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J Physiol. Feb 15, 2000; 523(Pt 1): 193–209.
PMCID: PMC2269788

Voltage-gated currents distinguish parvocellular from magnocellular neurones in the rat hypothalamic paraventricular nucleus

Abstract

  1. Magnocellular and parvocellular neurones of the hypothalamic paraventricular nucleus (PVN) differentially regulate pituitary hormone secretion and autonomic output. Previous experiments have suggested that magnocellular, or type I neurones, and parvocellular, or type II neurones, of the PVN express different electrophysiological properties. Whole-cell patch-clamp recordings were performed in hypothalamic slices to identify the voltage-gated currents responsible for the electrophysiological differences between type I and type II PVN neurones.
  2. Type I neurones, which display transient outward rectification and lack a low-threshold spike (LTS), generated a large A-type K+ current (IA) (mean ± s.e.m.: 1127.5 ± 126.4 pA; range: 250–3600 pA; voltage steps to −25 mV) but expressed little or no T-type Ca2+ current (IT). Type II neurones, which lack transient outward rectification but often display an LTS, expressed a smaller IA (360.1 ± 56.3 pA; range: 40–1100 pA; voltage steps to −25 mV), and 75 % of the type II neurones generated an IT (-402.5 ± 166.9 pA; range: −90 to −2200 pA; at peak).
  3. The voltage dependence of IA was shifted to more negative values in type I neurones compared to type II neurones. Thus, the activation threshold (−53.5 ± 0.9 and −46.1 ± 2.6 mV), the half-activation potential (−25 ± 1.9 and −17.9 ± 2.0 mV), the half-inactivation potential (−80.4 ± 9.3 and −67.2 ± 3.0 mV), and the potential at which the current became fully inactivated (−57.4 ± 2.1 and −49.8 ± 1.5 mV) were more negative in type I neurones than in type II neurones, respectively.
  4. IT in type II neurones activated at a threshold of −59.2 ± 1.2 mV, peaked at −32.6 ± 1.7 mV, was half-inactivated at −66.9 ± 2.2 mV, and was fully inactivated at −52.2 ± 2.2 mV.
  5. Both cell types expressed a delayed rectifier current with similar voltage dependence, although it was smaller in type I neurones (389.7 ± 39.3 pA) than in type II neurones (586.4 ± 76.0 pA).
  6. In type I neurones IA was reduced by 41.1 ± 7.0 % and the action potential delay caused by the transient outward rectification was reduced by 46.2 ± 10.3 % in 5 mm 4-aminopyridine. In type II neurones IT was reduced by 66.8 ± 10.9 % and the LTS was reduced by 76.7 ± 7.8 % in 100 μm nickel chloride, but neither IT nor LTS was sensitive to 50 μm cadmium chloride.
  7. Thus, differences in the electrophysiological properties between type I, putative magnocellular neurones and type II, putative parvocellular neurones of the PVN can be attributed to the differential expression of voltage-gated K+ and Ca2+ currents. This diversity of ion channel expression is likely to have profound effects on the response properties of these neurosecretory and non-neurosecretory neurones.

The hypothalamic paraventricular nucleus (PVN) is involved in the control of hormone secretion from the anterior and posterior lobes of the pituitary gland, and in the regulation of the autonomic nervous system (Swanson & Sawchenko, 1983; Liposits, 1993). The PVN consists predominantly of three classes of cells: the parvocellular neuroendocrine cells, which control hormone secretion from the anterior pituitary gland, the parvocellular preautonomic cells, which are involved in the regulation of the autonomic nervous system, and the magnocellular neuroendocrine cells, which are responsible for hormone secretion from the posterior pituitary (Swanson & Sawchenko, 1983; Liposits, 1993).

Previous work has shown that neurones in the region of the PVN can be divided into three groups, cell types I, II and III, based on differences in their electrophysiological properties (Poulain & Carette, 1987; Tasker & Dudek, 1991), and that cell types I and II correspond to putative magnocellular and parvocellular neurones of the PVN, respectively (Hoffman et al. 1991). The most salient differences between type I and type II neurones is the generation of a pronounced transient outward rectification in type I neurones, which is not found in type II neurones, and the capacity of some type II neurones, but not type I neurones, to generate a Ca2+-dependent low-threshold spike of variable amplitude and waveform (Tasker & Dudek, 1991; Hoffman et al. 1991).

Type I PVN neurones, like magnocellular neurones of the supraoptic nucleus, express a pronounced A-type K+ current (IA) (Bourque, 1988; Li & Ferguson, 1996). The transient outward rectification, as well as IA, in supraoptic magnocellular neurones is reduced by the K+ channel blocker 4-aminopyridine (Fisher et al. 1998), which suggests that the transient outward rectification in type I PVN neurones may also be generated by the activation of IA. The voltage dependence and pharmacological sensitivity of the low-threshold spike in type II PVN neurones suggests that it is caused by the activation of a T-type Ca2+ current (IT) (Tasker & Dudek, 1991; Luther & Tasker, 1997).

The temporal firing pattern of neuroendocrine cells has a profound impact on hormone release (Dutton & Dyball, 1979; Bicknell & Leng, 1981). Under stimulated conditions, vasopressin-secreting magnocellular neurones adopt a spiking pattern characterised by alternating periods of activity and quiescence, termed phasic firing (Poulain & Wakerly, 1982), which leads to enhanced vasopressin release compared to tonic firing (Dutton & Dyball, 1979; Bicknell & Leng, 1981). IA and IT exert a strong influence on repetitive firing patterns, IA through its effect on the inter-spike interval, action potential repolarisation, and postsynaptic responsiveness (Rogawski, 1985; Rudy, 1988; Magee et al. 1998) and IT through the generation of Ca2+-dependent burst firing and membrane potential oscillations (Jahnsen & Llinás, 1984; Huguenard, 1996). Differences in IA and IT are therefore likely to underlie differences in repetitive firing properties that impact on hormone release from magnocellular and parvocellular neuroendocrine cells, and autonomic output mediated by parvocellular preautonomic neurones. We conducted voltage-clamp and current-clamp experiments in hypothalamic brain slices in order to correlate the expression of voltage-gated currents to the specific electrophysiological properties displayed by type I and type II PVN neurones.

METHODS

Hypothalamic slice preparation

Male Sprague-Dawley rats (21–30 days old) (Charles River Laboratories, Raleigh, NC, USA) were used in our experiments with the approval of the Tulane University Animal Care and Use Committee and according to guidelines of the US Public Health Service. They were deeply anaesthetised with 50 mg kg−1i.p. pentobarbitone sodium (Abbott Laboratories, North Chicago, IL, USA) and decapitated in a rodent guillotine. The brain was removed and placed into ice-cold (≤ 1°C) artificial cerebrospinal fluid (ACSF), consisting of (mm): NaCl 140, KCl 3, MgSO4 1.3, NaH2PO4 1.4, d-glucose 11, Hepes 5, CaCl2 2.4, NaOH 3.25; pH 7.3, osmolarity 290–300 mosmol l−1, and bubbled with 100 % O2. A 1 cm3 block of hypothalamus was isolated from the rest of the brain by making razor cuts rostral to the optic chiasm, caudal to the median eminence, dorsal to the third ventricle, and lateral to the fornix. The block was glued to the chuck of a vibrating tissue slicer (World Precision Instruments, Sarasota, FL, USA) and three 400 μm slices containing the PVN were sectioned in cold, oxygenated ACSF. The slices were equilibrated for 1–2 h at room temperature in a tissue storage chamber containing ACSF saturated with 100 % O2. Slices were bisected along the third ventricle and a single hemi-slice at a time was placed on the ramp of an interface recording chamber and superfused with oxygenated ACSF maintained at 29–30°C. The other slices were kept in the storage chamber until needed.

Electrophysiology

Patch electrodes were pulled in multiple stages on a Flaming-Brown P-97 horizontal puller (Sutter Instruments Co., Novato, CA, USA) from borosilicate glass (1.2 mm i.d., 1.65 mm o.d., KG-33, Garner Glass Co., Claremont, CA, USA) to a resistance of 2–4 MΩ. They were filled with a solution consisting of (mm): potassium gluconate 130, Hepes 10, NaCl 1, CaCl2 1, EGTA 10, MgCl2 1, adenosine 5′-triphosphate (magnesium salt) 2, guanosine 5′-triphosphate (sodium salt) 0.5; pH was adjusted to 7.2 with KOH. The osmolarity of the internal solution was increased to 300–310 mosmol l−1 with D-sorbitol, which was found to decrease the series resistance of recordings without eliciting a significant osmotic response from the neurones. Slices were transilluminated and visualized under a dissecting microscope. Electrodes were positioned in the PVN under visual control and advanced through the tissue using a piezoelectric step motor (Nano-Stepper type B, Adams & List Associates, Ltd, Westbury, NY, USA). Recordings were made using an Axopatch 1-D amplifier (Axon Instruments, Foster City, CA, USA). Data were low-pass filtered at 2 kHz with the amplifier and sampled at 5–10 kHz using the pCLAMP 6 data acquisition and analysis software (Axon Instruments). The liquid junction potential (11 mV) was corrected for according to Neher (1992). All voltage-clamp traces represent an average of at least three separate trials. IT and delayed rectifier K+ current (IK) traces were leak subtracted using a P/8 protocol. IA traces were leak subtracted by digitally subtracting the current elicited from a conditioning step at which the IA was completely inactivated (-50 mV), which removed IK, leak, and capacitative currents. Data were recorded onto VHS video tapes using a Neuro-corder DR-484 digitising unit (NeuroData Instruments Corp., New York, NY, USA). Selected traces were saved to the hard drive of a personal computer using a Digidata 1200B interface (Axon Instruments).

Cells were excluded from analyses if they did not meet the following criteria: action potentials greater than or equal to 50 mV from threshold to peak, input resistance near resting potential of at least 500 MΩ, and resting potential negative to −50 mV. Series resistance compensation of 80 % or higher was routinely employed and changes in series resistance were monitored and compensated for throughout the course of experiments. The mean ( ± s.e.m.) series resistance at the start of the voltage-clamp analyses was 10.5 ± 0.4 MΩ. The mean voltage error due to series resistance for IA was 2.1 ± 0.3 mV (with steps to −25 mV), and for IT was 0.7 ± 0.2 mV (at peak). Recordings during which the series resistance increased to greater than 20 MΩ were discarded. The quality of the space clamp was assessed by measuring the rate of activation and inactivation of IA and IT activated by stepping from progressively more hyperpolarised conditioning steps to a constant test step. The rates of activation and inactivation of IA and IT are voltage dependent and should vary as a function of test step potential, whereas varying the conditioning step should affect only the amplitude of the current (i.e. more negative conditioning steps remove more inactivation and elicit larger currents). Plots of 10–90 % rise time and inactivation time constant against the range of conditioning steps used were judged by eye to be linear and horizontal (i.e. unchanging with conditioning steps of increasing amplitude); cells that did not meet this criterion were not included in the comparative analyses. Approximately 30 % of all recorded type I and type II neurones met the two criteria of a series resistance less than 20 MΩ and no relationship between current amplitude and kinetics. Of neurones that were used for analysis of voltage-dependent and kinetic properties, the activation and inactivation kinetics of IA did not differ between 100 % availability (0 % inactivation) and 50 % availability (50 % inactivation) of the current, as determined by using Student's paired t test. Thus, for IA in type I neurones, the 10–90 % rise time was 1.5 ± 0.12 ms at 100 % and 1.5 ± 0.14 ms at 50 % availability, and the inactivation time constant was 23.2 ± 3.6 ms at 100 % and 22.8 ± 3.6 ms at 50 % availability; for IA in type II neurones, the 10–90 % rise time was 2.7 ± 0.34 ms at 100 % and 2.9 ± 0.34 ms at 50 % availability, and the inactivation time constant was 23.0 ± 4.8 ms at 100 % and 20.2 ± 3.5 ms at 50 % availability.

Isolation of potassium and calcium currents

Potassium currents were studied using ACSF containing (mm): NaCl 140.5, KCl 3, MgCl2 3.7, D-glucose 11, Hepes 5, NaOH 3.25, tetrodotoxin 0.003 (to block voltage-gated Na+ currents), CaCl2 0 and CdCl2 0.2 (to block voltage-gated Ca2+ currents); osmolarity was 290–300 mosmol l−1, and pH was adjusted to 7.3 with NaOH. The K+ channel blocker 4-aminopyridine (5 mm) was added to the above solution in some experiments to block IA, in which case the NaCl concentration was reduced proportionately to maintain constant osmolarity. Since 4-aminopyridine is basic, it was necessary to replace the NaOH in the above solution with NaCl and to adjust the pH with HCl.

Calcium currents were not isolated completely since, in current-clamp mode, low-threshold Ca2+ spikes could not be studied in isolation without contamination from high-threshold Ca2+ potentials in solutions in which K+ channels were blocked completely. Consequently, K+ channels were blocked strongly but not completely (> 50 %) in order to isolate Ca2+ currents using ACSF containing (mm): NaCl 105.5, KCl 3, MgCl2 1.3, d-glucose 11, Hepes 5, CaCl2 10, tetrodotoxin 0.003, and 4-aminopyridine 5, CsCl 3, and tetraethylammonium chloride 20 (to block voltage-gated K+ currents); osmolarity was 290–300 mosmol l−1, and pH was adjusted to 7.3 with HCl. The Ca2+ channel blockers NiCl2 (100 μM) and CdCl2 (50 μM) were added directly to the above solution to block Ca2+ currents in some experiments. All chemicals were obtained from Sigma (St Louis, MO, USA) with the exception of tetrodotoxin, which was acquired from Alomone Labs (Jerusalem, Israel).

Current-clamp analyses and cell identification

Type I, putative magnocellular neurones were differentiated from type II, putative parvocellular neurones based on the expression of a transient outward rectification. Hoffman et al. (1991) demonstrated that the expression of transient outward rectification in type I PVN neurones is positively correlated to neurophysin immunoreactivity and large somatic diameter, suggesting that type I neurones are magnocellular neuroendocrine cells. In our experiments, the presence of transient outward rectification was qualitatively assessed with a current-clamp protocol consisting of a series of progressively more depolarising current injections from a hyperpolarisation to near −100 mV. The amplitudes of current injected depended on the input resistance of the cell. Transient outward rectification was identified as a pronounced dampening of the membrane charging curve that was associated with a delay in onset to the first action potential (Fig. 1A). Transient outward rectification is not observed in type II neurones (Fig. 1B). Some type II neurones generate a low-threshold spike in response to the same protocol, which varies in amplitude and waveform between cells (Fig. 1C) (Tasker & Dudek, 1991). All cells were tested for the ability to express transient outward rectification and a low-threshold spike (LTS) using this protocol.

Figure 1
Type I, putative magnocellular neurones and type II, putative parvocellular neurones were distinguished on the basis of the expression of a transient outward rectification and a low-threshold spike

Action potentials were analysed by depolarising cells from potentials at or near resting potential to a predicted membrane potential of −30 mV with 250 ms current injections. The current step amplitude necessary to depolarise cells from resting potential to −30 mV was calculated based on the cells' input resistance (Rin) and membrane potential (Vm) using Ohm's law: Istep = (-30 mV –Vm)/Rin. The second action potential in the train elicited by this current step was chosen for detailed analysis since any contribution to the action potential waveform by IA would be maximised following the hyperpolarising afterpotential of the first action potential. Each parameter measured was averaged over three separate trials for each cell. Action potential amplitude was measured from the threshold to the peak of the action potential. The 10–90 % rise time, 10–90 % decay time, and half-amplitude duration of the action potential were measured using the Clampfit spike statistics function (pCLAMP 6, Axon Instruments). The hyperpolarising afterpotential amplitude was defined as the membrane hyperpolarisation measured from the spike threshold to the negative peak of the hyperpolarising afterpotential.

Statistical methods and curve fitting

Values are expressed as means ± standard errors of the mean. Comparisons between two different normally or non-normally distributed data sets were made using Student's unpaired t test or the Mann-Whitney rank sum test, respectively, and comparisons between normally or non-normally distributed data sets from repeated measures were made with Student's paired t test or the Wilcoxon signed rank test, respectively (SigmaStat 2.0, Jandel Scientific Software, San Rafael, CA, USA). Differences were considered significant at P < 0.05. Boltzmann and exponential fits were used to fit data plots and current traces using the Simplex fitting method (Clampfit, pCLAMP 6, Axon Instruments). The goodness-of-fit for fitted exponentials and sums of exponentials were determined in pSTAT (pCLAMP 6, Axon Instruments) using the Simplex maximum likelihood estimate technique.

Biocytin histochemistry

Following experiments, the slices were fixed in 4 % paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) at 4°C for 48 h, then washed in PBS with 20 % sucrose for 1 h. The fixed slices were sectioned to 20–25 μm on a cryostat and washed in PBS for 30 min. Biocytin-filled neurones were labelled by incubating the sections for 3 h in a solution containing 5 μg ml−1 streptavidin conjugated with 7-amino-4-methyl-coumarin-3-acetic acid (AMCA) (Vector Labs., Burlingame, CA, USA) and 0.5 % Triton-X in PBS. The sections were then washed for 10 min in PBS and examined under a fluorescence microscope using a UV/420K filter combination to find the biocytin-filled, AMCA-labelled neurones. Cell diameter measurements were made with an ocular micrometer.

RESULTS

In total, 75 type I and 55 type II neurones were analysed for this study. Type I neurones had a more hyperpolarised mean resting membrane potential than type II neurones (-61.0 ± 0.1 and −56.9 ± 1.1 mV, respectively; P < 0.01, Mann-Whitney rank sum test), but the resting input resistances did not differ between the two cell types (1203.0 ± 7.0 MΩ and 1227.6 ± 76.4 MΩ in type I and type II neurones, respectively).

Activation of IA

Potassium currents were examined in isolation (see Methods) in 36 type I neurones and in 24 type II neurones. Currents were elicited by stepping to a range of potentials (-70 mV to +20 mV) from a 200 ms conditioning step to either −100 mV or −50 mV. With conditioning steps to −100 mV, a low-threshold, transient component to the outward current was revealed that was not observed with conditioning steps to −50 mV. When currents generated from −50 mV were subtracted from the currents generated from −100 mV, the transient, A-type K+ current (IA) was isolated (Fig. 2A). The mean peak amplitude of IA was three times larger in type I neurones than in type II neurones (1127.5 ± 126.4 and 360.1 ± 56.3 pA, respectively, when measured with voltage steps from −100 mV to −25 mV; P < 0.01, Mann-Whitney rank sum test) (Fig. 2B). The current density was also significantly larger for IA in type I neurones than in type II neurones (131.1 ± 13.1 and 50.3 ± 7.8 pA pF−1, respectively, measured with the same steps; P < 0.01, Mann-Whitney rank sum test). The peak amplitude of IA at −25 mV ranged from 250 to 3600 pA in type I neurones and from 40 to 1100 pA in type II neurones (Fig. 2C).

Figure 2
The IA was smaller and had a more depolarised voltage dependence of activation in type II neurones than in type I neurones

The voltage dependence of activation of IA in PVN neurones was examined in detail in 20 type I and 15 type II neurones by plotting normalised chord conductance (gchord = I/(VtestVK,rev)), where VK,rev is the K+ reversal potential, against test step potential. The voltage dependence of activation of IA was shifted to more negative values in type I neurones compared to type II neurones (Fig. 2D). The activation threshold was −53.5 ± 0.9 and −46.1 ± 2.6 mV (P < 0.05, Mann-Whitney rank sum test) and the half-activation voltage was −25.2 ± 1.9 and −17.9 ± 2.0 mV (P < 0.05, unpaired t test) in type I and type II neurones, respectively. The current apparently peaked near 20 mV in type I neurones and positive to 20 mV in type II neurones, although IA elicited with large depolarising voltage steps was subject to voltage-clamp error and determination of peak may not be accurate.

The rate of activation of IA was examined by measuring the 10–90 % rise time in 18 type I and 14 type II neurones (Fig. 3A). The rate of activation of IA was faster in type I neurones than in type II neurones (1.8 ± 0.1 and 2.5 ± 0.2 ms, respectively, with test steps to −25 mV; P < 0.01, unpaired t test) and was voltage dependent in both cell groups, becoming faster at more depolarised potentials (Fig. 3B).

Figure 3
Slower rate of activation of IA in type II neurones than in type I neurones

Inactivation of IA

The voltage dependence of inactivation of IA in PVN neurones was examined in 17 type I and 12 type II neurones by delivering a constant test step to −25 mV from a range of conditioning steps (-120 to −45 mV, 200 ms), which removed a variable amount of inactivation of IA (Fig. 4A). The voltage dependence of inactivation of IA was shifted in the negative direction in type I neurones compared to that in type II neurones (Fig. 4B). The half-inactivation potential was −80.4 ± 9.3 and −67.2 ± 3.0 mV (P < 0.01) and complete inactivation occurred at −57.4 ± 2.1 and −49.8 ± 1.5 mV (P < 0.05, Student's unpaired t test) in type I neurones and type II neurones, respectively.

Figure 4
The voltage dependence of inactivation of IA was more depolarised in type II neurones than in type I neurones

The rate of inactivation of IA in PVN neurones was examined by fitting current traces with exponential functions to determine the inactivation time constant (Fig. 5A). The decay phase of IA in type II neurones was best fitted with a single exponential function (τ = 21.0 ± 2.9 ms with test steps to −25 mV; n = 23) (Fig. 5A). The inactivation kinetics of IA in type I neurones, on the other hand, was heterogeneous, 51 % of the cells expressing an IA with a single exponential decay and 49 % of the cells showing an IA with a double exponential decay. The IA with a single exponential decay in type I cells had a rate of inactivation (τ = 19.9 ± 0.7 ms with steps to −25 mV; n = 16) that was similar to that of the IA of type II cells (Fig. 5B). The IA inactivation rate in both cell types was voltage dependent, becoming faster with increasing depolarisation, and did not differ over the test step range examined in 13 type I and 12 type II neurones (Fig. 5B).

Figure 5
The rates of inactivation and recovery from inactivation of IA did not differ between type I and type II neurones

The recovery of IA from inactivation was examined in 15 type I and 10 type II neurones. From a holding potential of −50 mV, conditioning steps to −100 mV of increasing duration were performed, followed by a test step to −25 mV to activate the current (Fig. 5C). IA recovered from inactivation with a time dependence that was similar in type I and type II neurones, recovering 100 % of peak current amplitude with conditioning steps lasting 160.6 ± 10.5 and 132.0 ± 13.0 ms, respectively (Fig. 5C). A plot of the mean normalised peak current versus the conditioning step duration was best fitted by a single exponential function, the time constant of which did not differ between type I (34.9 ± 5.1 ms, n = 15) and type II neurones (32.1 ± 4.6 ms, n = 10) (Fig. 5D).

Sensitivity of IA to 4-AP

Sensitivity to the K+ channel blocker 4-aminopyridine (4-AP) is a characteristic feature of IA in many cells (Rudy, 1988), including type I PVN neurones (Li & Ferguson, 1996). The IA in type I neurones was reversibly reduced by 5 mm 4-AP with variable effectiveness (range: 11–60 % block, mean: 41.1 ± 7.0 % block, with steps from −100 mV to −25 mV; n = 8; P < 0.05, Student's paired t test) (Fig. 6A and C). The effect of 4-AP on IA in type II neurones was also variable, ranging from 0 to 92 % block. IA was reversibly blocked by more than 40 % in 6 of 12 type II neurones (range: 42.0–92.1 % block, mean 69.2 ± 7.3 %, with steps from −100 mV to −25 mV; P < 0.05, Wilcoxon signed rank test); these cells were referred to as 4-AP sensitive. IA was relatively unaffected by 5 mm 4-AP (i.e. < 20 % block) in the other 6 type II neurones (range: 0–19.1 % block; mean: 8.2 ± 3.1 %; with steps from −100 mV to −25 mV; n.s.), referred to as 4-AP insensitive (Fig. 6B and C).

Figure 6
Sensitivity of IA to block by 4-AP

Activation of IT

Under conditions of partial isolation of Ca2+ currents (see Methods), depolarising voltage steps from −100 mV elicited an inward current in all of the 10 type I and 19 type II neurones tested. In some neurones the current consisted of two components, a low-threshold, small-amplitude current, and a high-threshold, large-amplitude current. In other neurones only a single high-threshold Ca2+ current was detected (data not shown). The low-threshold current, or T-type Ca2+ current (IT), peaked at potentials below the threshold for activation of the high-threshold current, which allowed us to study IT in isolation from high-threshold Ca2+ currents. High-threshold Ca2+ currents were not studied here.

IT was activated with depolarising test steps to between −70 and −20 mV delivered from a 500 ms conditioning step to −100 mV, which removed inactivation of the current (Fig. 7A). Fourteen of nineteen type II neurones (74 %) expressed an IT, whereas a small IT was detected in only 2 of 10 type I neurones (20 %). The peak IT amplitude ranged from −90 to −2200 pA (-402.5 ± 166.9 pA) in type II neurones, and was −40 pA and −60 pA in the two type I neurones (Fig. 7B). However, the incomplete block of K+ currents (see Methods) and the overlapping voltage dependence of low-threshold Ca2+ and K+ currents may have prevented detection of a smaller IT in type I neurones. The unblocked IA in type II neurones had a threshold that was positive to the peak of IT in 17 of the 19 cells analysed in medium containing K+ channel blockers, making it possible to study IT in isolation in these cells. The IT in type II neurones activated at a threshold of −59.1 ± 1.2 mV and peaked at −32.6 ± 1.7 mV (Fig. 7C).

Figure 7
IT was expressed predominantly in type II neurones

The activation rate of IT was examined in 9 type II neurones by measuring the 10–90 % rise time. The activation rate of IT was voltage dependent, becoming faster with increasing depolarisation (Fig. 7D). The 10–90 % rise time ranged from 18.0 ± 3.7 ms to 5.4 ± 1.7 ms with voltage steps ranging from −50 to −30 mV following a 500 ms conditioning step to −100 mV.

Inactivation of IT

The voltage dependence of inactivation of IT was examined in 13 type II neurones with a test step to −40 mV from a range of conditioning step potentials (-100 to −20 mV, 500 ms) (Fig. 8A). IT was completely inactivated at −52.2 ± 2.2 mV and half-inactivated at −66.9 ± 2.2 mV.

Figure 8
Inactivation properties of IT in type II neurones

The rate of inactivation of IT was measured in 9 type II neurones by fitting exponential functions to the decay phase of the IT traces to determine the inactivation time constant. The inactivation time constant ranged from 36.3 ± 10.8 to 11.7 ± 0.9 ms (voltage steps from −100 mV to between −50 and −30 mV, respectively), and was voltage dependent, becoming faster with increasing depolarisation (Fig. 8B).

The rate of recovery from inactivation of IT was examined in 6 type II neurones with conditioning steps to −100 mV of increasing duration from a −50 mV holding potential, followed by a test step to −40 mV to activate the current. IT recovered 100 % of its peak amplitude with conditioning steps lasting 523.3 ± 14.3 ms (Fig. 8C).

Sensitivity of IT to nickel and cadmium

T-type Ca2+ currents are generally characterised by a higher sensitivity to block by nickel than by cadmium (Fox et al. 1987; Huguenard 1996). The IT in type II neurones was reversibly inhibited by 66.8 ± 10.9 % in 100 μM nickel chloride, measured with steps from −100 mV to −35 mV (n = 7; P < 0.05), but was unaffected by 50 μM cadmium chloride (n = 6, Wilcoxon signed rank test) (Fig. 9).

Figure 9
IT was blocked by nickel

IK

Depolarising voltage steps from a holding potential of −50 mV elicited a sustained outward current that resembled the delayed rectifier K+ current (IK) in both type I (n = 12) and type II (n = 14) PVN neurones (Fig. 10A). IK activated much more slowly than IA, reaching its peak with a latency of about 100 ms in both cell groups (99.2 ± 14.6 mV in type I neurones and 101.5 ± 14.6 mV in type II neurones). Although the distribution of IK amplitudes was similar in both cell groups, the mean amplitude of IK was larger in type II neurones than in type I neurones (586.4 ± 76.0 pA vs. 389.7 ± 39.3 pA, respectively, measured at the end of a 150 ms voltage step to −10 mV from −50 mV; P < 0.05, Mann-Whitney rank sum test) (Fig. 10B). The current activated at a similar threshold (-27.7 ± 1.7 mV in type I neurones and −28.8 ± 1.1 mV in type II neurones) and peaked above 20 mV in both cell types (Fig. 10C).

Figure 10
The delayed rectifier current (IK) was similar in type I and type II neurones

The IK in both cell groups was equally sensitive to block by 5 mm 4-AP, being reduced by 32.7 ± 6.1 % in type I neurones (n = 7; P < 0.05) and by 36.5 ± 6.4 % in type II neurones (n = 11; P < 0.05, Student's paired t test) (measured at the end of a 150 ms step to −10 mV from −50 mV; data not shown).

Electrophysiological properties

The observed differences in the amplitude and voltage dependence of IA and IT in type I and type II neurones suggest that these currents play different roles in the firing properties of the two cell types. The respective roles of these currents in the generation of transient outward rectification in type I neurones, the low-threshold spike in type II neurones, and the action potential waveform in both cell types were examined with current-clamp experiments.

When type I neurones were depolarised from a membrane potential near −90 mV, they exhibited a delay to firing of the first action potential due to the transient outward rectification. Application of 5 mm 4-AP, which reduced IA by 41.1 ± 7.0 % in type I neurones, also decreased the transient outward rectification and reduced the delay to the first spike by 46.2 ± 10.3 % (n = 5; P < 0.05, Student's paired t test) (Fig. 11A). Additionally, the IA amplitude elicited in type I neurones with a voltage step to −25 mV from a holding potential of −100 mV was correlated with the delay to the first action potential evoked with a +50 pA current pulse from −90 mV to −100 mV (r = 0.82), suggesting a role for IA in the generation of the transient outward rectification (Fig. 11B).

Figure 11
Correlation of IA with transient outward rectification in type I neurones and IT with the low-threshold spike in type II neurones

Preliminary studies have suggested that the low-threshold spike in type II neurones is generated by the activation of an IT (Luther & Tasker, 1997). Application of 100 μM nickel chloride, which reduced the IT by 66.8 ± 10.9 %, also reduced the low-threshold spike amplitude by 76.7 ± 7.8 % (n = 10; P < 0.01, paired t test) (Fig. 11C). Application of 50 μM cadmium chloride affected neither the IT nor the low-threshold spike (n = 5) (data not shown). Additionally, the variability in IT corresponded qualitatively to the variability in low-threshold spike amplitude observed in type II neurones in current clamp (e.g. cells with a large-amplitude IT expressed a large-amplitude low-threshold spike). The peak amplitude of IT was significantly larger in type II neurones that expressed an LTS (-560.7 ± 276.6 pA, n = 7) than in type II neurones that did not (-100.6 ± 40.4 pA, n = 9; P < 0.05, Mann-Whitney rank sum test). The amplitude of IT elicited in type II neurones at −40 mV correlated to the amplitude of the maximal low-threshold spike generated when cells were depolarised from a membrane potential near −90 mV (r = 0.77; Fig. 11D). These findings together suggest a role for IT in the generation of the low-threshold spike in type II neurones.

IA and IK are believed to be important in regulating action potential repolarisation and in the timing of repetitive firing (Rudy, 1988). The action potential waveform and firing frequency elicited at resting membrane potential were examined and compared between 44 type I and 17 type II neurones, and were found to be similar between the two cell groups (Table 1). The action potential thresholds, amplitudes, and rise times of the two cell groups did not differ, but type I neurones had longer-lasting action potentials than type II neurones, as indicated by the slower decay and longer half-amplitude duration. Type II neurones generated a larger post-spike hyperpolarising afterpotential and exhibited a lower firing frequency than type I neurones.

Table 1
Properties of action potentials of PVN neurones

DISCUSSION

We conducted patch-clamp recordings of the A-type K+ current (IA), T-type Ca2+ current (IT), and delayed rectifier K+ current (IK) in type I and type II PVN neurones in order to understand the roles that voltage-gated currents play in the generation of the different electrophysiological properties of PVN neuronal subtypes. The transient outward currents recorded in both type I and type II PVN neurones resembled IA in terms of their hyperpolarised activation threshold, rapid activation and inactivation rates, steady-state inactivation at hyperpolarised potentials, and sensitivity to 4-AP (Rogawski, 1985; Rudy, 1988; Dolly & Parcej, 1996). The low-threshold, transient Ca2+ currents that we observed predominantly in type II neurones were considered to be IT because of their relatively hyperpolarised activation threshold, fast rate of inactivation, steady-state inactivation at hyperpolarised potentials, and their sensitivity to block by nickel (Fox et al. 1987; Huguenard 1996). The sustained K+ currents elicited in both type I and type II PVN neurones, except for their sensitivity to 4-AP, were identical to the delayed rectifier current (IK) in their depolarised activation threshold and low level of inactivation (Rudy, 1988; Dolly & Parcej, 1996).

Voltage-clamp considerations

Recordings from intact cells in brain slices are often subject to voltage-clamp error and dendritic filtering, which can lead to erroneous measurement of the voltage and kinetic properties of the recorded cells. The recordings included in our analyses of voltage-dependent currents in type I and type II neurones were selected on the basis of relatively rigorous criteria, including a series resistance of less than 20 MΩ, compared with a mean input resistance of ~1.2 GΩ, and consistent rates of activation and inactivation of IA and IT with varying levels of inactivation of the currents (see Methods). Additionally, support for the accuracy of our voltage-clamp data comes from studies performed in acutely dissociated magnocellular neurones of the supraoptic nucleus, which express an IA with voltage and kinetic properties similar to those reported here for type I neurones (Cobbett et al. 1989; Hlubek & Cobbett, 1997). Further, the voltage and kinetic properties of IT in type II PVN neurones reported here are similar to those measured in dissociated neurones from a variety of different brain tissues (Huguenard, 1996).

The morphologies of type I and type II neurones are typically bipolar, with relatively scant secondary dendrites (Rho & Swanson, 1989; Tasker & Dudek, 1991). The mean whole-cell capacitance of type I neurones measured during our recordings was 8.9 ± 0.67 pF and that of type II neurones was 6.6 ± 0.55 pF. We calculated the somatic surface area of biocytin-filled, streptavidin-labelled type I and type II cells by measuring the long and short axes of the cells and assuming an ovoid shape (i.e. A = π(r1×r2)). The calculated somatic surface area of type I neurones was 943.1 ± 75.7 μm2 and that of type II neurones was 672.3 ± 62.1 μm2. The specific capacitance, therefore, was 0.94 μF cm−2 for type I neurones and 0.98 μF cm−2 for type II neurones. These values differ from the accepted value of 1 μF cm−2 by 6 and 2 %, respectively. Since only the somatic surface area was used to calculate the specific capacitance of the two cell types, this suggests that the uncompensated whole-cell capacitance was probably due to uncompensated capacitance of the dendritic membrane, which implies that the currents we recorded were primarily somatic.

Most comparisons of kinetics and voltage dependence were made with voltage steps to −25 mV (i.e. 10–90 % rise time, inactivation time constant, and inactivation voltage dependence), a potential at which the voltage error was calculated to be relatively small (~2 mV). Some measures of the voltage dependence of IA were taken from the Boltzmann fits of the conductance-voltage relationships (e.g. half-activation voltage), and would therefore be subject to greater voltage errors due to the inclusion of currents elicited at more positive potentials. However, the differences between type I and type II neurones in these measures were similar to the difference in the threshold measurements (i.e. ~10 mV), which was not derived from the Boltzmann curves, but empirically. If we make the assumption that the threshold measurements are reasonably accurate because they were made at potentials relatively close to the resting potential, then the fact that these measurements and other measures of voltage dependence differ proportionally between the two cell groups suggests that the other measures are reasonably accurate, also. Additionally, we found a similar difference in the voltage dependence of inactivation of IA (i.e. 7–13 mV) between type I and type II neurones, which was calculated with test steps to −25 mV. This similarity in the voltage dependence of activation and that of inactivation is consistent with the coupling of the two processes for IA (Zagotta & Aldrich, 1990), and supports the accuracy of our IA activation voltage measures. Thus, while it may not be possible to completely eliminate voltage error in recordings from intact cells, we believe that the voltage and kinetic properties of the IA and the IT that we present here are reasonably accurate, and that the difference in voltage dependence of the IA between the two cell types is real.

Differential expression of potassium and calcium currents in PVN neurones

The amplitude of IA recorded in type I neurones was much larger than that in type II neurones. This difference is probably not due to the smaller somatic area of type II neurones since the current density of IA was also significantly larger in type I neurones than in type II neurones. Thus, the difference in the IA amplitudes between the two cell types is probably due to differences in the density of IA channels in the cell membrane. Another possibility is that the IA channels differ in single channel conductance between type I and type II neurones. Single channel recordings of IA in rat nodose neurones, avian sensory neurones, Drosophila neurones, rat posterior pituitary nerve terminals, and cultured rat neonatal hypothalamic neurones have revealed single IA channel conductances varying from 5 to 33 pS (Cooper & Shrier, 1985; Kasai et al. 1986; Solc et al. 1987; Florio et al. 1990; Bielefeldt et al. 1992; Wang et al. 1997), suggesting a wide variation in IA channel properties.

The voltage dependence of activation and inactivation of IA was approximately 10 mV more positive in type II neurones than in type I neurones. It is unlikely that this was due to variability in the voltage control of the two cell types because the rates of activation and inactivation of the IA measured at conditioning steps ranging from −120 to −60 mV did not differ, indicating good voltage control of both type I and type II neurones. A depolarised activation threshold for IA has been reported previously in neocortical neurones (-25 mV) (Andreasen & Hablitz, 1992; Zhou & Hablitz, 1996) and dorsal cochlear nucleus neurones (-45 mV) (Kanold & Manis, 1999). Expression of Kv3.4 K+ channels in Xenopus oocytes leads to a quickly activating and inactivating K+ current with a threshold above −20 mV (Schröter et al. 1991; Rettig et al. 1992). Thus the different activation and inactivation voltage dependence of IA in type I and type II neurones could be due to differential expression of IA channel subtypes. Additionally, Petersen & Nerbonne (1999) have shown that the same channels expressed in different cell lines can generate A-type currents that differ in kinetic properties and voltage dependence, suggesting that the cellular environment may influence the current. Thus, differences in the voltage dependence of IA may also arise from differential regulation by second messengers or post-translational processing of the IA channels between type I and type II neurones.

Our results showed a large variability in the sensitivity of IA in type I and type II PVN neurones to block by 5 mm 4-AP. Kanold & Manis (1999) reported a similar variability in the sensitivity of an A-type current to 1–2 mm 4-AP (12–100 % block) in pyramidal cells of the dorsal cochlear nucleus. Different IA channel subtypes have been shown to have variable sensitivities to 4-AP, suggesting that the variability in 4-AP sensitivity among PVN neurones could arise from differences in the subtypes of IA channels expressed (Rudy, 1988; Pongs, 1992; Dolly & Parcej, 1996). Type II PVN neurones comprise diverse neuronal phenotypes (i.e. parvocellular neurosecretory and non-neurosecretory cells), which could explain the variable functional expression and 4-AP sensitivity of IA in these neurones. Type I neurones, on the other hand, are relatively homogeneous (i.e. exclusively magnocellular neurosecretory cells), which may account for the lower variability of the IA observed in these neurones.

Other studies have shown that IA in magnocellular neurones of the supraoptic nucleus is dependent on extracellular Ca2+ (Bourque, 1988; Hlubek & Cobbett, 1997). We found that removal of extracellular Ca2+ and addition of 200 μM Cd2+ had no effect on the amplitude or voltage dependence of IA in type I PVN neurones (n = 4), which is in agreement with a previous report (Li & Ferguson, 1996). We did not examine the Ca2+ dependence of IA in type II neurones.

We found that IT was expressed in most type II neurones (14/19 cells), and in only 2/10 type I neurones under our recording conditions. The peak amplitude of IT was also larger in type II neurones than in the type I neurones. Other studies have found that magnocellular neurones of the hypothalamic supraoptic nucleus express a T-type Ca2+ current (Erickson et al. 1993; Fisher & Bourque, 1995; Seifert & Bourque, 1998). This discrepancy may be due to the incomplete block of K+ currents in our experiments, which could have caused residual K+ currents to obscure a small IT in type I neurones (i.e. less than about 30 pA), or may be the result of physiological differences between magnocellular neurones of the supraoptic nucleus and those of the PVN.

Both type I and type II neurones expressed an IK that was similar with regard to amplitude, activation threshold, activation rate, and sensitivity to block by 4-AP. Although IK in type II neurones had a somewhat larger amplitude than that in type I neurones, this difference is not likely to contribute substantially to the observed differences in electrophysiological properties between the two cell types.

Electrophysiological differences between PVN neurones are due to differential current expression

The differences in expression and voltage dependence of IA and IT between type I and type II PVN neurones can explain the different electrophysiological properties of these cell types reported by Tasker & Dudek (1991). Type I neurones are characterised by the expression of a transient outward rectification that causes a delay to action potential generation. IA in these neurones activates more than 10 mV negative to the action potential threshold, and is more than 20 % activated at action potential threshold. Thus, IA would be predicted to delay membrane depolarisation towards spike threshold. The large-amplitude and hyperpolarised voltage dependence of IA in type I neurones, therefore, can account for the pronounced transient outward rectification observed in these neurones. Indeed, application of 5 mm 4-AP reduced both IA and the transient outward rectification in type I neurones. The role of IA in the expression of transient outward rectification in type I neurones is therefore very similar to that seen in magnocellular neurones of the supraoptic nucleus (Fisher et al. 1998).

Type II neurones do not express a transient outward rectification (Tasker & Dudek, 1991). This lack of a transient outward rectification can be explained by the depolarised activation voltage dependence and the relatively small amplitude of IA expressed in these cells, as well as by the presence of an IT, which has a voltage dependence similar to that of IA. In type II neurones, IA begins to activate near the action potential threshold and would be predicted to peak during the action potential, facilitating repolarisation and contributing to the post-spike hyperpolarising afterpotential. This is supported by the observation that action potentials in type II neurones decay rapidly and have a large hyperpolarising afterpotential. IK is less likely to underlie the differences in action potential duration and hyperpolarising afterpotential amplitude since it was similar in its voltage dependence in type I and type II neurones. Variability in high-threshold Ca2+ currents may also account for differences in the action potential waveform in type I and type II neurones (Mason & Leng, 1984), although further experiments are necessary to confirm this. The lower firing frequency of type II neurones compared to type I neurones is probably due to differences in the expression of Ca2+-dependent K+ currents, which we did not analyse here, since IA and IK in type II neurones would deactivate too quickly during the post-spike hyperpolarising afterpotential to contribute significantly to the interspike interval.

IT is expressed predominantly in type II neurones in the PVN and is correlated with the expression of an LTS. The peak amplitude of IT was significantly larger in type II neurones that expressed an LTS than in cells that did not, and both the LTS and IT were reduced by 100 μM nickel chloride, but were unaffected by 50 μM cadmium chloride. We found that type I neurones generally did not express a detectable IT (8/10 cells) and that none of the type I neurones expressed an LTS.

IA, IT and IK have all been implicated in the control of temporal firing patterns and firing frequency in different areas of the nervous system (Jahnsen & Llinás, 1984; Rogawski, 1985; Rudy, 1988; Huguenard, 1996), including the hypothalamus (Fisher et al. 1998). Temporal firing patterns of hypothalamic neurosecretory neurones are particularly critical in the control of pituitary hormone output (Lincoln & Wakerly, 1974; Dutton & Dyball, 1979; Bicknell & Leng, 1981; Summerlee & Lincoln, 1981; Hastings, 1991). Bursting patterns of activity are responsible for the pulsatile release of the neurohypophyseal hormones, oxytocin and vasopressin, as well as that of the hypophysiotrophic hormones that control anterior pituitary hormone secretion. Differences in IA, IT and IK are likely to play a critical role in determining the specific patterned activities that control neurosecretory and autonomic output from subpopulations of PVN neurones.

Acknowledgments

We would like to thank Kriszta Szabó and Katalin Halmos for their technical assistance. This work was supported by grant NS34926 from the National Institutes of Health.

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