Biophysical Characterization of Rat Caudal Hypothalamic Neurons: Calcium Channel Contribution to Excitability

Yi-Ping Fan, Eric M. Horn, and Tony G. Waldrop

Department of Molecular and Integrative Physiology and Neuroscience Program, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Fan, Yi-Ping, Eric M. Horn, and Tony G. Waldrop. Biophysical Characterization of Rat Caudal Hypothalamic Neurons: Calcium Channel Contribution to Excitability. J. Neurophysiol. 84: 2896-2903, 2000. Neurons in the caudal hypothalamus (CH) are responsible for the modulation of various processes including respiratory and cardiovascular output. Previous results from this and other laboratories have demonstrated in vivo that these neurons have firing rhythms matched to the respiratory and cardiovascular cycles. The goal of the present study was to characterize the biophysical properties of neurons in the CH with particular emphasis in those properties responsible for rhythmic firing behavior. Whole cell, patch-clamped CH neurons displayed a resting membrane potential of -58.0 ± 1.1 mV and an input resistance of 319.3 ± 16.6 MOmega when recorded in current-clamp mode in an in vitro brain slice preparation. A large proportion of these neurons displayed postinhibitory rebound (PIR) that was dependent on the duration and magnitude of hyperpolarizing current as well as the resting membrane potential of the cell. Furthermore these neurons discharged tonically in response to a depolarizing current pulse at a depolarized resting membrane potential (more positive than -65 mV) but switched to a rapid burst of firing to the same stimulus when the resting membrane potential was lowered. The PIR observed in these neurons was calcium dependent as demonstrated by the ability to block its amplitude by perfusion of Ca2+-free bath solution or by application of Ni2+ (0.3-0.5 mM) or nifedipine (10 µM). These properties suggest that low-voltage-activated (LVA) calcium current is involved in the PIR and bursting firing of these CH neurons. In addition, high-voltage-activated calcium responses were detected after blockade of outward potassium current or in Ba2+-replacement solution. In addition, almost all of the CH neurons studied showed spike frequency adaptation that was decreased following Ca2+ removal, indicating the involvement of Ca2+-dependent K+ current (IK,Ca) in these cells. In conclusion, CH neurons have at least two different types of calcium currents that contribute to their excitability; the dominant current is the LVA or T-type. This LVA current appears to play a significant role in the bursting characteristics that may underlie the rhythmic firing of CH neurons.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The caudal hypothalamus (CH) in the rat is bordered medially by the third ventricle and laterally by the lateral hypothalamus (Paxinos and Watson 1986; Veazey et al. 1982). The function of neurons in this area has mainly been determined by electrical or chemical stimulation, which results in increases of respiratory output, sympathetic nerve discharge, arterial pressure, and heart rate (DiMicco and Abshire 1987; Kabat 1936; Spencer et al. 1990; Waldrop et al. 1988). In addition, results from this laboratory have shown that neurons in the CH have firing rhythms temporally related to either the cardiac or respiratory cycles in rats and cats (Barman 1990; Dillon and Waldrop 1993; Ryan and Waldrop 1995).

Of particular interest is the role that neurons in this region play in pathophysiological processes such as hypertension. Studies in a rodent hypertension model, the spontaneously hypertensive rat (SHR), have determined that sympathetic-related neurons in this region have a higher discharge rate than normotensive rats (Shonis and Waldrop 1993). In addition, a reduction in the GABAergic function of CH neurons in hypertensive rats compared with normotensive rats is strongly associated with an increased cardiovascular tone initiated by neurons in this region (Shonis et al. 1993). Thus the increased sympathetic tone in the SHR, which may play a role in the hypertensive state in these animals, is possibly due to an increased activity of CH neurons (Judy et al. 1976).

The CH has also been linked to the control of other functions including locomotion, antinociception, sleep/wakefulness, and the modulation of the hippocampal electroencephalogram (Bland et al. 1995; Carstens 1982; Eldridge et al. 1981; Nitz and Siegel 1996). For example, lesions in the caudal hypothalamus abolish or attenuate hippocampal theta activity (Anchel and Lindsley 1972; Robinson and Whishaw 1974), and high-frequency (100 Hz) stimulation in the CH has been shown to be particularly effective in eliciting hippocampal theta-activity (Bland and Vanderwolf 1972). Recent evidence indicates that theta-related tonic discharge in the CH is important in controlling hippocampal theta activity, which may be particularly important in hippocampal processing of spatial information (Kirk et al. 1996; Skaggs and McNaughton 1996).

Despite the extensive morphological and functional studies of the CH, little is known about the basic biophysical properties of neurons in this area. Of particular importance is the type of currents inherent to these neurons that underlie their rhythmic firing behavior. In view of a key role for the CH in various physiological and pathological states, we have analyzed the basic membrane and activity-related properties of neurons in the CH by using whole cell recordings in rat brain slices.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation of brain slices

All animal protocols and procedures described were performed in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Guidelines of the Laboratory Animal Care Advisory Committee at the University of Illinois. Male Sprague-Dawley rats (3-4 wk old, Harlan, Indianapolis, IN) were rapidly decapitated, and the brain was quickly removed and submerged in ice-cold artificial cerebrospinal fluid (ACSF) solution (see following text for composition). The section of the brain containing the caudal hypothalamus was cut into 400-µm-thick coronal slices with a tissue chopper. The slices were then transferred to an interface style recording chamber in which they were continuously perfused with a warmed (35°C), oxygenated ASCF solution. A 100% humidified gas (95% O2-5% CO2) flowed continuously over the slices, and the temperature was set at 35°C using a servo-controlled heating bath (Haake). The slices were allowed to stabilize for at least 1 h before electrical recording commenced.

Solutions and drugs

The composition of the ASCF solution was (in mM) 116 NaCl, 5.4 KCl, 0.81 MgSO4, 1.8 CaCl2, 1 NaH2PO4, 26.2 NaHCO3, 15 N-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and 10 glucose. All solutions were gassed with a mixture of 95% O2-5% CO2 and maintained at pH of 7.35-7.37. The osmolality was adjusted to 295-307 mOsm using a vapor pressure osmometer (Wescor). Due to the possible modulatory effects on several kinds of voltage-gated K+ channels, the common calcium channel blockers, such as Co2+, Cd2+, or Mn2+, were not used (Mayer and Sugiyama 1988). For Ca2+-free solution, Ca2+ was omitted and Mg2+ was raised to 5 mM. In a few experiments, 0.5 mM ethylene glycol-bis(beta -aminoethylether)-N, N,N',N'-tetra-acetic acid (EGTA) was added to chelate the residual calcium. In Ca2+-replacement solution, CaCl2 was replaced with 1.8 mM BaCl2 to enhance the current through specific calcium channels. In addition, some experiments utilized the specific low-voltage-activated (LVA) calcium channel blocker NiCl2 (0.3-0.5 mM). When Ba2+ or Ni2+ was added to the solution, NaH2PO4 and MgSO4 were omitted to prevent precipitation. Other drugs used included 1-2 µM tetrodotoxin (TTX, a sodium channel blocker), 10 mM tetraethylammonium chloride (TEA, a potassium channel blocker), 10 µM nifedipine (a specific L-type calcium channel blocker), 1 mM gamma -amino-n-butyric acid (GABA), and 0.1 mM bicuculline (a specific GABAA receptor antagonist). Concentrated GABA, TEA, and NiCl2 were first dissolved in perfusion solution, while concentrated TTX was first dissolved in citrate buffer. They were then added to the perfusion solution to reach the final concentration. When bicuculline was used, it was added to the GABA-containing solution. Nifedipine was dissolved in absolute ethanol and added to the perfusion solution to give the final concentration with a final alcohol content of 0.1%; a control solution contained only 0.1% alcohol. Drug and solution changes were performed by switching the inflow to reservoirs containing the desired test solution, and at least 15 min were allowed for equilibration before the data were collected or the effect of drug reached its plateau. All drugs were obtained from Sigma Chemical (St. Louis, MO).

Recording and data analysis

Blind, whole cell patch-clamp recordings were made using the technique described by Blanton et al. (1989). When filled with pipette solution [(in mM) 135 potassium gluconate, 5 KCl, 2 MgCl2, and 10 HEPES, pH 7.2 buffered with KOH, osmolality 275 mOsm], the DC resistance of the pipette was 4-7 MOmega . Recordings were obtained with an Axoclamp 2A (Axon Instruments) patch-clamp amplifier operating in bridge mode with the low-pass Bessel filter set at 3 kHz. Data were acquired on-line at 10 kHz for off-line analysis using analysis software (PClamp 5.5 and 6.0, Axon Instruments). Whole cell patch-clamp configuration was obtained following the formation of at least a 2-GOmega seal. Data were not collected for at least 3 min after obtaining the whole cell configuration to permit the pipette and cell interior to equilibrate.

In most neurons, several different protocols were utilized to obtain biophysical properties. To overcome any effects of the patch pipette on the membrane potential and thus the basal membrane properties, the membrane potential for each cell was set at -60 mV. Thus the resting membrane potentials of quiescent cells were recorded and then set at -60 mV, while the membrane potentials of spontaneously firing neurons were simply adjusted to -60 mV to inhibit spike activity. The input resistance (Rin) was measured by injecting a hyperpolarizing current pulse (-100 pA, 150 ms) and measuring the voltage response. The membrane time constants were calculated by injecting a hyperpolarizing current pulse (-100 pA, 150 ms) and fitting the induced membrane voltage transient to the exponential charging function [V = A1 × exp(-t/tau 0) + A2 × exp(-t/tau 1) + C] using the Chebyshev fitting method in the software package, where tau 0 represents the longest or somatic membrane time constant, tau 1 represents a shorter equalizing time constant, and C is a constant (Rall 1969). According to the relationship tau 0 RmCm, where Rm and Cm are the specific membrane resistance and capacitance respectively, we obtained Rm from tau 0 (Cm was assumed to be 1 µF/cm2). The rheobase, which was obtained with a 50-ms current pulse, was defined as the minimum current that produced an action potential (AP) in 100% of five trials. Depolarizing pulses of variable amplitude and duration were used to determine the following AP properties: AP amplitude (APamp), duration at half the AP peak (APW1/2) and afterhyperpolarization potential (AHP, postspike nadir subtracted from the prespike membrane potential).

Analysis of firing adaptation was determined by a procedure modified from a prior study in a separate laboratory (Viana et al. 1995). The procedure was modified because the strong adaptation of caudal hypothalamic neurons created difficulties in determining adaptation when a long (2 s) current pulse was employed. Thus repetitive firing was evoked by a series of 400-ms depolarizing current injections of increasing amplitude. The instantaneous firing frequency (fonset) was obtained from the first interspike interval. The maintained firing frequency (fmain) was the frequency of APs during the last 300 ms of the 400-ms depolarization. Frequency adaptation or acceleration was quantified by a spike frequency index (SFI), where SFI = fonset/fmain. For the analysis, if the SFI > 1, the firing pattern was considered to show adaptation; if the SFI < 0.1, the firing pattern was considered to show acceleration; and if the SRF = 1, there was no modulation of firing pattern.

A subset of neurons was tested for their pharmacological properties by first recording in ACSF, then adding a particular agonist or antagonist to the ACSF (see preceding text), and finally washing with ACSF. When the membrane potential was changed due to a drug or ion replacement, it was kept at the original resting potential using injection of bias current. In some neurons, a hypoxic stimulus (10% O2-5% CO2-85% N2) was presented by altering the gas flowing in the chamber for 90 s, as previously described (Dillon and Waldrop 1992).

The liquid junction potential (-12 mV) was measured using a separate set of experiments as previously described and subtracted from the recorded membrane potential (Horn et al. 1999). After a successful recording (membrane potential at least -40 mV and AP passing through 0), the position of the pipette was noted on a standard series of diagrams of the brain slices (Paxinos and Watson 1986). The recording depth varied between 5 and 230 µm, and all the data are presented as means ± SE. Statistical significance was determined by Student's paired and unpaired t-tests with P < 0.05 deemed significant.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Basic membrane properties of CH neurons

All cells were recorded within the caudal hypothalamus as identified using highly recognizable visual landmarks (mamillothalamic tracts, fornix, 3rd ventricle, etc.) according to a rat stereotaxic atlas (Paxinos and Watson 1986). The basal electrophysiological properties of the 50 recorded CH neurons are summarized in Table 1. Of the 50 cells tested, 33 were quiescent at their resting membrane potential and the other 17 spontaneously fired APs, thus only the quiescent cells were used for the determination of the average resting membrane potential in Table 1. For the spontaneously active cells, the resting membrane potential was adjusted to approximately -60 mV to calculate the input resistance (Rin) and time constants (tau 0 and tau 1). No differences were observed in any of the basal or active membrane properties between the quiescent and spontaneously active cells.


                              
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Table 1. Basic electrophysiological properties of caudal hypothalamic neurons

Postinhibitory rebound and burst firing of CH neurons

Postinhibitory rebound (PIR) refers to transient depolarization immediately after release from hyperpolarization that is manifested as a subthreshold depolarization, an AP or a burst of APs (Huguenard 1996; Jiang et al. 1999). In CH neurons, two different kinds of PIR were observed in 28/50 cells studied (Fig. 1), and it is important to note that there were no observed differences in basal membrane properties between cells that displayed PIR and those that did not display PIR. The first type of PIR (type I) was the most prevalent type (23 of the 28 cells exhibiting PIR were type I) and consisted of a burst of APs following the end of the hyperpolarizing current pulse (Fig. 1A, left). These APs were eliminated after perfusion with TTX, but a delayed transient depolarization (DTD) remained that increased in amplitude during perfusion with Ba2+ replacement solution (Fig. 1A, right). The PIR in type I cells was strongly dependent on the hyperpolarizing potential amplitude and duration as well as the level of prepulse membrane potential (see following text). The second type of PIR (type II, 5/28) showed a single AP following the end of the hyperpolarization pulse (Fig. 1B, left) that was eliminated when the cell was perfused with TTX. In contrast to the type I response, there was no DTD observed in these neurons following the hyperpolarizing pulse during TTX perfusion (Fig. 1B, right). Since the type I PIR was the most prevalent, the rest of the results refer to this type unless otherwise noted.



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Fig. 1. Two types of postinhibitory rebound responses. Type I (A), a burst discharge evoked at the end of a hyperpolarizing pulse (150 ms, -100 pA; left). In the presence of 1 µM tetrodotoxin (TTX), a delayed transient depolarization was exhibited at the end of hyperpolarizing pulse, which became larger and longer in the Ba2+-replacement solution (right). Type II (B), a single action potential evoked at the end of hyperpolarizing pulse (150 ms, -100 pA; left), which was eliminated in the presence of 1 µM TTX (right); no delayed transient depolarization was seen.

We found that type I PIR was affected by three factors, amplitude of the hyperpolarizing pulse, duration of the hyperpolarizing pulse, and prepulse membrane potential. As shown in Fig. 2A, a minimum amount of hyperpolarizing current at a constant duration was required to trigger the PIR. When the amplitude was kept constant in another cell, the duration of the hyperpolarizing pulse was directly related to its ability to elicit PIR (Fig. 2B). While increases in the amplitude of the hyperpolarizing pulse caused a graded enhancement of the PIR response, additional increases in the duration of the hyperpolarizing pulse had no further effect on the PIR once elicited. In general, the PIR was observed at the normal membrane potential for each cell but would completely disappear when the resting membrane potential was lowered (usually below -70 mV) prior to the hyperpolarizing pulse (Fig. 2C).



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Fig. 2. Postinhibitory rebound dependence on the voltage and duration of the hyperpolarizing pulse and the level of the prepulse potential in type I neurons. A: voltage dependence of activation on rebound response, note graded response. B: duration dependence of activation on rebound response, note lack of graded response. C: prepulse membrane potential dependence of activation on rebound response. The calibration bar applies to all panels.

In all of those neurons tested that showed Type I PIR, the pattern of AP firing in response to a depolarizing pulse was strongly influenced by the resting membrane potential, as illustrated in Fig. 3. When the neuron was at its normal resting membrane potential (-55 mV), the response to a depolarizing current pulse was characterized by regular repetitive firing throughout the entire pulse duration (Fig. 3A). Depolarization from a more hyperpolarized membrane potential (usually more negative than -65 mV) evoked a qualitatively different response, i.e., a burst discharge was induced (Fig. 3B). Five of these neurons were given the same depolarizing current step from the more hyperpolarized membrane potential in the presence of TTX, which abolished the fast spikes but not the DTD (Fig. 3C). In four separate neurons, hyperpolarizing pulses initiated from their normal resting membrane potentials elicited oscillatory activity that was dependent on the duration of the test pulse (Fig. 4A). Interestingly, bursting type firing responses and oscillatory membrane potentials were also elicited with a depolarizing current step in a separate group of cells with intrinsically lower resting membrane potentials such as the one shown in Fig. 4B. Furthermore, spontaneously occurring rhythmic burst firing in CH neurons of varying resting membrane potentials was encountered in three cells (Fig. 4C). This is a lower frequency than reported previously (~15%) in neurons recorded extracellularly from Wistar-Kyoto rats (Shonis and Waldrop 1993). Since the use of whole cell patch recording techniques can cause significant perturbations such as whole cell washout in neurons, the differences in discharge frequency between this study and the prior one may be due to different recording techniques.



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Fig. 3. Membrane voltage dependence of burst firing in type I neurons. A: tonic repetitive discharge evoked from resting membrane potential. B: bursting discharge in the same neuron evoked from a more hyperpolarized holding membrane potential. C: in the presence of 2 µM TTX, depolarizing pulse evoked a transient depolarization from the more hyperpolarized membrane potential.



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Fig. 4. Oscillatory activity in caudal hypothalamic neurons. A: rebound oscillation is dependent on the duration of hyperpolarizing potential (current step = 150 pA at 1st 150 ms, then 200 ms). B: bursting discharge evoked by a depolarizing current step in a neuron with a more hyperpolarized resting membrane potential. C: spontaneous bursting activity from a caudal hypothalamic neuron at its normal resting potential. A-C are from different cells.

Mechanism and modulation of PIR in CH neurons

The presence of a DTD following the hyperpolarizing pulse during perfusion with TTX suggests the entry of Ca2+ might be a key factor for the PIR. As shown in Fig. 5A, the DTD was eliminated when Ca2+ was removed from the bathing medium in all six neurons tested. Addition of 300-500 µM Ni2+ (specific LVA Ca2+ channel blocker) to the normal solution completely blocked the DTD in three neurons tested as shown in Fig. 5B. We also tested the effects of the L-type Ca2+ channel blocker nifedipine on PIR. As shown in Fig. 5C, 10 µM nifedipine caused a complete block of the DTD in the two neurons tested, while the solution containing 0.1% alcohol in which nifedipine was dissolved had little influence on the DTD. Thus LVA, or T-type Ca2+ channels, appear to be a major mechanism of PIR in these cells, although there may be a significant influence from L-type Ca2+ channels.



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Fig. 5. Pharmacological properties of type I postinhibitory rebound (PIR). A: block of rebound depolarization after perfusion with Ca2+-free solution. B: block by 300 µM Ni2+. C: block by 10 µM nifedipine. All recordings obtained in 1 µM TTX. The rebound depolarization was evoked by -100-pA, 150-ms pulse.

To determine how this PIR may be modulated, we explored the effects of GABA and hypoxia on the PIR in the presence of 2 µM TTX. In all three neurons tested, the DTDs were completely blocked by the application of 1 mM GABA in the normal bath solution, as shown in Fig. 6A. The effect of GABA on the PIR was almost completely reversed by application of 0.1 mM bicuculline in one cell tested. Furthermore exposure to 90 s of hypoxia (10% O2) in three cells tested caused an increase in the duration (30.2 ± 3.1 vs. 35.0 ± 3.3 ms, P < 0.05) and amplitude (15.7 ± 1.5 vs. 21.4 ± 1.8 mV, P < 0.05) of the PIR even though the input resistance decreased during this exposure, suggesting a possible direct effect of hypoxia on the channel (Fig. 6B).



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Fig. 6. Effects of GABA and hypoxia on rebound excitation. A: block by 1 mM GABA. B: membrane response to 10% hypoxia. Note the decrease of input resistance and the increase of rebound depolarization during 10% hypoxia. All recordings obtained in 2 µM TTX.

Influence of Ca2+ on membrane and firing properties in CH neurons

In the presence of the potassium channel blocker TEA (10 mM) and TTX, depolarizing current steps in two neurons tested elicited oscillatory potential responses and an afterdepolarizing potential (ADP) that were inhibited following perfusion in Ca2+-free solution (Fig. 7A). These depolarization responses needed a high-intensity stimulation to evoke in normal Ca2+ solution (with TEA); however, in four separate neurons perfused with Ba2+ replacement solution (without TEA), the Ca2+ oscillatory responses and larger ADP could be evoked with a smaller depolarizing current (Fig. 8B). Since Ba2+ is a better charge carrier than Ca2+ through L-type calcium channels (Bean 1989), the oscillations and ADP observed were most likely due to this channel type. Barium also decreased the voltage threshold and increased both the amplitude and duration of the DTD in these four neurons (all type I as shown in Fig. 1A, right), suggesting that Ba2+ is also a better charge carrier through T-type calcium channels in these neurons.



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Fig. 7. High-voltage-activated calcium channel responses. A: calcium responses recorded in the presence of 2 µM TTX and 10 mM tetraethylammonium (TEA). Note the block of calcium spikes and (ADP) in the Ca2+-free solution. B: calcium responses recorded in normal and Ba2+-replacement solution in the presence of TTX (without TEA). Note the calcium spikes and the larger, longer ADP in Ba2+ replacement solution. A and B are from different cells.



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Fig. 8. Effects of the removal of extracellular Ca2+ on action potential properties and adaptation. A: response to a depolarizing current pulse in normal solution. Note the strong adaptation [spike frequency index (SFI) = 2.5]. B: removing Ca2+ partially eliminated the adaptation (SFI = 1.6) and decreased the AP amplitude.

Considering the abundance of calcium channels in the CH neurons, we also evaluated the possible role of Ca2+-dependent K+ channels in repetitive firing and AP properties. In a total of 31 neurons tested, all but 2 showed a decrementing firing pattern, signifying adaptation in these cells. In addition, one cell had such a strong adaptation that the value for SFI could not be calculated (denominator = 0), thus the average SFI for the remaining 30 cells was 3.52 ± 0.71 (range = 0.92-18.23, Table 1) with the two cells not showing adaptation having SFI values of 0.92 and 0.96. The removal of Ca2+ in 11 of the neurons showing adaptation caused a significant amplitude decrease in the AP and AHP with no significant change in AP duration (Table 2, Fig. 8). The decrease in AP amplitude, however, may possibly be due to the more depolarized baseline and decreased sodium electrical gradient. Furthermore, in all eight of the neurons tested, Ca2+ removal significantly reduced the frequency adaptation (Table 2, Fig. 8).


                              
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Table 2. Effect of Ca2+ removal on action potential properties and frequency adaptation of caudal hypothalamic neurons


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Using whole cell recording methods and pharmacological manipulation, we have described the biophysical properties of CH neurons that have at least two different endogenous calcium channels, LVA and HVA. The neurons in CH are not electrophysiologically homogeneous with many neurons (23/50) showing PIR that is mediated through the activation of LVA calcium channels. Most of the neurons also showed adaptation and had firing patterns partly mediated by the interaction of calcium current and calcium-dependent potassium current. The significance of these properties is discussed in relation to the known function of neurons in this brain region.

LVA Ca2+ current involvement in PIR

A major finding from this work was that most of the CH neurons showed PIR. The major type of PIR encountered was characterized by the ability of a previously quiescent neuron to fire in a transient burst mode following a hyperpolarizing current injection and is referred to as type I PIR. The bursts of firing and rebound depolarizations were time and voltage dependent, requiring a sufficiently hyperpolarized membrane potential for several hundred milliseconds to obtain the full effect. After exposure to TTX, the cells still showed a DTD after being released from hyperpolarization, suggesting a mechanism different from voltage-dependent sodium conductance. In fact, the DTD was strictly dependent on the presence of extracellular calcium and was fully blocked by low concentrations of Ni2+. At a more depolarized membrane potential (more positive than -65 mV), direct stimulation did not trigger a burst discharge, suggesting that the ionic conductance underlying its generation was inactivated at these potentials (Llinas and Yarom 1981). De-inactivation of the conductance occurred as the membrane potential hyperpolarized as evidenced by the generation of the DTD. The properties of this DTD are similar to those found in neurons of the adult inferior olivary complex (Llinas and Yarom 1981), thalamus (Jahnsen and Llinas 1984a,b), dorsal raphe nucleus (Burlhis and Aghajanian 1987), neonatal hypoglossal nucleus (Viana et al. 1993), and many other central and peripheral neuronal types (Huguenard 1996). Taken together, the LVA calcium channel, or T-type channel, significantly affects the DTD and may underlie the PIR observed in these cells, although definitive proof needs assessment via voltage-clamp.

Interestingly, the LVA Ca2+ channels in the CH neurons were extremely sensitive to the organic channel blocker, nifedipine, which has also been shown to block these channels in mouse embryonic dorsal root ganglion neurons and isolated neurons from the ventromedial hypothalamus (Akaike et al. 1989; Richard et al. 1991). Recent evidence has indicated that dihydropyridine-sensitive L-type channels on CA3 neurons can be activated at potentials much more negative than other HVA calcium channels and thus contribute to the LVA currents (Avery and Johnston 1996). This challenges the traditional designation of L-type channel as exclusively HVA calcium channels and the T-type channel as the only LVA calcium channels. Thus it is unknown whether the nifedipine was affecting the PIR via LVA or HVA current in the CH neurons in the present study.

Barium is known to be a better charge carrier than calcium in certain types of HVA calcium channels, such as the L-type calcium channel (Bean 1989). This increased conductance by barium through putative HVA channels was observed in CH neurons. In contrast, LVA calcium current amplitude is normally unaffected or reduced by Ba2+ except in the rat thalamic reticular nucleus, where Ba2+ was shown to increase the LVA current (Akaike et al. 1989; Fox et al. 1987; Huguenard and Prince 1992). In type I neurons in the CH, Ba2+ replacement decreased the voltage threshold for the PIR-induced DTD, while increasing both its amplitude and duration. Although this suggests CH neurons have LVA current more sensitive to Ba2+ than Ca2+, voltage-clamp studies are needed to definitively attribute this response to the LVA and not HVA current.

Role of calcium on the AP and repetitive firing activity

In a majority of the cells tested, the blockade of Ca2+ entry caused a decrease in the AP amplitude without changing its duration. These properties have also been found in rat superior cervical ganglion and reflect the involvement of large conductance Ca2+-activated K+ channels activated during the AP in these cells (Davies et al. 1996). Although these cells were in the peripheral nervous system, a similar mechanism may underlie the response to decreased Ca2+ in the CH neurons.

The role of calcium in the repetitive firing qualities of CH neurons was also highlighted after perfusion with calcium-free solution. The loss of Ca2+ led to a significant decrease in the afterhyperpolarization, increase in firing frequency and reduction in adaptation (as determined from the SFI). These responses can be explained by the large presence of Ca2+-activated K+ channels that modulate the adaptive abilities of these CH neurons. However, the adaptation was never completely blocked by calcium-free solution, suggesting that other, unknown factors are involved in the adaptation of these neurons.

Functional role of LVA calcium channels in CH neurons

The physiological function of the LVA calcium channels in CH neurons has yet to be determined. In many of the CH neurons studied, LVA was the dominant calcium channel, which is similar to many areas in the hypothalamus (Akaike et al. 1989; Alonso and Llinas 1992). Our present data indicate a strong influence of LVA calcium channels on the firing pattern of CH neurons and suggest that this channel plays a significant role in burst discharge. The role of CH neurons in neuronal rhythms significant to physiological function may be twofold. First, there is compelling evidence that neurons in the CH have theta rhythm-related discharge implicating them in memory function (Bland et al. 1995; Kirk et al. 1996; Skaggs and McNaughton 1996). Second, neurons in this area have been shown to modulate cardiorespiratory activity and display rhythms related to sympathetic discharge (Barman 1990; Dillon and Waldrop 1993; Waldrop and Porter 1995).

The role of the caudal hypothalamus in memory function was first shown after lesions of the CH abolished or attenuated hippocampal theta activity (Anchel and Lindsley 1972; Robinson and Whishaw 1974). A recent anatomical study has further shown that the CH has dense projections to the medial septum and vertical limb of the diagonal band of Broca that are important relays in the generation of hippocampal theta activity (Vertes et al. 1995). Together with the results from the above-mentioned neuronal recordings, these findings implicate the CH in the formation of the theta rhythm found in hippocampal neurons. The LVA calcium channels in CH neurons may be one of the underlying ionic mechanisms for the theta-related tonic discharge in these cells.

Neurons in the CH discharge with rhythms related to the cardiovascular and sympathetic cycles in both rats and cats (Barman 1990; Dillon and Waldrop 1993; Ryan and Waldrop 1995). Furthermore neurons in this region send dense projections to other cardiovascular modulating areas such as the periaqueductal gray, ventrolateral medulla and nucleus tractus solitarius (Barman 1990; Ryan and Waldrop 1995; Vertes and Crane 1996). Because a hyperpolarization is required to activate the LVA Ca2+ current, the pattern of inhibitory input may be a critical factor in determining the role of LVA current in sympathetic-related CH neuronal discharge. One of the primary functions of GABAergic input to the caudal hypothalamus is to depress the activity of sympathoexcitatory neurons involved in cardiovascular control (DiMicco and Abshire 1987; Shonis et al. 1993; Waldrop and Bauer 1989). GABA completely abolished the LVA calcium current in these neurons, suggesting an effect of GABA on this channel. This GABAergic effect has been previously shown in hippocampal cells, where the GABAB agonist baclofen inhibited the LVA current (Fraser and MacVicar 1991). Since intracellular messenger systems are known to regulate calcium channel function, the effect of GABAB receptor activation on calcium channel function is not surprising (Gray et al. 1998). Our data suggest, however, that the affect of GABA on calcium current is mediated through the GABAA channel because it is inhibited by bicuculline. A similar mechanism of GABA action on ion channel properties has been demonstrated in astrocytes, where GABAA-induced chloride flux modulated potassium channel function over long durations (Bekar and Walz 1999). Thus the relative amount of GABA through the involvement of GABAA receptors in the CH may greatly affect the function of this current.

Since the LVA calcium channel is modulated by GABA, a differential level of GABA in the CH of the spontaneously hypertensive rat (SHR) may influence this current and thus significantly alter the firing properties of these neurons. Recent work from this lab has found that there is an elevated discharge rate of neurons in the CH of the SHR, and a higher percentage of these neurons have a bursting-type firing pattern of when compared with normotensive rats (Shonis and Waldrop 1993). It has been proposed that these bursting-type cardiovascular neurons may exert a more efficient influence than other types of cells in eliciting the vasoconstrictor response and thus contribute to the occurrence of hypertension (Chan et al. 1991; Shonis and Waldrop 1993). Indeed, it has been suggested that neuronal bursts are the best stimuli for exciting a cell and have been shown to make unreliable synapses reliable (for review, see Lisman 1997). Interestingly, the most obvious function of LVA calcium channels is to promote burst-type firing. Since the LVA calcium channel is found in a large number of CH neurons, it raises the possibility that the higher number of burst-type firing CH neurons in the SHR might be due to either a greater expression or a disinhibition of this channel in the CH of the SHR. The latter explanation might be more possible because the LVA channel is directly inhibited by GABA and the GABAergic input to CH neurons is reduced in the SHR (Horn et al. 1998). However, more work is needed to determine whether there is a greater function of the LVA channel in CH neurons of the SHR.

Several studies have suggested CH neurons function as putative oxygen-sensing neurons that modulate the cardiorespiratory response to hypoxia (Dillon and Waldrop 1992, 1993; Horn and Waldrop 1997; Horn et al. 1999). The response of these neurons to a hypoxic stimulus is a membrane depolarization accompanied by a significant increase in firing frequency. The specific ionic mechanism by which hypoxia causes this response in CH neuron has not been fully understood, but several channels are candidate oxygen sensors as shown in other studies. Work in hippocampal neurons has demonstrated that voltage-dependent sodium channels decrease their conductance during hypoxia, and human cardiac L-type Ca2+ channels also decrease their conductance during hypoxia (Fearon et al. 1997; O'Reilly et al. 1997). Furthermore both LVA and HVA calcium currents as well as persistent sodium currents are increased during hypoxia in rat rostral ventrolateral medullary neurons responsible for sympatho-excitatory output (Kawai et al. 1999; Sun and Reis 1994). In the present study, the duration and magnitude of the PIR-induced DTD in CH neurons increased during the hypoxic stimulus. Thus the LVA current may be directly affected by oxygen and help in the cardiorespiratory changes attributed to these and other neurons during hypoxia.

In conclusion, neurons in the caudal hypothalamus of rats contain LVA calcium channels that are essential to the postinhibitory rebound properties inherent in these cells. These channels, along with calcium-activated potassium channels, help establish the bursting firing properties of caudal hypothalamic neurons. Undoubtedly, the bursting properties of these neurons underlie the theta and sympathetic rhythms inherent in the caudal hypothalamus. Further work is needed to determine if the same cells are responsible for both of these rhythms or if there are subpopulations of caudal hypothalamic neurons separately supplying each rhythm and ultimately contributing to memory and/or sympatho-excitatory function.


    ACKNOWLEDGMENTS

This work was supported by National Heart, Lung, and Blood Institute Grant HL-06296 and an American Heart Association Grant-in-Aid to T. G. Waldrop and an American Heart Association Predoctoral Fellowship to E. M. Horn.

Present addresses: Y.-P. Fan, Dept. of Bioengineering, University of California, San Diego, La Jolla, CA 92093; E. M. Horn, Division of Neurosurgery, Barrow Neurological Institute, 350 Thomas Rd., Phoenix, AZ 85013.


    FOOTNOTES

Address for reprint requests: T. G. Waldrop, Dept. of Molecular and Integrative Physiology, University of Illinois, 524 Burrill Hall (MC-114), 407 S. Goodwin Ave., Urbana, IL 61801 (E-mail: twaldrop{at}uiuc.edu).

Received 29 November 1999; accepted in final form 16 August 2000.


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