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1Neuroscience Program, Ottawa Health Research Institute and University of Ottawa, Ottawa, Ontario K1Y 4E9; and 2Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario N6A 5C1, Canada
Submitted 6 January 2004; accepted in final form 12 February 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-conotoxin GVIA, and cadmium. Acting at postsynaptic GABAB receptors, baclofen hyperpolarized a majority of MnPO neurons by increasing a G protein–coupled inwardly rectifying potassium conductance and suppressing an N-type high-voltage–activated calcium conductance. The latter contributed to reduction in action potential afterhyperpolarization and enhanced cell firing and spike frequency adaptation when tested with a depolarizing stimulus. All baclofen-induced effects were blockable with CGP52432 CGP52432alone had no significant effect on SFO-evoked postsynaptic current amplitudes or paired-pulse ratios, but did induce an increase in miniature inhibitory postsynaptic current (mIPSC) frequency in 2/4 cells tested, indicating that ambient levels of GABA could activate presynaptic GABAB receptors on undefined inputs. These observations indicate that MnPO neurons receive both a GABAergic and glutamatergic innervation from SFO. Both forms of rapid neurotransmission are subject to modulation via pre- and postsynaptic GABAB receptors. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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; McKinley et al. 1996, 1999). Three cell groups are located within this region. Two of these are circumventricular organs (CVOs), the organum vasculosum lamina terminalis (OVLT) situated ventrally and the subfornical organ (SFO) located dorsally, where fenestrated capillaries permit central neurons access to blood-born molecules (McKinley et al. 1991; Simon 2000). Information received by neurons residing in these CVOs is believed to be transmitted and integrated within the third cell group, the median preoptic nucleus (MnPO, also called MnPN, POMe, or nucleus medianus) located around the anterior commissure at the midpoint along the lamina terminalis. Indeed the connectivity of MnPO neurons suggests their suitability for reception and integration of messages derived from hemal and/or neural origins, based on reciprocal connections with neurons in SFO and OVLT (Miselis et al. 1979; Oldfield et al. 1992), afferent and efferent connections with neurons involved in hypothalamic neuroendocrine regulation and hydromineral balance (Armstrong et al. 1996; Oldfield et al. 1992; Silverman et al. 1981; Swanson 1976), sleep-waking cycles (Chou et al. 2002), and brain stem cardiovascular centers (Bester et al. 1997; Kawano and Masuko 1993; Lind and Swanson 1984; Saper and Levisohn 1983; Zardetto-Smith and Johnson 1995). Lesions centered on MnPO result in profound disruptions in cardiovascular regulation, fluid balance, angiotensin-induced drinking, and salt appetite (Cunningham et al. 1991; Gardiner and Stricker 1985; Gardiner et al. 1985, 1986; Mangiapane et al. 1983), attesting to the importance of this nucleus in maintaining homeostasis in autonomic, neuroendocrine, and cardiovascular systems.
It has long been known that the ability for circulating angiotensin to elicit a drinking response in the rat depends on the integrity of the SFO and its connectivity with MnPO (Eng and Miselis 1981; Lind and Johnson 1982). Whereas the intrinsic properties and neuropharmacology of SFO neurons have been revealed in some detail (reviewed in Washburn and Ferguson 2001), relatively little is known about the nature of the SFO projection to MnPO. In this study, we used patch-clamp recording techniques in brain slice preparations to characterize the responses elicited in MnPO neurons by electrical stimulation along the ventral edge of the SFO. Our observations indicate that both GABAA and glutamate ionotropic receptors mediate rapid neurotransmission from SFO to MnPO. Recent reports also identify MnPO with moderate to high levels of GABABR1 immunoreactivity and mRNA (Bischoff et al. 1999; Margeta-Mitrovic et al. 1999), consistent with our data indicating the presence of both pre- and postsynaptic GABAB receptors that may modulate SFO and other GABAergic and glutamatergic transmission to MnPO neurons.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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) Briefly, animals were decapitated, and the brain was quickly removed and immersed in oxygenated (95% O2–5% CO2) cooled (<4°C) artificial cerebrospinal fluid (ACSF) of the following composition (in mM): 127 NaCl, 3.1 KCl, 1.3 MgCl2, 2.4 CaCl2, 26 NaHCO3, and 10 glucose, pH 7.3, osmolality 300–310 mOsm/kg. The brain was sliced in the sagittal plane with a vibratome, and a single midline slice 400–500 µm in thickness that contained the SFO and MnPO was preincubated in gassed ACSF for >1 h at room temperature and transferred to a submerged chamber and superfused (2–3 ml/min) with oxygenated ACSF at 22–5°C. Using the blind patch-clamp technique, recordings were obtained from MnPO neurons with borosilicate thin-walled micropipettes filled with (in mM) either 130 K-gluconate, 10 KCl, 10 NaCl, 2 MgCl2, 10 HEPES, 1 EGTA, 2 Mg-ATP, and 0.3 Na-GTP or cesium salts (e.g., Cs-methylsulfonate or CsCl) substituted for K-gluconate and KCl (pH adjusted with KOH or CsOH, respectively, to 7.3) when recordings required amplification of spontaneous and miniature inhibitory postsynaptic potentials (mIPSPs). Pipettes had resistances of 3–7 M
. Correction for liquid junction potential was applied to recorded membrane currents and voltages. Access resistance <15 M was considered acceptable. Input resistances were determined from the linear slope (i.e., between –50 and –80 mV) of the current-voltage (I-V) relationships. Whole cell current-clamp and voltage-clamp recordings were obtained with an Axopatch 200B (Axon Instruments, Union City, CA). Data were filtered at 2 kHz, continuously monitored, and stored on videotape. Digidata 1200 interface and clampex (pClamp8) software (Axon Instruments) were used on-line to generate current and voltage commands. Afferents to MnPO arising from the SFO were activated with a concentric bipolar electrode (25 µm tip diam; FHC, Bowdoinham, ME) positioned at the ventral aspect of the SFO and connected to a stimulus isolation unit that delivered voltage pulses (1–30 V, 0.1 ms) under program control.
Off-line analyses were performed using Clampfit version 8 (Axon Instruments). Statistical comparisons between control and experimental values (P < 0.05 and better) were determined using both the paired or unpaired Student t-test and ANOVA. Results are expressed as means ± SE. For assessment of miniature postsynaptic events, 2–3 min of recordings (minimum of 100 events) were analyzed using Mini Analysis software from Synaptosoft (Leonia, NJ). Miniature events were defined as those recorded in the presence of 1 µM TTX and detected with an adjustable threshold that was maintained at a constant level in a given neuron. The analysis was performed by using cumulative probability plots, and statistical comparisons were evaluated with Kolmogorov-Smirnov (K-S) test.
Drugs were bath applied at the concentrations indicated. These included (–)-bicuculline methochloride (BIC), D(–)-2-amino-5-phosphonovaleric acid (APV), 2,3-dioxo-6nitro-1,2,3,4-tetrahydrobenzo [f] quinoxaline-7-sulfoamide disodium (NBQX), (R)-4-amino-3-(4-chlorophenyl) butanoic acid (Baclofen), guvacine, and CGP52432from Tocris Cookson (Ballwin, MO), -conotoxin GVIA from Bachem (Torrance, CA), norepinephrine, Mg-ATP, Na-GTP, NO711, and cytochrome C from Sigma and/or Sigma-RBI (St. Louis, MO), and TTX from Alomone Laboratories (Jerusalem, Israel).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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To assess the nature of synaptic transmission from SFO to MnPO, we applied electrical stimulation at the ventral edge of the SFO, a location likely to activate the majority of axons coursing toward MnPO. Postsynaptic responses were evident in 28/40 neurons tested. Since these featured both excitatory and inhibitory components, their further characterization was conducted in the presence of selective pharmacological receptor antagonists. In ACSF containing NBQX (5 µM) and APV (20 µM), SFO stimulation evoked IPSPs at a latency of 8.9 ± 1.2 ms, reversing polarity at –65 mV and reversibly blockable on addition of BIC (20 µM; n = 10; Fig. 1, A and B). In any given neuron, IPSPs displayed a constant latency over a range of stimulation intensities and ability to follow three pulses at 20 Hz, features deemed consistent with a monosynaptic connection. In ACSF containing BIC, SFO stimulation evoked excitatory postsynaptic potentials (EPSPs) at a latency of 7.9 ± 1.3 ms (n = 7). EPSPs also displayed constant latencies over a range of stimulation intensities and reliably followed three pulses at 20 Hz.
At resting membrane potentials, EPSPs displayed fast and slow components. The amplitude of the "slow" component measured at 200 ms after the stimulus artifact was reduced by membrane hyperpolarization and by the addition of APV (20 µM; Fig. 1C; n = 10), consistent with an N-methyl-D-aspartate (NMDA) receptor-mediated component. The remaining fast component of the EPSP was abolished by further addition of NBQX (5 µM; Fig. 1C). Under voltage clamp in the presence of ACSF containing NBQX and APV, SFO-evoked inhibitory postsynaptic currents (IPSCs; VH, –45 mV) were recorded as outward currents that reversed at –66.5 ± 3.5 mV (n = 11) and were blockable with BIC (n = 3). IPSC amplitudes (but not rise-time or decay time constants) differed significantly (P < 0.01) depending on the cell's location: 38.3 ± 11.5 pA for cells recorded in the dorsal MnPO (n = 8) versus 16.8 ± 1.8 pA for cells in the ventral MnPO (n = 20). In ACSF containing BIC, SFO-evoked excitatory postsynaptic currents (EPSCs; VH, –55 mV) were similar among ventral (38.9 ± 8.8 pA; n = 15) and dorsal MnPO cells (33.5 ± 12.6 pA; n = 4) and blockable with a cocktail of NBQX and APV (n = 4). Thus under our experimental conditions, amino acids appear to be the sole mediators of rapid neurotransmission along the SFO pathway to MnPO.
Because the activity of SFO neurons is reported to vary from slow firing in vivo (0.1–1.2 Hz; Tanaka et al. 1987) to bursting patterns when recorded in vitro as isolated cell preparations (Washburn et al. 2000), we were prompted to test responsive MnPO neurons to SFO stimulation presented in a brief burst (20 Hz for 0.5 s). When applied in the presence of bicuculline, all seven cells responded with a slow membrane depolarization, triggering a burst of action potentials (data not shown). After addition of NBQX to block AMPA and kainate receptors, 5/7 cells still responded to the stimulus barrage with slow membrane depolarization lasting up to 2 s (Fig. 1D). Since this response was either totally abolished or substantially attenuated after further addition of APV, it would appear that NMDA receptors mediated most or all of this slow depolarization.
GABAB receptor activation suppresses SFO inhibitory and excitatory inputs to MnPO
The preceding observations imply that GABAA and glutamate ionotropic receptors mediate virtually all rapid neurotransmission from SFO to MnPO. Two recent reports also identified MnPO with moderate to high levels of GABABR1 immunoreactivity and mRNA (Bischoff et al. 1999; Margeta-Mitrovic et al. 1999), so we next investigated a role for GABAB metabotropic autoreceptors. Within minutes of the addition of a GABAB receptor agonist baclofen (0.3–10 µM) to the bath, both inhibitory and excitatory SFO-evoked postsynaptic currents underwent a significant concentration-dependent and reversible reduction in their amplitudes (Fig. 2). In 0.3 µM baclofen, the mean IPSC amplitude was reduced to 70.8 ± 9.6% of control (n = 4; P < 0.01); in 3 µM baclofen, the reduction was to 32.4 ± 5.1% of control (n = 11; P < 0.01). Similarly, for EPSCs, 0.3 µM baclofen reduced their mean amplitude to 81 ± 9.6% of control (n = 4; P < 0.05), 3 µM baclofen effected a reduction to 39.3 ± 5.6% of control (n = 6; P < 0.01), and 10 µM baclofen caused a reduction to 16.3 ± 7.5% of control (n = 3; P < 0.001). Given that baclofen can also influence postsynaptic membrane conductances (see Fig. 5), we examined the kinetics of normalized IPSCs and EPSCs and found no changes (Fig. 2). While barium (1 mM) does influence postsynaptic GABAB receptor function, its presence did not influence baclofen's ability to suppress SFO-evoked IPSCs (n = 4 cells) or EPSCs (n = 3 cells). Efforts to evaluate the influence of conotoxin were impeded by its ability to block synaptic transmission. Baclofen-induced effects on IPSCs (n = 3) or EPSCs (n = 2) were completely blockable when CGP52432(1–3 µM), a potent GABAB receptor antagonist (Lanza et al. 1993), was added prior to application.
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To determine whether the baclofen-induced reduction in SFO-evoked IPSCs and EPSCs was mediated by a presynaptic mechanism that reduced transmitter release, we examined effects on paired-pulse protocols. SFO-evoked postsynaptic currents displayed varying degrees of paired-pulse depression (PPD), and less commonly facilitation (PPF), when tested at interstimulus intervals between 100 and 200 ms. In 19 cells, where a mean P2/P1 ratio of 0.81 ± 0.02 for paired IPSCs reflected a strong PPD, this ratio changed from a trend toward an increase in 0.3 µM baclofen (from 0.88 ± 0.05 to 1.04 ± 0.25; n = 4; P > 0.05) to a significant increase in 3 µM baclofen (from 0.92 ± 0.04 to 1.16 ± 0.06; n = 8; P < 0.01). This was due to baclofen's suppression of the amplitude of both the P1 and P2 responses, with the larger action affecting the P1 response (see Fig. 3A). For EPSCs, an overall average P2/P1 ratio of 1.08 ± 0.08 (n = 15) included four cells displaying PPF (1.36 ± 0.19) and six cells displaying PPD (0.75 ± 0.04). Among the cells displaying PPD, the P2/P1 ratio changed from 0.82 ± 0.14 to 1.01 ± 0.27 (n = 4; P > 0.05) in 0.3 µM baclofen, and 1.52 ± 0.32 (n = 5; P < 0.05) in 3 µM baclofen, again reflecting an action on both P1 and P2 responses with the greatest change involving the amplitude of the P1 response. These features are consistent with a modulating action of presynaptic GABAB receptors.
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GABAB receptor depression of TTX-independent transmitter release: mechanisms
MnPO neurons display spontaneous inhibitory and/or excitatory amino acid-mediated postsynaptic potentials. Investigation of the effects of baclofen on calcium- and TTX-independent miniature events that represent quantal release of transmitter can provide insight into their signaling mechanisms. In ACSF containing APV and NBQX, mIPSCs were amplified using a CsCl-based internal solution. As noted by Lenz et al. (1997), such a recording arrangement significantly reduced the outward current induced by 10 µM baclofen compared with data obtained using a potassium-based internal solution (7.8 ± 2.6 pA, n = 5, vs. 17.4 ± 2.1 pA, n = 18; P < 0.05). Under these conditions, addition of baclofen decreased mIPSC frequency to 31.7 ± 7.04% of control (from 1.3 ± 0.25 to 0.39 ± 0.12 Hz; P < 0.05) without affecting their amplitude distribution (9.57 ± 4.12 vs. 8.47 ± 4.04 pA, P > 0.05; n = 6; Fig. 4, A and B), rise, or decay times. Baclofen also significantly reduced mEPSC frequency (from 1.8 to 0.3 Hz; P < 0.05) without a change in their amplitude (n = 3; data not shown). Interestingly, in 2/4 cells tested, application of CGP52432alone induced an increase in mIPSC frequency (161.9 ± 1.2% of control) without affecting amplitude distribution, rise, or decay times. Therefore by contrast with the negative findings on evoked responses mentioned above, ambient levels of GABA may be sufficient to activate some presynaptic GABAB receptors under resting conditions in this preparation.
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). As noted below in the section on postsynaptic GABAB receptors, barium and -conotoxin GVIA alter receptor coupling to potassium and calcium channels, respectively. However, when tested for an influence on baclofen-induced actions on mIPSCs, each agent alone nonsignificantly increased mIPSC frequency, but neither altered the response to baclofen (Fig. 4B). To evaluate possible coupling to high-voltage–activated (HVA) calcium channels other than the N-type, we applied cadmium at a concentration (100 µM) that completely blocked postsynaptic calcium channels in MnPO neurons (see Kolaj and Renaud 2001). The result was a nonsignificant increase in mIPSC frequency (to 115.8 ± 8% of control). Cadmium did significantly decrease mIPSC amplitude (to 69.6 ± 6.3% of control; n = 5; P < 0.001; Fig. 4B) and decay times (from 37.13 ± 1.01 to 33.06 ± 2.06 ms; P < 0.05), an action likely due to its effect on postsynaptic GABAA receptors (LeFeuvre et al. 1997). Postsynaptic GABAB-mediated responses in MnPO neurons
Baclofen depressed excitability in 80% of cells tested in the dorsal and ventral MnPO. Under current clamp, applications of baclofen (5–10 µM) induced a gradual membrane hyperpolarization (12.8 ± 1.1 mV) that peaked after 30–150 s and lasted 5.6 ± 1.1 min, accompanied by a cessation in spontaneous action potential discharges (Fig. 5A; n = 14 cells). When recorded under voltage clamp (VH, –55 mV) and in the presence of 0.5 µM TTX, the response to baclofen was a slowly rising outward current that persisted for 7.1 ± 0.8 min at the 10 µM concentration (Fig. 5B; n = 14 cells). This response was concentration-dependent (EC50 = 0.36 µM), associated with a proportionate increase in membrane conductance (110.7 ± 3.9% with 0.3 µM baclofen; 139.3 ± 7.1% with 10 µM baclofen; P < 0.05), and was completely blockable in the presence of CGP52432(1–3 µM; n = 13). Whereas the addition of GTP-
-S to the internal solution yielded responses to 10 µM baclofen that were similar in magnitude to those under control conditions (peak outward current of 16.3 ± 3.6 pA and conductance increase of 142.1 ± 9.1%; n = 4), the effect was not reversible by 20 min. These data are consistent with mediation through G protein–coupled receptors.
In hippocampus and other preparations, electrical stimulation may evoke sufficient release of GABA to reveal a late slow IPSC due to activation of postsynaptic GABAB receptors, an event that can be enhanced by addition of GABA uptake inhibitors (Isaccson et al. 1993). When tested in the presence of NO711 (25–50 µM) or guvacine (50–100 µM) to block GABA uptake, and in ACSF containing GABAA and glutamate receptor blockers, no late slow IPSCs were observed to follow a burst of SFO stimuli (5–10 pulses at 20 Hz; n = 5). In contrast with the situation in hippocampal slice preparations and despite the evidence for functional GABAB receptors in MnPO, it would appear that GABA released by SFO stimulation was not sufficient to activate postsynaptic GABAB receptors. Other investigators have reported difficulties in evoking postsynaptic GABAB responses (e.g., Jensen et al. 2003; Overstreet and Westbrook 2001). In MnPO, one might speculate that this reflects a combination of the extrasynaptic location of GABAB receptors and the particular anatomical features of the synapses arising from SFO afferents. Other identified inputs to MnPO neurons have yet to be examined.
Postsynaptic GABAB receptors increase a potassium conductance
The baclofen-induced membrane hyperpolarization and increased conductance in MnPO neurons was mediated by potassium channels, consistent with data from other CNS neurons (Misgeld et al. 1995). I-V plots revealed intersections and/or net baclofen-induced conductances that reversed at –97.7 ± 2.6 mV (Fig. 6, A–C; n = 16), approximating the potassium equilibrium potential (EK+) of –98 mV under these experimental conditions. In ACSF containing 10 mM potassium, this value shifted from –93.2 ± 1.7 to –63.3 ± 5.4 mV (Fig. 6D; P < 0.01; n = 6), a 29.8 ± 6.1-mV difference consistent with the calculated EK. Conductance plots also displayed inward rectification, with a coefficient for the net baclofen-induced current (calculated as the ratio between currents at –110 and –20 mV), increasing significantly from –1.27 ± 0.38 in control ACSF to –6.40 ± 1.11 in ACSF containing 10 mM potassium (P < 0.05; n = 6); virtually all of this difference was attributable to increased currents at hyperpolarized levels. The addition of barium (0.1–1 mM) to block potassium channels induced an inward current of –5.6 ± 1.8 pA coupled with a decrease in membrane conductance (to 80.3 ± 5.6% of control), with reversal of polarity at –90.3 ± 5.6 mV. In the presence of barium, reductions were noted in both the baclofen-induced outward current (Fig. 6E; from 15.2 ± 2.3 to 3.6 ± 1.3 pA; P < 0.001) and conductance (from 130.8 ± 4.6% to 107 ± 4.1% of control; P < 0.05; n = 5). The baclofen-induced outward current was unchanged by further addition of cadmium (200 µM; n = 3).
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). However, this treatment failed to significantly alter the baclofen-induced outward current (13 ± 1.9 pA in control vs. 11.8 ± 2.9 pA in ZD7288) or resting conductance (125 ± 9.6% in control vs. 118.3 ± 5.9% in ZD7288). Thus whereas the membrane hyperpolarization evoked by baclofen appears to be more pronounced in cells with a strong IH, the baclofen-induced current is largely due to opening of potassium channels of the GIRK type. Postsynaptic GABAB receptors suppress a calcium conductance
Activation of GABAB receptors is also known to suppress calcium conductances (Misgeld et al. 1995). We therefore used cesium-filled pipettes, added TTX to the ACSF, and replaced calcium with barium as a charge carrier, a choice based on barium's ability to block both the baclofen-induced activation of GIRK channels as well as several other potassium channels. With neurons held at –60 mV and exposed to slow depolarizing ramp commands (from –90 to +50 mV), the evoked calcium current had a mean amplitude of –274 ± 43 pA (n = 9), peaking between –10 and 0 mV. Cadmium (100 µM), a broad-spectrum calcium channel blocker, virtually abolished this current (1.8 ± 0.6% of control; n = 3). As illustrated by the example in Fig. 7, application of 10 µM baclofen reversibly suppressed peak inward current to 70.4 ± 3.6% of control (n = 9; P < 0.01). Baclofen also produced a small outward current (4.5 ± 2.2 pA; n = 9), but its contribution to calcium currents was negligible after using leak subtraction protocol.
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2 receptors selectively suppressed N-type HVA calcium channels (Kolaj and Renaud 2001). When tested on four cells (see example in Fig. 7A), we observed a comparable suppression in the HVA calcium current with both 10 µM baclofen (65.9 ± 6.5%) and 30 µM norepinephrine (68.4 ± 9.3%), but saw no additive effect when baclofen and norepinephrine were administered together (71.8 ± 6.2%; P > 0.05). After application of -conotoxin GVIA (2 µM), a specific blocker of N-type calcium channels that irreversibly suppressed peak calcium currents to 67.9 ± 4.6% (P < 0.05), baclofen had no further effect, implying that GABAB receptors and 2 adrenoceptors in MnPO neurons both act to suppress N-type HVA calcium channels. Postsynaptic GABAB receptors modulate IAHP and increase cell excitability
Physiological consequences of Ca2+ entry through Ca2+ channels include the activation of Ca2+-dependent K+ currents that underlie and contribute to action potential repolarization, afterhyperpolarization (AHP), and spike-frequency adaptation (Bevan and Wilson 1999; Meech 1978). Given the suppressant effect of baclofen on Ca2+ channels, we examined membrane currents underlying the AHP (IAHP) under voltage clamp. Tail currents following 200-ms depolarizing voltage steps (to +10 mV from a holding potential of –55 mV) were depressed by baclofen (10 µM) from 41.1 ± 12.6 to 28 ± 9.9 pA (i.e., to 64.9 ± 4.9% of control; P < 0.01; Fig. 7, B and C) in 8/10 cells tested. As expected, IAHP was also significantly reduced by barium (1 mM; to 50.9 ± 14.9% of control) and by -conotoxin GVIA (2 µM; to 12.2 ± 5.2% of control). The nonselective Ca2+ channel blocker cadmium (200 µM) reduced IAHP to 5.1 ± 1.9% of control (Fig. 7, B and C).
Since AHP can have a profound influence on the firing pattern of a neuron (Storm 1990), we next examined how baclofen might affect the firing pattern of MnPO neurons based on response to a test pattern consisting of 2-s depolarizing current pulses of 35 pA prior to, and during, the peak of the baclofen-induced response. To offset the hyperpolarizing effect of baclofen, current was injected to maintain the cell at the resting potential level (51.4 ± 1.9 mV in control vs. 50.9 ± 1.6 mV during baclofen; 5 µM; n = 13). As shown in the example in Fig. 8, A and B, application of such tests at the peak of the baclofen-induced response revealed both an increase in firing frequency (118.4 ± 5.5% over control; P < 0.05) and a shortening of the mean initial (1st 5) instantaneous frequencies (123.1 ± 4.4% over control; P < 0.01). In contrast, no change was detected in the last five instantaneous frequencies in the burst (103.9 ± 7%). Therefore spike frequency adaptation (calculated as the ratio between the initial and last interspike frequencies) increased in the presence of baclofen (from 1.71 ± 0.25 to 2.03 ± 0.32; P < 0.05). There was no obvious correlation between the size of baclofen-induced outward current and changes in these firing properties. Analysis of first spike during the 35-pA pulse in 13 cells revealed no significant change in amplitude (60.1 ± 4.1 mV in control vs. 60.3 ± 4 mV in baclofen; measured from threshold), width (2.35 ± 0.24 vs. 2.35 ± 0.22 ms), threshold (31.7 ± 1.4 vs. 31.3 ± 1.4 mV), or spike delay (26.4 ± 3.6 vs. 27.4 ± 4.8 ms; measured as time from the beginning of pulse to the 1st peak). However, in keeping with the data for IAHP presented above, there was a significant decrease in the amplitude of the AHP (14.7 ± 1.6 vs. 12.3 ± 1.5 mV; P < 0.05), supporting a role of the AHP in defining the firing properties of these neurons. In cadmium, which blocked calcium channels and decreased IAHP, mean firing frequency increased by 179.5 ± 24.9% during a 35-pA pulse (P < 0.05), and the AHP decreased from 15.4 ± 3.5 to 11.6 ± 3.1 mV (P < 0.05; n = 4), without a change in spike amplitude, width, or threshold.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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In earlier extracellular studies in vivo, Tanaka et al. (1987) reported that stimulation in SFO evokes inhibitory and excitatory responses in MnPO neurons. The present intracellular analysis convincingly demonstrates mediation of rapid neurotransmission in this pathway by GABAA and glutamate receptors. Interestingly, SFO-evoked excitation in MnPO neurons has been proposed to be mediated by the peptide angiotensin (Tanaka et al. 1987). Consistent with this notion are reports of angiotensin-like immunoreactivity in SFO neurons and their axonal projections (Jhamandas et al. 1989; Lind et al. 1984), a high-density of angiotensin AT1 receptors in MnPO (Lenkei et al. 1998), and activation of a population of MnPO neurons by exogenous angiotensin (Bai and Renaud 1998a; Travis and Johnson 1993). It seems reasonable to propose angiotensin as a coexisting neuropeptide whose co-release might contribute to "delayed amplification" of synaptic transmission (see Hökfelt et al. 2000). If so, one might anticipate that SFO stimulation might evoke both an early event related to the release of rapidly acting neurotransmitters and a "later prolonged" increase in neuronal excitability due to the effects of a co-released neuropeptide. In fact, such has been observed in vivo in extracellular recordings from hypothalamic supraoptic nucleus neurons, also known targets of SFO efferent fibers, where the late component of the SFO-evoked response could be partially reduced by local application of saralasin (Jhamandas et al. 1989). Conceivably, the late response was the "signature" of endogenous angiotensin release in an SFO efferent pathway. In vitro slice preparations may present conditions (e.g., room temperature; need for peptidase inhibitors) less favorable for eliciting/detecting endogenous release of the peptide. Notably few in vitro mammalian brain slice preparations have in fact yielded observations attributable to their synaptic release. It is anticipated that future studies will clarify the features related to endogenously released angiotensin within the SFO projection to MnPO.
Presynaptic GABAB receptors: a calcium independent mechanism?
Activation of presynaptic GABAB receptors can reduce GABA and/or glutamate release, thereby reducing the efficacy of neurotransmission at many CNS sites; this is commonly attributed to inhibition of presynaptic calcium channels (reviewed in Misgeld et al. 1995), although other mechanisms have been implicated (Capogna et al. 1996). Our observations concur with presynaptic GABAB receptor modulation at both inhibitory and excitatory inputs to MnPO neurons, including those arising from SFO. In the present situation, it is interesting that the baclofen-induced effects lacked sensitivity to barium, -conotoxin GVIA, or cadmium. While the nature of these receptors and their physiological role in MnPO will require further analysis, our investigation suggests a mechanism of action that does not involve either presynaptic reduction in calcium conductance and/or an increase in potassium conductance. Baclofen remained effective in the presence of cadmium at concentrations that blocked HVA calcium currents in these slices (Fig. 7) and in dissociated MnPO neurons (Kolaj and Renaud 2001). Since inhibition induced by presynaptic GABAB receptors can involve both calcium-dependent and calcium-independent mechanisms (Capogna et al. 1996; Wu and Saggau 1995), the signaling mechanism that effects the baclofen-induced decrease in mIPSC frequency in MnPO neurons may occur downstream from calcium influx, possibly through a direct interaction with exocytotic processes (Dittman and Regehr 1996; Sakaba and Neher, 2003).
Postsynaptic GABAB receptors couple with potassium and calcium channels
In contrast with observations in hippocampus where GABAB receptors can mediate a late slow IPSP (reviewed in Misgeld et al. 1995), the SFO-evoked IPSPs in MnPO neurons were completely blockable with bicuculline. In hippocampus, postsynaptic GABAB receptors are located extrasynaptically, and their activation is under tight control of GABA uptake mechanisms (Scanziani 2000). While the latter was not investigated in detail here, effects of their activation by exogenously applied agonists resembled that reported in other neurons: suppression of neuronal firing consequent to membrane hyperpolarization through increase in a G protein–coupled inwardly rectifying potassium conductance and suppression of voltage-gated calcium channels. The latter action of GABAB receptor activation has been noted to differ among neurons in terms of channel subtypes involved: L-, N-, and P/Q-type in hippocampal inhibitory neurons (Lambert and Wilson 1996); N- and P/Q-type in supraoptic neurons (Harayama et al. 1998); and L-type in cerebellar granule cells (Wojcik et al. 1990). Observations in MnPO neurons are consistent with a selective effect on N-type channels since the magnitude of the baclofen-induced reduction of calcium channel function was comparable with that induced by -conotoxin GVIA, and most of the baclofen-induced suppression was completely abolished by -conotoxin GVIA. In acutely dissociated MnPO neurons, we previously reported suppression of N-type channels by norepinephrine acting via 2 adrenoceptors (Kolaj and Renaud 2001). Given that the action of baclofen in these slice preparations could be occluded by norepinephrine (Fig. 7), it is possible that similar downstream mechanism(s) may mediate suppression of N-type calcium channels subsequent to activation of both GABAB and 2 adrenoceptors.
In MnPO neurons, a correlation observed between the magnitude of the baclofen-induced outward current and the resting conductance suggest a possible contribution of this current to resting membrane conductance. On the other hand, we saw no correlation between baclofen-induced suppression of SFO-evoked responses and the change in resting conductance. Collectively, these findings suggest a lack of correlation between the presynaptic and postsynaptic effects of baclofen, in keeping with the evidence that pre- and postsynaptic GABAB receptors in MnPO neurons utilize different mechanisms and/or receptor subtypes, as proposed for other central sites (Yamada et al. 1999). Interestingly, it has been suggested that presynaptic GABAB receptors contain R1a subunits while the postsynaptic receptors contain R1b subunits (Bischoff et al. 1999; Kaupmann et al. 1998a,b).
GABAB receptors: a role in modulation of firing patterns?
The GABAB receptor-induced decrease in neuronal excitability and spontaneous firing was seen to involve but a few tens of picoamps of outward current. However, because of the high-input resistance (low resting conductance) of MnPO neurons (
1 nS), this could result in a significant change in membrane potential (10 pA will cause a change of 10 mV with a conductance of 1 nS), especially important if the membrane potential were close to threshold for action potential generation. The fact that cadmium did not change the amplitude of the baclofen-induced outward (potassium) current implies little contribution of calcium channels toward the magnitude of membrane hyperpolarization. However, should the calcium currents measured in our configuration be of somatic origin, it is conceivable that they may couple with calcium-activated potassium channels that contribute toward the magnitude of the AHP whose reduction could lead to increased firing rate and longer burst duration during transient depolarization (Matsushima et al. 1993). Furthermore, we did not find correlation between baclofen-induced outward current and change in firing properties suggesting heterogeneous coupling between GABAB receptors and potassium versus calcium channels. By analogy with recent observations in retinal ganglion cells (Zhang et al. 1998), it is possible that GABAB receptors could serve as discriminators, effectively reducing the influence of weak signals while boosting responses to strong signals. In this regard, one might speculate that SFO neurons might be able to provide such strong signals to MnPO neurons, as might arise under conditions where threats to body fluid homeostasis (e.g., raised plasma osmolality or circulating levels of angiotensin) would raise impulse traffic in the SFO to MnPO pathway.
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Address for reprint requests and other correspondence: L. P. Renaud, Neuroscience Program, Ottawa Health Research Institute, 725 Parkdale Ave., Ottawa, Ontario K1Y 4E9, Canada (E-mail: lprenaud{at}ohri.ca).
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  B. Nahir, C. Bhatia, and C. J. Frazier Presynaptic Inhibition of Excitatory Afferents to Hilar Mossy Cells J Neurophysiol, June 1, 2007; 97(6): 4036 - 4047. [Abstract] |
) determined by subtraction of these I-V values displays reversal of approximately –90 mV. C: mean net baclofen current for 16 cells indicates reduction at more hyperpolarized levels with reversal of approximately –90 mV and mild rectification. D: plots of mean net baclofen currents from 6 neurons with different extracellular potassium concentrations show the shift in reversal potential from approximately –90 mV in normal ACSF (
), in agreement with values calculated from the Nernst equation. Note the larger rectification with higher potassium concentrations. E: plots of the mean net currents from 4 cells where baclofen (3 µM for 30–50 s) was applied 1st in normal ACSF (
) and again 20 min later after preincubation in ACSF containing barium (
; 0.5–1 mM for 5–7 min). The net barium current (shaded diamond symbol) is in the opposite direction to the net baclofen current, showing that the barium-induced inward current was due to a closing of potassium channels.
