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
h
and or TDR was observed in 87 of phasically firing Electrophysiological recording followed by fluorescent
POA neurons. histochemistry was performed in cells from any one of
51 of POA neurons expressed a low threshold spike four slices through the POA see Methods. The dis-
LTS in current clamp on the off-step of a hyperpolarizing tribution of cells from which electrophysiological record-
current pulse Fig. 5. The LTS was associated with a ings were acquired ranged from the anteroventral region of
transient inward current like that shown in Fig. 6, which the POA to the medial, periventricular region of the more
appeared immediately following the off-step of a hy- caudal POA. This latter region contains the somata of A
perpolarizing voltage command Fig. 6A and was marked-
14
dopamine neurons [32], an example of which is illustrated ly attenuated by NiCl
200 mM; Fig. 6B and C. The
2
by the dual biocytin TH immunohistofluoresence shown in transient inward currents elicited immediately following
Fig. 1A and B. The majority of the recordings were the onset of depolarizing voltage commands from a
performed in the intermediate slices two and three, and holding potential of 2100 mV reached maximum am-
the documented cell locations within these slices, including plitude at |245 mV, and a resultant fit of the normalized
the TH-positive neuron mentioned above, are shown in currents to the Boltzmann equation revealed a half-maxi-
Fig. 1C and D. mal activation voltage V
of |270 mV Fig. 7.
1 2
The present study included a total of 79 cells, 29 of Coupled with its transient nature, this current has the
21
which were recorded from intracellularly using sharp characteristics of the T-type Ca
current I . As with the
T
electrodes and 50 of which were recorded from using the I , the I and or LTS was detected in a high percentage
h T
whole-cell configuration. POA neurons exhibit a resting 87 of phasically firing POA neurons. Most importantly,
membrane potential of 250.060.7 mV from sharp elec- all phasically firing POA neurons displayed either an
trode and whole-cell recordings, as well as a R of
I TDR or an I LTS, and many of these neurons 73
in h
T
445664 MV and a t of 15.661.6 ms from sharp
expressed both conductances Fig. 5. electrode recordings. We also observed that 62 of POA
29 of POA neurons exhibited a transient outward neurons spontaneously fire in a bursting pattern. This was
current similar to that shown in Fig. 8. This current characterized empirically by the intermittent clustering of
appeared immediately following the onset of depolarizing action potentials .3 Hz interspersed among periods of
voltage commands from a holding potential of 2100 mV quiescence 200 ms as shown in the intracellular, sharp
Fig. 8A. This transient outward current also was sensitive electrode recording in Fig. 2A.
to a high dose of 4-AP 3 mM; Fig. 8B and C, indicative The remaining 38 of POA neurons either fired action
of the expression of the I . Alternatively, it could be
A
potentials at uniform, regular intervals or were silent at observed upon a depolarizing voltage step to 230 mV
rest. Evaluation of the ISI histograms revealed a bimodal given subsequently to a series of pre-pulses ranging from
distribution for burst firing POA neurons and a unimodal 2100 to 240 mV Fig. 8D. We used these two protocols
distribution for nonbursting POA neurons Fig. 2B. The to construct the inactivation and activation curves for the
mean firing frequency of burst firing POA neurons was I , which are shown in Fig. 8E. It should be noted that the
A
significantly greater than that observed in nonbursting expression of this current and that of the I were, in most
T 2
POA neurons 4.761.2 Hz vs. 1.060.7 Hz; P,0.05. In cases, mutually exclusive
x 56.4; P,0.05. addition, the majority of POA neurons 75 regardless of
92 of POA neurons 54 of 59 responded to the their firing pattern displayed an AHP on the off-step of a
application of m-opioid peptides. As observed with sharp depolarizing current pulse 100 pA delivered at rest in the
electrode recording,
met-enkephalin 300
nM and
presence of 2 mM TTX Fig. 3. The mean AHP amplitude DAMGO 1 mM elicited a hyperpolarization in current
was larger in burst firing POA neurons than in nonbursting clamp 8.261.6 mV; n511 or outward current in voltage
POA neurons 4.661.1 mV vs. 1.660.6 mV; P,0.05. clamp 210 pA; n51 that was reversible upon drug
70 of POA neurons expressed a hyperpolarization- discontinuation not shown. Similar results, albeit smaller
activated, rectifying inward current I similar to that in magnitude, were obtained with whole-cell patch record-
h
shown in Fig. 4. This current had the appearance of a sag ing
Fig. 9.
The opioid-induced
outward current
in the membrane current that slowly developed after the 26.963.6 pA; n542 was accompanied by an increase in
E .J. Wagner et al. Brain Research 879 2000 29 –41
33
Fig. 1. A Confocal image of the biocytin–streptavidin–FITC labeling of a neuron in the periventricular zone of the POA. B Confocal image of TH immunoreactivity in the soma of the cell in A as visualized with CY-3. Note the multiple spines that exist along the processes radiating from the cell body.
The scale bar equals 25 mm for both A and B. C Camera lucida drawing through slice two showing the distribution of documented recording sites in this slice. D Camera lucida drawing illustrating the distribution of documented cell locations in slice three. The location of the double-labeled neuron in
A and B is denoted by the filled circle. The documented cell locations in these two slices were determined primarily from sharp electrode, intracellular recordings and, in some cases, from whole-cell patch recordings. LV5lateral ventricle, AC5anterior commissure, III V5third ventricle, OC5optic chiasm.
conductance Fig. 9A–C that reversed polarity near the together, these data demonstrate that m-opioid peptides
1
equilibrium potential for K E
; Fig. 9D. The I V plot inhibit POA neurons via an increase in I
.
K1 Kir
shown in Fig. 9D revealed that the DAMGO-induced Likewise, POA neurons 84; 38 of 45 responded to
conductance increase is more pronounced at membrane the bath application of the GABA
receptor agonist
B
potentials more negative than E Furthermore, the
baclofen 100 mM with a reversible hyperpolarization in
K1.
conductance increase produced by DAMGO was sensitive current clamp 9.461.7 mV; n56 or an outward current in
to antagonism by BaCl 100 mM; Fig. 9E. Taken
voltage clamp 80660 pA; n52. As with the m-opioid
2
34 E
Fig. 2. A A representative example of a POA neuron firing action potentials in a bursting manner. Its resting membrane potential was 251 mV, and its mean firing frequency was 5.8 Hz. The action potentials were not fully reproduced by the chart recorder. B Histograms illustrating the distribution of the
ISI for bursting and non-bursting POA neurons. The histogram of the burst firing POA neuron was obtained from the cell in A. ISI distributions were derived from digitally captured segments of spontaneous firing. The curves were generated from a user-defined transform that shaded the ISI bin-width 50
ms count pairs to their respective axes. The curve for the bursting POA neuron revealed a bimodal distribution. The first mode occurred at 138.5 ms, which reflected the ISI determined during a burst denoted by the solid arrows in A. The other mode occurred at 415.4 ms, which represented the
interburst interval denoted by the dashed arrow in A. By contrast, the distribution of the non-bursting POA neuron mean firing frequency50.5 Hz is unimodal in nature, with the mode occurring at 1429 ms.
peptides, we observed qualitatively similar responses in in Fig. 10E, BaCl
100 mM completely abolished the
2
whole-cell patch recordings. The baclofen-induced out- increase in slope conductance produced by baclofen.
ward current 19.261.9 pA; n532 was accompanied by a Coadministration of baclofen and DAMGO elicited no
reversible increase in conductance Fig. 10A–D, and this additional outward current or increase in slope conduct-
current reversed polarity near E Fig. 10D. As depicted
ance when compared to that obtained with either of the
K1
Fig. 3. Representative examples illustrating the AHP in bursting A and nonbursting B POA neurons. In both cases the AHP denoted by the arrows was elicited in the presence of TTX 2 mM on the off-step of a depolarizing current pulse 100 pA; 1 s duration. Note the larger AHP amplitude in the
bursting neuron 7 mV compared to the nonbursting neuron 3 mV.
E .J. Wagner et al. Brain Research 879 2000 29 –41
35
Fig. 4. A Current record from a whole-cell recording of a POA neuron demonstrating the hyperpolarization-activated inward current I . This current has
h
the appearance of a sag following the instantaneous current observed with the onstep of the hyperpolarizing voltage command. The cell was held at 250 mV, and delivered a series of hyperpolarizing voltage commands 10 mV, 1 s down to 2150 mV. B I V plot of the instantaneous inst. vs. the
steady-state membrane current of the cell in A showing the inward rectification of the I and an activation threshold of 250 mV. Instantaneous currents
h
solid circles and steady-state currents open circles were measured from the points along the traces in A indicated by their respective symbols. C and D, current record and I V plot of the instantaneous inst. vs. the steady-state membrane current showing the cesium 3 mM block of the I observed in the
h
cell in A. Instantaneous currents solid triangles and steady-state currents open triangles were measured from the traces in C at the points indicated by their respective symbols.
agonists alone not shown. Thus, m-opioid and GABA burst firing neurons show a more robust AHP than do
B
receptors couple to a common pool of inwardly-rectifying non-bursting POA neurons. The AHP is mediated by a
1 21
1
K channels.
small conductance SK, Ca -dependent K
current [40,41], and three SK channel subtypes have been cloned
[27]. In addition, apamin-sensitive SK2 and SK3 channel
4. Discussion subtypes are highly expressed throughout the hypo-
