INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The cellular mechanisms regulating the sleep-wakefulness cycle remain largely unknown. Certain brain regions have, however, been implicated in this regulation, particularly the ventrolateral preoptic nucleus (VLPO) and the tuberomammillary nucleus (TMN) (Gallopin et al. 2000; Saper et al. 2001; Sherin et al. 1998).

The VLPO contains both GABAergic and galaninergic neurons which project to, and innervate, the TMN (Sherin et al. 1996, 1998; Steininger et al. 2001). The TMN predominantly contains histaminergic neurons whose activation promotes physiological arousal (Lin et al. 1996; Monti et al. 1991; Wada et al. 1991). Electrical stimulation in the preoptic area causes -aminobutyric acid-A (GABA_A) receptor-mediated hyperpolarization and inhibition of TMN neurons (Yang and Hatton 1997) and this inhibition plays a pivotal role in causing sleep (Sherin et al. 1998). For example, lesions of the VLPO have been reported to cause insomnia (Lu et al. 2000). These results suggest that the GABAergic projections from the VLPO to TMN neurons play an important role in regulation of sleep and wakefulness.

The VLPO is innervated by a variety of afferent inputs, including monoaminergic, GABAergic, and galaninergic. These afferent inputs arise from a number of different neuronal regions, including a dense innervation from the TMN itself (Chou et al. 2002). These different neurotransmitter systems are thought to work together in harmony to modulate the excitability of VLPO neurons. However, little is known about how these different neurotransmitters regulate the activity of VLPO neurons. Specifically, little is known about how GABA or monoamines modulate the excitability of VLPO and what receptor subtypes may mediate these possible actions. Therefore, in the present study, we focused on investigating whether VLPO neurons were innervated by GABAergic terminals and how VLPO neuronal excitability was modulated by noradrenaline (NA).

NA is one of the neurotransmitters implicated in regulation of sleep-wakefulness. In the medial preoptic area, local application of the ₂-adrenoceptor agonist, clonidine, produces arousal, while its antagonist, yohimbine, produces sleep (Ramesh et al. 1995). Adrenergic innervation of the VLPO from the locus coeruleus and other regions has been reported (Chou et al. 2002) and NA has been demonstrated to directly modulate the excitability of VLPO neurons (Gallopin et al. 2000). These reports further implicate noradrenergic inputs to the VLPO as playing an important role in regulating sleep-wakefulness.

To examine the role of NA in VLPO neurons, we used mechanically dissociated VLPO neurons to which functional presynaptic nerve terminals remain adherent (the "synaptic bouton" preparation, Rhee et al. 1999). These preparations enabled us to focus selectively on the nature of these proximal presynaptic nerve terminals and also to examine the modulation of transmitter release. The preparation is ideally suited for such studies as it is devoid of possible complications arising from both enzymatic effects on various membrane and/or cytoplasmic proteins (Armstrong and Roberts 1998) and from indirect actions on surrounding neurons and/or glia.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Preparations

Wistar rats (15-20 days postpartum) were decapitated under pentobarbital sodium anesthesia (50 mg kg¹ ip). The brain was quickly removed and sliced in the coronal plane at a thickness of 330 µm using a microslicer (VT1000S; Leica, Germany). The slices were bathed in an incubation medium (see Solutions) saturated with 95% O₂-5% CO₂ at room temperature (21-24°C) for 1 h. For dissociation, the slices were transferred into a 35-mm culture dish (Primaria 3801; Becton-Dickinson, NJ) containing the standard external solution (see Solutions). The VLPO neurons were identified under a binocular microscope (SMZ-1; Nikon, Tokyo). Details of the mechanical dissociation have been described previously (Koyama et al. 1999; Rhee et al. 1999). Briefly, the tip of a fire-polished glass pipette was lightly placed on the surface of the VLPO region using a micromanipulator. The tip of the glass pipette was vibrated horizontally at 30-50 Hz using a custom-built vibration device for about 2 min. The slices were then removed and the mechanically dissociated neurons, with some of their native presynaptic nerve terminals adherent, were left for about 20 min to settle and adhere to the bottom of the dish. By using a fine pipette for dissociation, this procedure also enabled us to obtain neurons from a small area of the brain, such as the VLPO, and also to select different cells according to their shape.

Every time we performed electrophysiological experiments, we carefully consulted the brain map and Saper group's papers (Lu et al. 2000; Sherin et al. 1998) and dissociated neurons from the region they presented in their papers. Slices we could get, which contain VLPO region, from one rat was no more than two pieces. After the dissociation, we had two populations of neurons: smaller neurons that are <12 µm, and relatively larger neurons that soma size about 12-20 µM. We selected the larger size neurons that are supposed to be VLPO neurons according to the reports by Gallopin et al. (2000).

We stained dissociated neurons with anti-galanin antibody to make sure that dissociated neurons in this study really do belong to the VLPO neurons. Normally we dissociate neurons from one piece of slice in one dish; however, only in the immunocytochemistry study we used three pieces of slice from three rats so that we can compensate for the neurons washed away during the procedure and to minimize the consequent amount of animals we used.

All experiments conformed to the guiding principles for the care and use of animals approved by The Council of The Physiological Society of Japan. Efforts were made to minimize the number of animals used and any suffering.

Immnofluorescent microscopy

To determine whether the neurons we used for electrophysiological measurements are galanergic VLPO neurons, we performed immunocytochemistry using anti-galanin antibodies as described below. Neurons were mechanically dissociated on the glass coverslips coated with polyethylenimine (PEI). For PEI-coating, glass coverslips were washed with alkalized ethanol (more than 5 h), neutralized with ethanol hydrochloride (more than 30 min), and rinsed with sterilized water (more than 5 times). After sterilizing by autoclaving, coverslips were coated with 0.1% PEI overnight and washed extensively with sterilized water (5 times, more than 5 min for every wash) to remove the possible toxicity of PEI for neurons. PEI-coated coverslips were kept in PBS until used.

Brain slices were mechanically dissociated on the PEI-coated glass coverslips placed in a culture dish. After most neurons were settled down and attached to the coverslips, each coverslip was moved to a parafilm sheet for immunocytochemistry. Neurons were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min, permeabilized with 0.25% Triton X-100 in PBS for 5 min, and blocked with 5% BSA in PBS for 30 min. Then they were treated with polyclonal antiserum against galanin [Peninsula Labs; rabbit, 1:10,000 diluted in PBS with 1% bovine serum albumin (BSA)] (Elmquist et al. 1992; Sherin et al. 1998) for 1 h. After being rinsed with 1% BSA in PBS (3 times for 10 min), they were incubated in fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit secondary antibodies (Jackson ImmunoResearch; 1:200) to visualize galanin immunoreactivity. As a control, some coverslips were treated only with the secondary antibodies. Cells were then washed with PBS extensively (4 times, 15 min for every wash) and mounted in the vectorshield mounting medium (VectorStain). Images were obtained using an Axioskop2 plus (Carl Zeiss, Germany) epifluorescence microscope equipped with AXIOCAM (Carl Zeiss Microimaging) and a ×20 objective lens. All procedures were performed at room temperature.

Electrical measurements

Electrical measurements were performed using nystatin perforated patch recording (Akaike and Harata 1994) and conventional whole-cell patch recording under voltage-clamp conditions. Current recordings and voltage control were obtained using a patch-clamp amplifier (CEZ-2300; Nihon Kohden, Tokyo) and all recordings were made at a holding potential (V_H) of 70 mV, except where indicated. Patch pipettes were pulled from borosilicate capillary glass (1.5 mm OD, 0.9 mm ID; G-1.5 Narishige, Tokyo) in two stages on a vertical pipette puller (PB-7; Narishige). The resistance of the recording pipettes filled with internal solution was 6-8 M. Neurons were visualized under phase-contrast optics on an inverted microscope (Diaphot, Nikon, Tokyo). Current and voltage were continuously monitored on an oscilloscope (Textronix 5111A; Sony, Tokyo) and a pen recorder (Linearcorder WR3320; Graphtech, Tokyo). Stored currents were filtered at 1 kHz (E-3201A Decade Filter, NF Electronic Instruments, Tokyo) and digitized at 4 kHz using a Digidata 1200 and pCLAMP software (version 8.0, Axon Instruments, CA). All experiments were performed at room temperature (21-24°C).

Data analysis

Miniature inhibitory postsynaptic currents (mIPSCs) were collected in preset epochs before, during, and after each experimental condition. Synaptic currents were detected and analyzed using MiniAnalysis software (Synaptosoft, Decatur, GA). The amplitude of each mIPSC was measured from the initial point of deflection to the peak of the current response. All detected events were visually checked before undergoing further analysis to avoid the inclusion of obvious experimental artifacts. Numerical data are presented as the means ± SE. Differences in mIPSC amplitude and frequency distributions were compared using nonparametric analysis [Kolmogorov-Smirnov test (K-S test)] with P < 0.05 considered significant. Mean mIPSC amplitude and frequency were normalized to the respective control values and statistical differences under different experimental conditions were analyzed using Student's two-tailed t-test. Kolmogorov-Smirnov test was performed using MiniAnalysis software and the other statistical analysis was performed using Microsoft Excel software (Microsoft).

Solutions

The ionic composition of incubation medium was as follows (in mM): 124 NaCl, 5 KCl, 1.2 KH₂PO₄, 24 NaHCO₃, 2.4 CaCl₂, 1.3 MgSO₄, and 10 glucose, equilibrated with 95% O₂-5% CO₂. The standard external solution was as follows (in mM): 150 NaCl, 5 KCl, 2 CaCl_2, 1 MgCl₂, 10 glucose, and 10 HEPES. Ca²⁺-free external solution contained the following (in mM): 146 NaCl, 5 KCl, 5 MgCl₂, 10 glucose, 10 HEPES, and 2 EGTA. The external solution with Cd²⁺ was made by adding 100 µM CdCl₂ to the standard external solution. The pH of these external solutions was adjusted to 7.4 with tris (hydroxymethyl) aminomethane (Tris-OH). To isolate mIPSCs, external solutions routinely contained 300 nM TTX to block voltage-dependent Na⁺ channels and 10 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 20 µM D-2-amino-5-phosphovaleric acid (AP5) to block glutamatergic currents. The ionic composition of the internal (patch-pipette) solution for whole cell recording was as follows (in mM): 80 Cs-methanesulfonate, 70 CsCl, 2 EGTA, 10 HEPES, and 4 ATP-Mg with pH adjusted to 7.2 with Tris-OH. The internal (patch-pipette) solution for the nystatin perforated patch recording was as follows (in mM): 150 KCl and10 HEPES with pH adjusted to 7.2 by adding Tris-OH. Nystatin was dissolved in acidified methanol at 10 mg ml¹. This stock solution was diluted with the internal solution just before use to a final concentration of 100-200 µg ml¹.

Drugs used in the present study were as follows: PEI, TTX, AP5, CNQX, bicuculline, nystatin, prazosin, clonidine, s-propranolol, yohimbine, forskolin, N-ethylmaleimide (NEM), Rp-cAMP, SQ22536 (all from Sigma); L-noradrenaline (from Tokyo Kasei, Tokyo); WB4101 (from TOCRIS); paraformaldehyde (from Ishizu Seiyakua, Osaka, Japan), and CdCl₂ from Katayama Chemical Co. (Osaka, Japan). The drugs that were insoluble in water were first dissolved in dimethylsulfoxide (DMSO) and were then diluted in the external solution. Final concentrations of DMSO were always <0.1%, at which concentration DMSO had no effect on the membrane potential or electrical activities. Antibodies used in the present study were Rabbit Anti-Galanin (Rat) (from Peninsula Laboratories, CA) and FITC-conjugated Affinipure donkey anti-rabbit IgG (from Jackson ImmunoResearch, West Grove, PA).

All drugs were applied using a rapid application system termed "The Y-tube method" (Akaike and Harata 1994). By using this perfusion technique, the external solution surrounding a neuron could be exchanged within 20 ms.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Mechanically dissociated VLPO neurons and GABAergic miniature inhibitory postsynaptic currents

After the mechanical dissociation, we had two populations of neurons, smaller neurons (soma <12 µm in diameter) and relatively larger neurons (soma 12-20 µM) (Fig. 1A). To confirm that these dissociated neurons contain galanin, which is the specific substance observed in VLPO neurons (Sherin et al. 1996, 1998; Steininger et al. 2001), we stained neurons with anti-galanin antibody. As shown in Fig. 1A, almost 90% (56/64) of larger neurons (both multipolar neurons and bipolar ones) distinguished for the electrophysiological experiment showed galanin-immunoreactivity. On the contrary, <10% (9/138) of smaller neurons were stained positive in the same conditions. These soma sizes of neurons were also compatible with reports by Gallopin et al. (2000). Thus, from these results, we employed larger neurons as galanin-immunoreactive VLPO neurons in the following studies.

View larger version (19K): [in this window] [in a new window]   Fig. 1. Mechanically dissociated 2 types of ventrolateral preoptic nucleus (VLPO) neurons and GABAergic miniature inhibitory postsynaptic currents (mIPSCs). A: typical example of multipolar and bipolar neurons obtained from the VLPO region after mechanical dissociation. Bottom panels are photoimages of galanin-immunoreactive VLPO neurons. B: summary of the differential postsynaptic effect of noradrenaline (NA) on multipolar and bipolar VLPO neurons. In 10 of 12 multipolar neurons investigated, NA (100 µM) hyperpolarized the membrane potential, while it depolarized all 6 bipolar neurons. C: typical traces of GABAergic mIPSCs in the absence (top) and presence (bottom) of 10 µM bicuculline, recorded from a multipolar cell. All recordings were performed in the presence of 300 nM TTX, 10 µM CNQX, and 20 µM AP5.

We also investigated the electrophysiological properties of these neurons using nystatin-perforated patch recordings under current-clamp conditions. All neurons had resting membrane potentials ranging from -60 to -65 mV and exhibited spontaneous action potentials firing (data not shown); these properties did not differ between the two morphological subtypes. In 10 of 12 multipolar neurons investigated, NA (100 µM) hyperpolarized the membrane potential by -5.7 ± 1.3 mV (P < 0.05, n = 10, Fig. 1B), resulting in a decrease in the frequency of action potential firing. In contrast, NA depolarized all bipolar neurons investigated (+5.8 ± 0.4 mV, P < 0.05, n = 6, Fig. 1B) and increased the frequency of action potential firing. These results are very similar to the study by Gallopin et al. (2000) and indicate that NA directly, but differentially, modulates the excitability of the two types of VLPO neurons.

When recording under voltage-clamp conditions using conventional whole cell patch-clamp recordings, and in the presence of TTX (300 nM), CNQX (10 µM), and AP-5 (20 µM), spontaneous postsynaptic currents were recorded from all VLPO neurons. These events were completely and reversibly blocked by the addition of 10 µM bicuculline, a selective GABA_A receptor antagonist (Fig. 1C). The reversal potential of these events estimated from the current-voltage (I-V) relationship was 23.4 ± 1.5 mV (n = 6), which was very close to the theoretical Cl equilibrium potential (E_Cl; 21.3 mV) calculated from the Nernst equation using the extra- and intracellular Cl concentrations in our recording solutions (161 and 70 mM, respectively). Thus the synaptic currents were identified as spontaneous mIPSCs mediated by activation of GABA_A receptors.

Adrenergic modulation of GABAergic transmission onto VLPO neurons

Application of NA (1 µM) rapidly and reversibly decreased the frequency of these GABAergic mIPSCs recorded from multipolar VLPO neurons (58.6 ± 7.5% of the control, P < 0.01, n = 10, Fig. 2, A and B), but had no significant effect on the frequency of GABAergic mIPSCs recorded from bipolar VLPO neurons (92.7 ± 7.0% of the control, P = 0.47, n = 5, Fig. 2B). The mIPSC frequency distribution of mIPSCs recorded from multipolar cells was also significantly (P < 0.01, K-S test, Fig. 2Ca) shifted to the right by NA (1 µM), indicating longer inter-event intervals. In this same set of multipolar neurons, the mean mIPSC amplitude was unaffected by NA (92.1 ± 5.9% of the control, P = 0.16, Fig. 2B) as was the amplitude distribution (P = 0.094, K-S test, Fig. 2Cb). These results indicate that NA acts presynaptically to reduce the probability of miniature GABA release at these synapses. As GABA release from terminals adherent to bipolar cells was unaffected by NA, the following experiments were restricted to the multipolar VLPO neurons.

View larger version (22K): [in this window] [in a new window]   Fig. 2. Effect of NA on GABAergic transmission in multipolar VLPO neurons. A: a typical trace of mIPSCs observed before, during, and after the application of 1 µM NA. Insets represent an expanded portion of the top trace, as indicated by the dashed bars. B: averaged effects of NA on mIPSC amplitude and frequency. Application of NA (1 µM) reversibly decreased GABAergic mIPSC frequency in the multipolar VLPO neurons; however, NA had no significant effect on mIPSCs recorded from bipolar VLPO neurons. Each column represents the relative effect of NA, normalized to the value observed in the absence of NA [means + SE, n = 10 and 5, respectively]. C: cumulative distributions for inter-event intervals (a) and current amplitudes (b) of mIPSCs obtained from the same neuron as shown in A.

₂-Adrenoceptors have been reported to reduce the probability of release of a wide range of neurotransmitters at various central and peripheral synapses (see Bylund et al. 1994; Docherty 1998). Thus we investigated whether this subtype of adrenoceptor was also involved in NA-induced inhibition of mIPSC frequency in the multipolar VLPO neurons using the selective ₂-adrenoceptor antagonist, yohimbine. The application of yohimbine (300 nM) alone had no effect on mIPSC amplitude or frequency, but completely occluded the inhibitory effect of NA (1 µM) on mIPSC frequency. The GABAergic mIPSC frequency, in the presence of NA and yohimbine, was 100.4 ± 12.3% of that observed in the presence of yohimbine alone (P = 0.68, n = 6, Fig. 3, A and B). In addition, clonidine (1 µM), a selective ₂-adrenoceptor agonist, also significantly decreased mIPSC frequency to 70.5 ± 3.0% of the control (P < 0.01, n = 65, Fig. 3, C and D) without affecting the mean current amplitude (98.0 ± 2.0% of the control, P = 0.11, n = 65, Fig. 3, C and D). The number of the subject here is the summary of all the following experiments that used clonidine as a control. In contrast, however, neither the ₁-adrenoceptor antagonists, WB4101 (100 nM, n = 9) and prazosin (1 µM, n = 5), nor the -adrenoceptor antagonist, s-propranolol (100 nM, n = 5), affected the inhibitory action of NA on GABAergic mIPSC frequency (data not shown). These results clearly indicate that the inhibitory effect of NA on GABA release from terminals adherent to multipolar VLPO neurons is mediated via ₂-adrenoceptor activation. Due to its high selectivity for ₂-adrenoceptors, the following experiments were conducted using clonidine.

View larger version (19K): [in this window] [in a new window]   Fig. 3. 2-Adrenoceptor-mediated presynaptic inhibition of GABAergic mIPSCs. A: typical traces of mIPSCs observed before, during, and after the application of 1 µM NA, all in the continued presence of 300 nM yohimbine. B: group data illustrating how the inhibitory effect of NA on GABAergic mIPSC frequency was completely blocked in the presence of 300 nM yohimbine, a selective 2-adrenoceptor antagonist (100.4 + 12.3% of the yohimbine condition, P < 0.01, n = 6). C: typical traces of mIPSCs observed before (left) and during (right) the application of 1 µM clonidine (1 µM), a selective 2-adrenoceptor agonist. D: averaged data illustrating how clonidine (1 µM) significantly decreased mIPSC frequency without affecting the mean current amplitude (means + SE, n = 65). The number of the subject here is the summary of all the following experiments that used clonidine as a control. In this figure, and subsequent figures, all mIPSC frequencies and amplitudes have been normalized to the original control values.

Effect of NEM

Adrenoceptors are coupled to guanosine 5'-triphosphate (GTP)-binding proteins (G-proteins) (Bylund et al. 1994; Docherty 1998; Wickman and Clapham 1995). Pertussis toxin (PTX) is frequently used to identify whether the PTX-sensitive G-proteins (G_i/G_o) mediate effects of G-protein-coupled receptors. However, in our acutely dissociated neuronal preparations, cells typically only remain viable for patch-clamp recordings for a maximum of 5 to 6 h, which may be an insufficient period to observe the full effects of PTX. Therefore NEM, a sulfhydryl-alkylating agent (Asano and Ogasawara 1986), was used to test whether ₂-adrenoceptor activation is coupled to a PTX-sensitive G-protein (G_i/G_o) in these GABAergic presynaptic terminals. Pretreatment with NEM (10 µM) for 15 min markedly increased mIPSC frequency to 435.9 ± 95.8% of the control (n = 11) without affecting the mean mIPSC amplitude (103.9 ± 11.4% of the control, n = 11, Fig. 4, A and B). In the presence of NEM, the inhibitory effect of clonidine on GABAergic mIPSCs was totally abolished, and there was even a trend for some slight further mIPSC frequency enhancement (mIPSC frequency was 123.0 ± 12.2% of the NEM control frequency, P = 0.16, n = 11, Fig. 4, A and B). Thus ₂-adrenoceptors on the GABAergic presynaptic nerve terminals projecting to multipolar VLPO neurons seem to be coupled to G_i/G_o proteins.

View larger version (21K): [in this window] [in a new window]   Fig. 4. Effect of N-ethylmaleimide (NEM) on clonidine-mediated inhibition of the frequency of GABAergic mIPSCs. A: typical traces of mIPSCs observed before (top) and during (bottom) the application of 1 µM clonidine in the continued presence of 10 µM NEM. B: summary of data illustrating the effects of NEM on basal mIPSC amplitude and frequency, and on the effects of clonidine (means + SE, n = 11).

Involvement of adenylate cyclase-cAMP pathway in the ₂-adrenoceptor action

Activation of ₂-adrenoceptors is typically associated with inhibition of adenylate cyclase (AC), resulting in a decrease in cytosolic cAMP levels and an inhibition of Ca²⁺ influx (Bylund et al. 1994; Docherty 1998). We therefore examined whether a similar signal transduction pathway was responsible for the inhibition of miniature GABA release following ₂-adrenoceptor activation. Stimulation of AC with 10 µM forskolin (Seamon et al. 1981) significantly increased mIPSC frequency to 193.3 ± 35.2% of the control (P < 0.05, n = 9, Fig. 5, A and B) without affecting the mean mIPSC current amplitude (98.1 ± 9.5% of the control, Fig. 5, A and B). This forskolin-mediated enhancement of GABA release was significantly attenuated by clonidine (1 µM), with mIPSC being decreased to 71.5 ± 4.3% of the mIPSC frequency observed in the presence of forskolin alone (P = 0.012, n = 9, Fig. 5, A and B), although mIPSC frequency was still somewhat higher than the original control frequency being 138.2 ± 18.7% of the original control.

View larger version (15K): [in this window] [in a new window]   Fig. 5. 2-Adrenoceptor-mediated presynaptic inhibition of GABAergic mIPSCs is related to the adenylate cyclase-cAMP signal transduction pathway. A: typical traces of mIPSCs observed before (top) and during (bottom) the application of 1 µM clonidine, all in the presence of 10 µM forskolin, an adenylate cyclase (AC) activator (Aa), or all in the presence of 1 µM SQ22536, an AC inhibitor (Ab). B: summary of the group data illustrating the effects of forskolin or SQ22536 (n = 9 and 6, respectively) on basal mIPSC amplitude and frequency and on the effects of clonidine on mIPSC amplitude and frequency. C: typical traces of mIPSCs in the presence of 100 µM Rp-cAMP, a protein kinase A inhibitor (top traces) and in the presence of Rp-cAMP and clonidine. Rp-cAMP significantly occluded the inhibitory effect of clonidine on mIPSC frequency. D: averaged group data for the effects of Rp-cAMP.

Pretreatment of multipolar VLPO neurons for 10 min with the AC inhibitor SQ22536 (1µM) (Evans et al. 2001) decreased mIPSC frequency to 80.1 ± 6.0% of the control (P < 0.05, n = 6, Fig. 5B) without affecting the mean mIPSC amplitude (Fig. 5B). In the presence of SQ22536, clonidine had no further effect on mIPSC amplitude or frequency; the mean mIPSC frequency was 110.7 ± 20.0% of that observed in the presence of SQ22536 alone, which was not significantly different (P = 0.77, n = 6, Fig. 5B), while the mean current amplitude was 106.4 ± 8.2% of that in SQ22536 alone (n = 6, Fig. 5B). Furthermore, pretreatment with 100 µM Rp-cAMP, a membrane permeant protein kinase A inhibitor (Van Haastert et al. 1984), for 10 min also significantly occluded the inhibitory effect of clonidine (mIPSC frequency was 105.5 ± 12.5% of that observed in Rp-cAMP alone, P = 0.35, n = 8, Fig. 5, C and D). In this case, the interpretation of the lack of clonidine effect was not complicated by any effects of Rp-cAMP on basal mIPSC frequency (Fig. 5D). Neither Rp-cAMP, nor the combination of clonidine and Rp-cAMP, had any effect on mean mIPSC amplitude (Fig. 5D), with mean mIPSC amplitude in the presence of clonidine and Rp-cAMP being 106.0 ± 3.9% of that observed in Rp-cAMP alone (n = 8). These results strongly suggest that the AC/cAMP/protein kinase A (PKA) pathways mediated the clonidine-induced presynaptic inhibition of GABA release.

Ca²⁺ influx and ₂-adrenoceptor-mediated inhibition of GABAergic transmission

In an attempt to clarify whether ₂-adrenoceptor activation also causes a subsequent inhibition of extracellular Ca²⁺ influx, we examined the effect of Cd²⁺, a nonspecific blocker of voltage-dependent Ca²⁺ channels (VDCCs). In the presence of 100 µM Cd²⁺, the frequency of GABAergic mIPSCs was significantly decreased to 30.1 ± 6.2% of the control (P = 0.009, n = 12, Fig. 6, Aa and B), and this decrease in mIPSC frequency was accompanied by a reduction of mIPSC amplitude (75.2 ± 8.0% of the control, P = 0.005, data not shown). The results suggest that VDCCs contribute to the spontaneous release of GABA at these synapses. When clonidine was added in the presence of Cd²⁺, mIPSC frequency was even further and significantly decreased to 53.7 ± 8.8% of the mIPSC frequency observed in the presence of Cd²⁺ alone (P = 0.012, n = 12, Fig. 6, Aa and B; 16.1 ± 2.8% of the original control frequency). We also investigated the effect of 0 Ca²⁺ external solution on IPSC frequency. Again, 0 Ca²⁺ external solution markedly decreased mIPSC frequency (to 26.5 ± 5.3% of the control P < 0.01, n = 14, Fig. 6, Ab and B). When clonidine was added in the continued presence of the 0 Ca²⁺ solution, a further statistically significant inhibition of mIPSC frequency was observed to 64.2 ± 11.3% of the mIPSC frequency observed in the presence of the 0 Ca²⁺ solution alone (P = 0.034, n = 14) or, alternatively, to 17.0 ± 3.9% of the original control mIPSC frequency (Fig. 6, Ab and B).

View larger version (17K): [in this window] [in a new window]   Fig. 6. Ca2+ influx is not related to the 2-adrenoceptor-mediated presynaptic inhibition of GABAergic transmission in the multipolar VLPO neurons. A: typical traces of mIPSCs observed before (top) and during (bottom) the application of 1 µM clonidine, all in the continued presence of 100 µM Cd2+(Aa) or in the continued presence of 0 Ca2+ external solution (Ab). B: summary data of the effect of Cd2+ and 0 Ca2+ external solution on mIPSC frequency (n = 12 and 14, respectively).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Different morphology and adrenergic modulation of mechanically dissociated VLPO neurons

After mechanical dissociation of the VLPO, we obtained two populations of neurons, smaller neurons and relatively larger neurons. Of those, we selected larger size neurons. Within larger neurons, the one subset of cells was bipolar and responded to NA (100 µM) with a depolarization, while the other group of cells was multipolar and responded to NA (100 µM) with a hyperpolarization. This observation is very similar to that observed in brain slices by Gallopin et al. (2000). Also in immunocytochemistry studies, both multipolar neurons and bipolar ones showed galanin-immunoreactivity. These results are compatible with the reports suggesting VLPO neurons contain galanin (Sherin et al. 1996, 1998; Steininger et al. 2001).

Furthermore, our results show that both groups of cells receive GABAergic inputs from proximally located terminals and that this GABAergic transmission is differentially regulated by presynaptic ₂-adrenoceptors. GABAergic inputs to the bipolar cells were unresponsive to NA (1 µM), while GABA releases to multipolar cells were inhibited by NA (1 µM). Hence these results demonstrate that the different morphology is correlated not only to different postsynaptic NA responses but also to different presynaptic NA responses. These different responses to NA observed in this study are probably due to the difference in adrenoceptor subtypes activated by NA in different concentrations. As high concentration of NA (100 µM) reduced the amplitude of IPSCs in supraoptic neurons and no effect was observed in lower concentration (NA, 1 µM) (Wang et al. 1998), it is more likely to presume that also in the VLPO neurons, NA activated postsynaptic ₁-adrenoceptor and/or -adrenoceptor at a concentration of 100 µM and presynaptic ₂-adrenoceptor was activated in a lower concentration (NA, 1 µM).

The physiological reason and significance of the selective presynaptic ₂-adrenoceptor-mediated inhibition of GABAergic transmission to multipolar neurons is unclear. Presumably the specificity arises due to a differential distribution of ₂-adrenoceptor on the GABAergic terminals synapsing on to multipolar neurons. The bipolar and multipolar neurons are distributed throughout the VLPO, as is the adrenergic innervation arising from a variety of neuronal regions (Chou et al. 2002). Hence it is unclear whether there are specific adrenergic axo-axonic synapses on to the terminals synapsing on to multipolar neurons or even whether there is specific postsynaptic adrenergic innervation of multipolar VLPO neurons. It may be that the source of NA for activation of presynaptic receptors in vivo is spillover from these postsynaptic synapses. Clearly, however, this study has demonstrated that presynaptic noradrenaline actions may potentially also contribute to the importance of this neurotransmitter in regulating the activity of VLPO neuron.

Cellular mechanisms of ₂-adrenoceptor-mediated inhibition of GABAergic transmission

Adrenoceptors, especially presynaptic ₂-adrenoceptors, modulate the release probability of a wide range of neurotransmitters, including GABA, acetylcholine, serotonin, and glutamate (Bertolino et al. 1997; Boehm 1999; Boehm and Huck 1996; Frankhuijzen et al. 1998; Miyazaki et al. 1998; Wang et al. 1998). In the present study, application of NA to multipolar VLPO neurons reversibly decreased GABAergic mIPSC frequency without affecting the current amplitude (Figs. 1D and 2, A and B), indicating that NA acts presynaptically to inhibit GABA release. This inhibition was replicated by clonidine and, in the presence of blockade of ₂-adrenoceptors with yohimbine, there was no effect of NA on GABA release. This indicates that ₂-adrenoceptors are totally responsible for NA presynaptic inhibitory actions, although the specific ₂ subtypes responsible remains to be elucidated. These findings are also consistent with the distribution of adrenoceptors in the hypothalamic region where the VLPO neurons are located (see Nicholas et al. 1996).

The ₂-adrenoceptor that mediates the inhibition of GABA transmission to multipolar VLPO neurons are coupled to NEM-sensitive G_i/G_o proteins, as has also been observed in a number of tissues including the rat cerebral cortex (Kitamura and Nomura 1987) and rat vas deferens (Browne et al. 1994). Our results further suggest that ₂-adrenoceptor activation acts via the AC/cAMP/PKA transduction pathway. This cascade has been shown in a range of tissues to play an important role in regulation of transmitter release (Bukharaeva et al. 2002). The most likely scenario is that ₂-adrenoceptor is coupled to a G-protein (G_i/o) that inhibits adenylate cyclase and the subsequent decrease in cAMP reduces the activity of PKA. While we did not elucidate the nature of the secretory proteins that responded to the decreased PKA activity, we did demonstrate, rather interestingly, that inhibition of voltage-dependent Ca²⁺ channels were not wholly responsible for the inhibition of release (although they may have contributed to some small degree). The fact that we still observed an ₂-adrenoceptor-mediated inhibition in the presence of Cd²⁺, and when Ca²⁺ was removed from the external solution, indicates that the transduction mechanisms must act at a site that is subsequent to Ca²⁺ influx.

Physiological significance

Our study sheds light on the GABAergic projection to VLPO neurons and their modulation by presynaptic ₂-adrenoceptors. We show that all VLPO neurons receive inhibitory GABAergic inputs (Figs. 1C and 2, A and B), although the exact origin of the GABAergic innervation is not clear at present. We also confirmed that VLPO are differentially modulated by postsynaptic NA actions. Furthermore we demonstrate that these GABAergic inputs to multipolar neurons are inhibited by presynaptic ₂-adrenoceptors.

VLPO neurons are reportedly active during REM sleep and inactive during wakefulness, in vivo, and the inhibition of VLPO neurons by monoaminergic inputs during daytime are proposed to be responsible for this change in VLPO activity (Gallopin et al. 2000; Saper et al. 2001). VLPO is also known to contain GABA and galanin and to control excitability of TMN neurons (Sherin et al. 1996, 1998; Steininger et al. 2001). Our results suggest that monoaminergic inputs to multipolar VLPO neurons can have two effects, postsynaptic hyperpolarization and a reduction in GABAergic inhibition. The postsynaptic hyperpolarization will lead to a reduction in GABA release from the terminals of multipolar VLPO neurons onto TMN neurons, and consequently, to an increase in histamine release from TMN terminals, thus maintaining wakefulness in the daytime. These speculations are compatible with the fact that NA neurons are most activated in the waking state (Hobson et al. 1975). Interestingly, however, NA's presynaptic actions on multipolar VLPO neurons will have an opposite effect on the output of VLPO and TMN neurons. In this sense, it can be proposed that, in the multipolar VLPO neurons, NA acts in a reciprocal manner on the pre- and postsynaptic adrenoceptors. These reciprocal actions may play an important role in preventing VLPO neurons becoming either over-excited or over-inhibited. Furthermore, if specific axo-axonic adrenergic synapses exist in vivo, perhaps even arising from a different origin to the postsynaptic adrenergic synapses, it provides an additional mechanism for adrenergic regulation of VLPO output.

In conclusion, our studies have demonstrated that morphological differences in VLPO neurons correlates not only with different postsynaptic NA responses, but also with different NA presynaptic responses in the GABAergic terminals projecting to these neurons. We found that NA, via ₂-adrenoceptors coupled to G-proteins and inhibition of the AC/cAMP/PKA transduction pathway, acts presynaptically to inhibit GABA release onto multipolar VLPO neurons. This action demonstrates another potential mechanism by which NA may play an important role in the regulation of the excitability of the VLPO neurons and the consequent regulation of sleep/wakefulness.