber
and xylazine anesthesia 33 and 6 mg kg, respectively; perfused with warmed 358C, oxygenated aCSF. Artificial
s.c. 5–9 days before experimentation in order to remove CSF and all drugs diluted with aCSF were perfused via a
the confounding influence of gonadal steroid hormones on peristaltic pump 1.5 ml min or via gravity feed. Sharp
synaptic transmission in hypothalamic neurons [26]. The electrode intracellular recording in current and voltage
surgical and experimental procedures described in the clamp was performed as described previously [14,26,29].
present study are in accordance with institutional guide- Briefly, microelectrodes were assembled from borosilicate
lines based on NIH standards. glass pipettes 1.2 mm O.D.; Sutter Instrument Co.,
E .J. Wagner et al. Brain Research 879 2000 29 –41
31
Novato, CA, USA pulled on either a P-87 or a P-97 the application of further suction or a transient voltage
Flaming Brown puller Sutter Instrument Co.. They were pulse via the preamplifier. Membrane currents were re-
filled with a 3 biocytin solution in 1.75 M KCl and 0.025 corded in voltage clamp with access resistances of 25–40
M Tris pH 7.4. Electrode resistances varied from 100 to MV, and underwent analog–digital conversion via a
225 MV. The membrane potential of POA neurons was Digidata 1200 interface coupled to either pClamp 6.0 or
measured in current clamp. Potentials were amplified and 7.0 software. Low-pass filtering of the currents was
current was passed through the electrode using an Axo- conducted at a frequency of 2 KHz. Series resistance was
clamp 2A preamplifier Axon Instruments, Foster City, compensated up to 80. The liquid junction potential was
CA, USA. Current and voltage traces were stored on a 211 mV, and was corrected in subsequent data analysis.
digital oscilloscope Tektronix 2230, Tektronix, Beaverton, Voltage command protocols used to characterize the
OR, USA, and were recorded on a chart recorder Gould current voltage I V relationships of the prominent intrin-
2200, Gould Inc., Glen Burnie, MD, USA. They also sic conductances I , I
and I are described in the
h T
A
underwent analog–digital conversion with a CyberAmp legends
to their
respective figures.
Pharmacological 320 signal conditioner for amplification connected to a
characterization of these conductances was ascertained DigiData 1200 A D converter sampling frequency: 62 Hz
following the application of CsCl 3 mMor ZD 7288 30 for the gap-free tape mode, 10–50 KHz for the oscillos-
mM; I , NiCl 200 mM; I or 4-AP 3 mM; I 15 min
h 2
T A
cope mode and subsequent storage on a computer con- before and during a second protocol run for the appropriate
taining Axoscope software Axon Instruments. Following conductance. Responses elicited by m-opioid or GABA
B
successful impalement, action potentials were collected for receptor activation were evaluated by applying met-en-
the subsequent determination of firing pattern, frequency kephalin 300 nM, DAMGO 1 mM or baclofen 100
and the interspike interval ISI. Slices then were perfused mM, respectively, until a new steady-state membrane
with 2 mM TTX for at least 6 min to block spontaneous potential or holding current was obtained 4–7 min. I V
firing, and all subsequent drug solutions were sup- relationships were generated before and near the end of
plemented with 1 mM TTX. Pre-drug voltage-current V I agonist application according to the voltage command
relationships were established by giving hyperpolarizing protocol described in the legends for the appropriate
and depolarizing current pulses 0.2 Hz; 1 s duration of figures. Pharmacological verification that these agonist
varying magnitudes, and monitoring the resultant voltage effects were mediated via an I
was determined by
Kir
deflections. These were performed in order to determine applying BaCl 100 mM in the presence of agonist 15
2
both the input resistance R , measured by linear regres- min prior to and during the generation of a final I V
in
sion between 260 and 280 mV and the magnitude of the relationship.
AHP. The membrane time constant t was calculated as the time necessary for a voltage deflection |10 mV to
2.5. Histology reach 63 of its maximum. For some experiments, single
electrode voltage clamp was performed using the dis- Following recording with biocytin-filled electrodes,
continuous mode with a switching frequency of 1–2 KHz slices were fixed with 4 paraformaldehyde in Sorensen’s
and 30 duty cycle. The optimization of clamp parameters phosphate buffer pH 7.4 for 90–180 min [39]. They were
and the monitoring of the headstage output was done on a immersed overnight in 20 sucrose dissolved in Soren-
separate oscilloscope. sen’s buffer, and then frozen in Tissue-Tek embedding
For whole-cell recordings, electrodes were fabricated medium Miles, Inc., Elkhart, IN, USA. Coronal sections
from thin wall borosilicate glass A-M Systems, Carlsborg, 16 mm were cut on a cryostat, and were mounted on
WA; 1.5 mm O.D. pulled on a Model 720 vertical pipette slides coated with poly-
L
-lysine. These sections were puller David Kopf Instruments, Tujunga, CA. Resultant
washed with 0.1 M sodium phosphate buffer pH 7.4, and electrodes were then filled with an internal solution
then processed
with streptavidin–fluorescein
isothio- containing 0.5 biocytin and consisting of the following:
cyanate FITC as described previously [39]. After localiz-
1
K gluconate, 128; NaCl, 10; MgCl , 1; EGTA, 11;
ing the biocytin-filled neuron, the slides containing the
2
HEPES, 10; ATP, 1; GTP, 0.25; in mM; pH adjusted to 7.3 appropriate sections were processed with a monoclonal
with 1 N KOH; 292 mOsm. Voltage pulses were amplified tyrosine hydroxylase TH antibody DiaSorin, Stillwater,
and passed through the electrode using an Axopatch 1D MN, USA at a 1:3000 dilution using fluorescence im-
preamplifier Axon Instruments. munohistochemistry [39].
The resultant current deflections were monitored using a digital oscilloscope Tektronix TDS 340A. Upon the
2.6. Statistical analyses reduction of the current deflection, negative pressure was
applied via a 10 ml syringe connected by polyethylene Comparison of the mean between two groups was
tubing to the electrode in order to form a seal .1 GV. performed using a two-tailed Student’s t-test. Homogeneity
After seal formation the cell membrane was ruptured by of variance was analyzed using Bartlett’s test. If variances
32 E
were not homogeneous, then the comparisons between two instantaneous current, and reached steady-state within 1 s.
groups were performed using the Mann–Whitney U-test. The threshold for activation was 50 mV, and the mag-
Evaluation of the frequency of occurrence was accom- nitude of this current increased with progressively hy-
plished using the Chi-square test in conjunction with a two perpolarizing voltage commands. Bath application of CsCl
by two contingency table. Differences were considered 3 mM; Fig. 4C and D or ZD 7288 30 mM; not shown
statistically significant if the probability of error was less completely blocked the expression of this current. The I
h
than 5. was associated with a time-dependent rectification TDR
in current clamp that was manifested by a depolarizing relaxation of the membrane potential in response to
3. Results hyperpolarizing current pulses Fig. 5. In addition, the I
