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. 2002 Nov 1;545(Pt 3):855–867. doi: 10.1113/jphysiol.2002.030049

Cellular mechanisms of orexin actions on paraventricular nucleus neurones in rat hypothalamus

Matthew J Follwell 1, Alastair V Ferguson 1
PMCID: PMC2290730  PMID: 12482891

Abstract

Using whole-cell patch clamp techniques we have examined the cellular mechanisms underlying the effects of orexin A (OX-A) on electrophysiologically identified magnocellular and parvocellular neurones in the rat hypothalamic paraventricular nucleus (PVN). The majority of magnocellular neurones (67 %) showed concentration-dependent, reversible depolarizations in response to OX-A. These effects were abolished in tetrodotoxin (TTX), suggesting them to be indirect effects on this population of neurones. OX-A also caused increases in excitatory postsynaptic current (EPSC) frequency and amplitude in magnocellular neurones. The former effects were again blocked in TTX while increases in mini-EPSC amplitude remained. Depolarizing effects of OX-A on magnocellular neurones were also found to be abolished by kynurenic acid, supporting the conclusion that these effects were the result of activation of a glutamate interneurone. Parvocellular neurones (73 % of those tested) also showed concentration-dependent, reversible depolarizations in response to OX-A. In contrast to magnocellular neurones, these effects were maintained in TTX, indicating direct effects of OX-A on this population of neurones. Voltage clamp analysis using slow voltage ramps demonstrated that OX-A enhanced a non-selective cationic conductance with a reversal potential of -40 mV in parvocellular neurones, effects which probably explain the depolarizing effects of this peptide in this subpopulation of PVN neurones. These studies have identified separate cellular mechanisms through which OX-A influences the excitability of magnocellular and parvocellular PVN neurones.

Since the initial studies describing orexins (OX) (Sakurai et al. 1998) and hypocretins (de Lecea et al. 1998), which suggested important roles in the control of feeding behaviour, a number of reports have suggested additional roles for the involvement of these peptides in the control of narcolepsy (Chemelli et al. 1999) and diverse autonomic functions including hormone secretion, energy metabolism and cardiovascular control (Samson et al. 1999). The immunocytochemical identification and mapping of OX-projecting fibres throughout the brain (Peyron et al. 1998) directed attention to a number of important brain nuclei as the likely sites underlying the physiological actions of these peptides. In addition to the demonstration of projections to the locus coeruleus, zona incerta, central grey and substantia nigra (all suggested to be involved in maintenance of the arousal state), the demonstration of orexinergic projections to the paraventricular nucleus of the hypothalamus (PVN), nucleus of the solitary tract (NTS), parabrachial nucleus and spinal cord (Peyron et al. 1998; van den Pol, 1999) identified potential targets at which orexins may act to exert such diverse influences over central autonomic control.

Immunocytochemical studies have identified OX-R1 receptors on magnocellular and parvocellular neurones of the PVN (Backberg et al. 2002) emphasizing the potential importance of orexins in controlling the excitability of neurones in this nucleus, which is uniquely positioned to influence not only hormone secretion from the pituitary, but also control of autonomic output, as a consequence of descending projections to medullary and spinal autonomic centres.

Intriguingly, intracerebroventricular (i.c.v.) injection of OX-A and OX-B into the lateral cerebral ventricle of conscious, unrestrained rats resulted in an increase in blood pressure and heart rate, suggesting a stimulation of sympathetic function (Samson et al. 1999). The exact site of action of OX-A in the brain that mediates these cardiovascular effects is not known. The rapid onset of action following lateral ventricle administration of the peptide also points to the hypothalamic PVN as a likely site of action. Anatomical mapping indicating dense innervation by orexin-positive fibres of the PVN, combined with up-regulation of fos-like immunoreactivity within this nucleus following i.c.v. administration of the peptide (Edwards et al. 1999), provide the framework for further analysis of the cellular mechanisms of OX actions on PVN neurones.

The PVN consists of magnocellular (MNC - neurohypophysial oxytocin and vasopressin) and parvocellular (PARVO - corticotrophin releasing hormone (CRH) as well as other tuberoinfundibular) neurones, as well as glutamate and GABA interneurones which are now recognized as playing essential roles in regulating the excitability of these neurones (Decavel & van den Pol, 1990; Wuarin & Dudek, 1991; Bains & Ferguson, 1997; Daftary et al. 1998, 2000). Recent studies demonstrating OX influences on PVN neurones (Shirasaka et al. 2001; Samson et al. 2002) have neither identified specific actions on separate subpopulations of PVN neurones, nor described the specific cellular and membrane events underlying such effects. The present studies were therefore undertaken to determine the specific ion channels and synaptic events underlying OX actions on electrophysiologically identified subpopulations of PVN neurones.

Methods

Slice preparation

Experiments were performed using hypothalamic slices prepared as previously described (Li & Ferguson, 1996). Male Sprague-Dawley rats (150-250 g, Charles River, Quebec, Canada) were decapitated, and the brain quickly removed from the skull and immersed in cold (1-4 °C) artificial cerebrospinal fluid (aCSF). The hypothalamus was blocked and 400 μm slices including the PVN were cut using a vibratome. Slices were incubated in oxygenated aCSF (95 % O2-5 % CO2) for at least 90 min at room temperature. Thirty minutes prior to recording, the slice was transferred into an interface-type recording chamber and continuously perfused with oxygenated aCSF (see below for solution composition) at a rate of 1 ml min−1. All techniques were carried out in accordance with the guidelines of the Canadian Council for Animal Care and were approved by Queen's University Animal Care Committee.

Electrophysiology

Whole-cell patch recordings were obtained using the whole-cell configuration of the ‘blind’ patch clamp technique to record from PVN neurones (see Li & Ferguson, 1996). Patch pipettes were pulled to a resistance of 5-8 MΩ and filled with internal solution (see below for solution composition). Following establishment of a 1 GΩ seal as monitored by voltage response to hyperpolarizing current pulses, a brief suction was applied to rupture the membrane and achieve whole-cell configuration. Signals were processed with an Axoclamp-2A amplifier. A Ag-AgCl electrode connected to the bath solution via a KCl-agar bridge served as reference. A 3-8 mV junction potential correction was applied to all data presented. When recording excitatory postsynaptic currents (EPSCs), the membrane was clamped at -80 mV to eliminate Cl−-mediated currents (thus GABA antagonists were not used), such as inhibitory postsynaptic currents (ISPCs). This is slightly more positive than the reversal potential for Cl− (ECl = -83.2 mV) predicted by the Nernst equation, as the passage of other anions (e.g. CO3−) would result in a reversal potential for IPSCs that is more depolarized than ECl (Hille, 1992). Drugs were applied by switching perfusion from aCSF to a solution containing the desired drug. All signals were digitized using the CED 1401 plus interface (CED, Cambridge, UK) and stored on computer for off-line analysis. Data were collected using the Signal (episode based capture) or Spike2 (continuous recording) packages (CED).

Solutions

The internal pipette solution contained (mm): potassium gluconate, 140; CaCl2, 0.1; MgCl2, 2; EGTA, 1.1; Hepes, 10; Na2ATP, 2; adjusted to pH 7.25 with KOH. The aCSF composition was (mm): NaCl, 124; KCl, 2; KPO4, 1.25; CaCl2, 2.0; MgSO4, 1.3; NaHCO3, 20; and glucose, 10. Osmolarity was maintained between 285 and 300 mosmol l−1 and pH between 7.3 and 7.4.

Peptides and drugs

OX-A (Phoenix Pharmaceuticals, Belmont, CA, USA) was prepared fresh on the day of experiment from 100 μl aliquots of a 10 μm stock solution stored at -70 °C, to concentrations ranging from 1 to 500 nm in aCSF. Na+ channels were blocked by adding 1-5 μm tetrodotoxin (TTX) in aCSF. Blockade of Na+ channels was confirmed when depolarizing current pulses up to 60 pA (cell depolarizes to 0 mV) failed to elicit fast spikes. The glutamate receptor antagonist kynurenic acid (KA) was prepared fresh on the day of experiment to 50 μm in aCSF. All drugs were dissolved in aCSF and applied directly through the bath perfusion system.

Analysis

A series of hyperpolarizing current pulses was applied to determine the identity of each neurone based on its electrophysiological fingerprint (Tasker & Dudek, 1991; Bains & Ferguson, 1997). Following electrophysiological identification, a neurone was required to maintain action potentials of at least 50 mV in magnitude throughout the recording to be included for further analysis.

Neurones were allowed to maintain a stable baseline for 60-180 s prior to application of the agents. Following peptide application, responses were assigned to three groups. (1) Depolarization - characterized as an increase in membrane potential of at least 3 mV followed by a return to baseline. (2) Hyperpolarization - characterized as a decrease of at least 3 mV followed by a return to baseline. (3) No response - characterized as the failure to generate a change in membrane potential greater than ±3 mV. Changes were characterized by measuring the peak membrane potential change maintained for a minimum of 10 s (this excludes transient changes such as action potentials and postsynaptic potentials) following peptide application. In all cases where single neurones were tested sequentially with more than a single concentration, the order of application was randomly assigned, and the second test was never initiated until recovery of membrane potential to baseline levels was observed.

We also assessed changes in the current-voltage relationship (I-V curve) or input resistance (Rin) in response to OX-A application. Changes in Rin in response to OX-A were compared using Student's t test, with P < 0.05 being set as the level of significance. I-V curves were plotted from data obtained following a series of current pulses run prior to OX-A application, following OX-A application, and following the return of the membrane potential to baseline. If a response was observed the membrane potential was adjusted to the original baseline using hyperpolarizing current prior to running protocols for the construction of I-V curves.

EPSCs were analysed using the Mini Analysis Program 4.1.1 (Synaptosoft Inc., Decatur, GA, USA) and quantified based on amplitude (minimum 5 pA) and shape (fast rising phase followed by a slow decay). Each detected event was inspected visually to exclude obvious false EPSCs. The data generated from these analyses were used to graph frequency and cumulative probability amplitude plots. Changes in EPSC frequency in response to different conditions were compared using Student's paired t test, with P < 0.05 being set as the level for significance. All values were plotted as means ± s.e.m. Each amplitude distribution was normalized to a maximum value of one. In order to determine whether two cumulative distributions differ by chance alone, the difference between data obtained during each condition was evaluated for significance using the Kolmogorov-Smirnov (K-S) statistic (Lupica, 1995).

Results

Whole-cell patch clamp recordings were obtained from a total of 149 PVN neurones. Using previously described techniques (Tasker & Dudek, 1991; Bains & Ferguson, 1997), cells that possessed a linear I-V relationship and a prominent transient potassium current (IA) were classified as MNC neurones (n = 29). In contrast, those exhibiting either an inward rectification at hyperpolarized potentials and a low threshold-activated potential (n = 82), or a low threshold rebound action potential and rapid spike adaptation in response to depolarizing current pulses (n = 28) were classified as PARVO (n = 110). The remaining 10 neurones could not be accurately classified as either MNC or PARVO and were therefore excluded from further analysis.

OX-A depolarizes magnocellular neurones

Twenty-one MNC neurones were tested for the effects of bath application of OX-A (1-500 nm, applied for 60 s) in aCSF under current clamp configuration. These neurones had a mean resting membrane potential of -57.2 ± 1.4 mV and a mean input resistance of 943 ± 18 MΩ. The majority of these cells (n = 14) depolarized in response to 10-500 nm OX-A application, while the remaining cells showed no response. OX-A (100 nm) evoked depolarizations (4.9 ± 1.1 mV, 73 % responsive) which usually occurred within 30 s of OX-A reaching the slice and lasted for 3-15 min prior to a return to baseline membrane potential, as shown in Fig. 1. The concentration of OX-A utilized in this experiment did not desensitize these neurones as multiple applications of peptide to the same cell resulted in comparable responses (Fig. 1A). Cells were tested with concentrations ranging from 1 to 500 nm and effects on membrane potential were found to be concentration dependent with an EC50 of 51 nm, as shown in the concentration-response relationship presented in Fig. 1B. Two cells tested with 1 nm OX-A did not show any change in membrane potential but were included to complete the concentration-response relationship.

Figure 1. OX-A depolarizes MNC PVN neurones.

Figure 1

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A, bath application of OX-A resulted in depolarization and increased frequency of action potentials (upper trace). These responses were repeatable since the same cell responded to additional applications of 100 nm OX-A (middle trace and inset). Inset (top): summary of membrane potential response following repeated application of 100 nm OX-A (1st application, 5.2 ± 1.5 mV vs. 2nd application, 5.0 ± 1.3 mV; n = 3). This response was no longer observed in the presence of 5.0 μm TTX suggesting that synaptic input to magnocellular neurones drives the OX-A-mediated response (bottom trace). OX-A application is represented by the horizontal bar above each trace. The horizontal dotted lines indicate resting membrane potential. Inset (bottom): membrane response to applied current pulses (-50 to -10 pA) during control (•) and TTX (▪) conditions (n = 3). B, graph illustrating the mean change in membrane potential in response to different concentrations of OX-A (1, 10, 50, 100 and 500 nm). Each point represents the mean ± s.e.m. with the number of trials in each group denoted in parentheses. Data were fitted to a sigmoid concentration- response function (Graphpad Prism) and the resulting curve was overlaid. C, the membrane potential response was abolished in the presence of TTX. **P < 0.01.

In order to determine whether the observed actions of OX-A were due to direct effects on MNCs, five neurones that responded to 100 nm OX-A were tested with OX-A during the blockade of action potentials by bath administration of TTX (5.0 μm). Following such pre-treatment with TTX, bath administration of OX-A failed to elicit a significant response (OX-A, 4.8 ± 1.6 mV vs. OX-A + TTX, 1.0 ± 0.8 mV; P < 0.01, n = 5, paired t test; Fig. 1C).

The depolarization in response to OX-A application was not a function of changes in passive membrane properties, as there was not a significant change in Rin during 100 nm OX-A application (control, 947 ± 51 MΩ vs. 100 nm OX-A, 938 ± 63 MΩ; P > 0.05, n = 6, paired t test), assessed by measuring membrane potential changes in response to hyperpolarizing current injection.

Orexin increases EPSCs in magnocellular neurones

The observation that TTX abolished effects of OX-A on membrane potential in MNC neurones suggested that these effects were the result of modified input from other neurones in our hypothalamic slice. Using continuous whole-cell voltage clamp recordings, we examined the effects of OX-A administration on postsynaptic currents (PSCs) in six MNCs. OX-A increased the frequency of EPSCs in five of the neurones tested, as illustrated in Fig. 2 (control, 1.6 ± 0.12 Hz vs. OX-A, 4.2 ± 0.16 Hz, n = 5, P < 0.001, paired t test). In addition, analysis of EPSC amplitude changes using cumulative probability plots indicated an increase in amplitude (control, 14.5 ± 0.9 pA vs. OX-A, 22.1 ± 1.1 pA, P < 0.01 in four of six cells tested, K-S test) accompanied this increase in frequency (Fig. 2C). The increase in EPSC frequency was no longer observed following bath application of 5.0 μm TTX (control, 0.45 ± 0.09 Hz vs. OX-A, 0.48 ± 0.06 Hz, n = 4, P > 0.1, paired t test, Fig. 2D). However, cumulative probability plots still indicated an increase in miniature EPSC (mEPSC) amplitude (TTX, 7.6 ± 0.5 pA vs. TTX + OX-A, 10.4 ± 0.4 pA, P < 0.05 in four of four cells tested, K-S test) during bath application of 100 nm OX-A in the presence of 5.0 μm TTX, as illustrated in Fig. 2E. These data suggest that OX-A-mediated depolarizations in MNC neurones are likely to be the result of both an action potential-dependent increase in excitatory input and modulation of a postsynaptic receptor causing an increase in EPSC amplitude.

Figure 2. OX-A application increase in EPSC frequency and amplitude in MNC neurones.

Figure 2

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A, continuous whole-cell voltage clamp recording from a MNC neurone illustrating that bath application of 100 nm OX-A (as indicated by the horizontal bar above the trace) resulted in an increase in EPSC (using 5 pA minimum cutoff) frequency. B, data summarizing the mean (± s.e.m.) increase in EPSC frequency in response to 100 nm OX-A (0 time point) observed in four MNC neurones (presented as 30 s mean frequencies). Note that the peak effect was observed at approximately 240 s, followed by a return to control values by 960 s, after OX-A administration. C, analysis of changes in EPSC amplitude using cumulative probability plots identified a shift in this distribution towards larger amplitude events following OX-A application (P < 0.01 in four of six cells, K-S test). D, analysis of a single neurone following OX-A application under control (•) and TTX (○) conditions demonstrating no increase in mEPSC frequency following bath application of OX-A in the presence of 5.0 μm TTX. This suggests that the increase in EPSC frequency was driven by a spike-dependent increase in vesicle release. E, cumulative distribution amplitude plots of mEPSCs recorded in TTX and OX-A indicate a shift towards larger amplitude events, suggesting a modification in postsynaptic receptor function (P < 0.05 in four of four cells, K-S test).

Evidence for a presynaptic glutamatergic neurone

The potential role for an excitatory input mediating MNC responses to OX is in accordance with previous reports suggesting a role for glutamate interneurones in mediating responses of this population of neurones to noradrenaline (Daftary et al. 1998). We therefore examined the involvement of glutamate in mediating MNC responses to OX in a further set of experiments using the broad-spectrum glutamate antagonist kynurenic acid (KA). In these experiments we were able to demonstrate that 50 μm KA, which abolished EPSCs, also blocked OX-A (100 nm)-induced depolarizations (OX-A, 5.2 ± 1.6 mV vs. OX-A + KA, 0.6 ± 0.4 mV; n = 4, P < 0.01, paired t test; Fig. 3).

Figure 3. Kynurenic acid blocks OX-A effects on MNC neurones.

Figure 3

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A, recording from a MNC neurone illustrating that while under control conditions bath application of 100 nm OX-A resulted in depolarization (top trace), perfusion of the slice with 50 μm kynurenic acid (KA) before and during a second OX-A application (bottom trace) abolished the depolarizing effects of OX-A. Peptide application is indicated by the horizontal bar above each trace. The horizontal dotted lines indicate resting membrane potential. Inset: bar graph summarizing the membrane potential effects following application of 100 nm OX-A (5.2 ± 1.6 mV, n = 4) and 100 nm OX-A in the presence of KA (0.6 ± 0.4 mV, n = 4). B, voltage clamp traces showing the increase in EPSC frequency and amplitude (middle panel) following a 60 s application of 100 nm OX-A. As with current clamp recordings, the OX-A-mediated increase in EPSC frequency and amplitude was abolished during application of 50 μm KA (bottom panel).

OX-A increases glutamate current in magnocellular neurones

This potential enhancement of postsynaptic glutamate receptor function by OX-A in MNC neurones was examined further in voltage clamp experiments carried out in the presence of 5.0 μm TTX. A 10 s bath application of 100 μm glutamate induced rapid reversible inward current in MNCs (49.1 ± 4.3 pA), an effect that was found to be enhanced in the presence of 100 nm OX-A (76.8 ± 12.2 pA, n = 5, P < 0.05, paired t test; Fig. 4A and B), and showed full recovery 20 min after return to aCSF in both cases where recordings were maintained for sufficient time (48.8 pA, n = 2; Fig. 4A).

Figure 4. OX-A potentiates glutamate current in MNC neurones.

Figure 4

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A, bath application of 100 μm glutamate (10 s) resulted in a whole-cell inward current in this MNC neurone (top trace). Subsequent glutamate application in the presence of 100 nm OX-A resulted in an increase in the peak glutamate-evoked inward current (middle trace). Following a 20 min wash with aCSF, a third application of glutamate induced an inward current similar to control conditions (bottom trace). Glutamate application is indicated by the horizontal bar above each trace. The dashed lines indicate resting membrane potential. B, summary of peak current response to glutamate under control conditions and in the presence of 100 nm OX-A (n = 5, P < 0.01, paired t test).

OX-A depolarizes parvocellular neurones

The observation that OX-A exerted indirect effects on MNC neurones of the PVN suggests that this peptide probably exerts these effects as a result of direct actions on an additional subpopulation of PVN neurones. PARVO neurones of the PVN represent one such subpopulation, which can also be identified electrophysiologically. We therefore obtained recordings from 52 PARVO neurones that were tested for the effects of bath application of OX-A using current clamp recording techniques. These neurones had a mean resting membrane potential of -56.1 ± 1.6 mV and a mean input resistance of 864 ± 21 MΩ. A total of 38 cells showed rapid reversible depolarizations in response to such peptide application, as illustrated in Fig. 5. OX-A (100 nm)-evoked depolarizations (8.4 ± 1.6 mV) usually occurred within 30 s of OX-A reaching the slice and lasted between 4 and 20 min prior to a return to baseline membrane potential. OX-A-mediated depolarizations of PARVO neurones were also found to be concentration dependent at concentrations ranging from 1 to 500 nm, with an EC50 of 67 nm (Fig. 5B). In order to examine the possibility that the observed actions of OX-A on PARVO neurones were also due to modifications in synaptic input, an additional five neurones that responded to 100 nm OX-A were tested with OX-A during the blockade of action potentials by bath administration of TTX. OX-A elicited TTX-resistant depolarizations in all five neurones tested as shown in Fig. 5A (aCSF, 8.6 ± 1.1 mV vs. aCSF + TTX, 7.9 ± 1.6 mV; n = 5, P > 0.5, paired t test).

Figure 5. OX-A directly activates PARVO neurones in a concentration-dependent manner.

Figure 5

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A, current clamp recordings from PARVO neurones demonstrating that this population of PVN cells also showed reversible concentration-dependent depolarizing responses to bath application of OX-A. The top trace shows the typical membrane potential changes in a PARVO neurone following bath application of 100 nm OX-A. The bottom trace shows a similar depolarization in the same neurone in response to 100 nm OX-A in the presence of 5.0 μm TTX. The duration of OX-A perfusion is indicated by the horizontal bar above each trace. The horizontal dotted lines indicate resting membrane potential. Inset: histogram summarizing the mean membrane potential changes recorded in response to 100 nm OX-A in aCSF (8.6 ± 1.1 mV) and aCSF + TTX (7.9 ± 1.6 mV). B, graph summarizing the mean changes in membrane potential measured in PARVO neurones in response to 1, 10, 50, 100 and 500 nm OX-A. Each point indicates the mean change in membrane potential (± s.e.m., n values in parentheses). Data were fitted to a sigmoid concentration- response function and the resulting curve overlaid. C, OX-A also caused consistent decreases in input resistance summarized in the mean I-V relationships obtained from six cells in control (•) and during application of 100 nm OX-A (○). Successive hyperpolarizing pulses (-10 to -60 pA) were delivered and the peak changes in membrane potential were measured. The reversal potential was approximately -45.5 mV, as indicated by the arrow.

The depolarizations were also accompanied by a decrease in Rin as measured by the maximum voltage response to a hyperpolarizing current pulse (control, 834 ± 72 MΩ vs. OX-A, 637 ± 83 MΩ, P < 0.01, n = 7; Fig. 5C). The remaining two cells did not exhibit a significant change in input resistance and did not depolarize following OX-A application. Extrapolation of complete I-V curves constructed for six of these cells indicated a reversal potential for the effects of OX-A of -45.5 ± 2.3 mV (n = 6; Fig. 5C) suggesting the potential involvement of a non-selective cationic conductance (NSCC).

OX-A induced inward current

In the voltage clamp configuration, the effects of OX-A on specific ionic conductances were investigated by applying slow (10 mV s−1) depolarizing voltage ramps (-80 to 20 mV) in aCSF containing TTX (5.0 μm). Bath application of OX-A to PARVO neurones caused an increase in conductance over the full voltage range tested (n = 6), as shown in Fig. 6A. The actual current induced by OX-A is shown in the difference current obtained by subtracting the control current from that obtained during application of 100 nm OX-A (Fig. 6A, inset). The difference current (IOX-A) was linear throughout the voltage range tested (r2 = 0.97) indicating a lack of voltage dependence to the current. The mean reversal potential of the OX-A (100 nm)-sensitive current (EOX-A) was found to be -40.5 ± 1.2 mV (n = 6; Fig. 6B).

Figure 6. Ramp currents activated by OX-A in PARVO neurones.

Figure 6

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A, instantaneous I-V relationships elicited by slow voltage ramps from -80 to +20 mV in control, and during bath application of 100 nm OX-A and recovery (Wash). OX-A increased conductance throughout the voltage range (n = 6). Inset: current activated by OX-A (IOX-A), obtained by subtracting the control and OX-A currents. B, the current was voltage independent, as indicated by the linearity (r2= 0.97). The reversal potential for the average current was -40.5 ± 1.2 mV, suggesting a non-selective cation conductance. Average IOX-A over the entire voltage range from six cells; data presented as means ± s.e.m.

Discussion

The hypothalamic distribution of OX-positive fibres has focused investigation on feeding, energy homeostasis, cardiovascular regulation and endocrine functions (Mondal et al. 2000). The prominent role of the PVN in the integration of these functions, when combined with studies demonstrating the PVN as a likely site of action for OX in control of food intake (Dube et al. 1999), cardiovascular regulation (Samson et al. 1999; Shirasaka et al. 1999) and control of the hypothalamic-pituitary- adrenal (HPA) axis (Jaszberenyi et al. 2000; Kuru et al. 2000; Russell et al. 2001; Samson & Taylor, 2001), further focuses attention on the regulatory roles of OX within this nucleus. In addition, two studies have reported excitatory effects of OX on PVN neurones without determining the specific mechanisms underlying such effects (Shirasaka et al. 2001; Samson et al. 2002). Our experiments were designed to elucidate the specific cellular mechanisms underlying the effects of this peptide on electrophysiologically identified subpopulations of PVN neurones.

Cell identification

Considerable focus over the past 20 years has been directed towards the development of techniques to allow the definitive phenotypic and/or functional identification of subpopulations of neurones within the PVN (Ferguson & Renaud, 1987; Bains & Ferguson, 1995, 1997; Stern & Armstrong, 1996; Luther & Tasker, 2000). In vitro, it is now well accepted that electrophysiologically identified Type 1 neurones represent MNC vasopressin and oxytocin-secreting cells of the PVN (Hoffman et al. 1991; Tasker & Dudek, 1991). Although studies have attempted to differentiate further between these two peptidergic populations of cells (Stern & Armstrong, 1996), the fact that a clear consensus has not emerged has led us to the conservative approach of not attempting to further subdivide this subpopulation.

PARVO neurones of the PVN represent a more diverse amalgamation of subpopulations of neurones including phenotypic cell groups synthesizing CRH, thyrotropin-releasing hormone, glutamate, GABA or somatostatin, as well anatomically defined cell groups projecting to the median eminence, spinal cord or medulla (Swanson & Sawchenko, 1983). Again, despite significant effort using a combination of cell labelling, in situ hybridization, as well as attempts at single cell RT-PCR, a consensus has not yet emerged as to methods for the electrophysiological differentiation of these subpopulations of PARVO neurones of the PVN. To date, most workers have again chosen to group all non-MNC (Type 1) cells together as PARVO (Type II), while at the same time providing as much information as possible about subpopulations of this group with common unifying electrophysiological properties. This is the approach we have taken in the present study.

Magnocellular neurones

Here we report that bath application of OX-A resulted in concentration-dependent, depolarization of the majority of MNC neurones tested, effects which were rapid, reversible and repeatable. These effects were abolished by TTX, observations which are in contrast to both our own preliminary findings (Samson et al. 2002) and those of Shirasaka et al. 2001 suggesting effects to be maintained in synaptic isolation. In our studies the reason for this discrepancy is probably that we did not separate MNC from PARVO neurones in our analysis (Samson et al. 2002), while there are several differences in Shirasaka's study (Shirasaka et al. 1999) including the use of neonatal rats, OX-B, and the application of 10-fold higher peptide concentrations.

The existence of a TTX-sensitive persistent sodium current, as previously described in these neurones (Tanaka et al. 1999), suggests an alternative site for OX-A modulation of neuronal activity. However, the lack of significant change in input resistance (no change in whole-cell conductance) in response to 100 nm OX-A argues against this possibility.

Importantly in our studies, in addition to the depolarizing effects of OX-A on PVN neurones, an increase in excitatory synaptic transmission was observed following bath application of the peptide, supporting the hypothesis that increased synaptic input made an important contribution to these responses. Such observations are in accordance with previous studies describing OX-A-induced increase in excitatory synaptic activity in locus coeruleus (Horvath et al. 1999) and dorsal raphe nucleus (Brown et al. 2001) neurones. Although this barrage of synaptic activity apparently contributes to membrane depolarization, it was not associated with a sustained inward current. Such observations are similar to those of Kombian et al. (2000), and may be due to the fact that these excitatory events were not of sufficient frequency or duration to summate as a sustained change in whole-cell current.

In MNC PVN neurones, this increase in EPSC frequency was abolished in TTX, supporting a role for increases in presynaptic action potential frequency in mediating these effects. Intriguingly, the observed increase in mEPSC amplitude in TTX suggests that OX-A may also play a postsynaptic modulatory role enhancing the excitatory effects of these inputs in MNC neurones, and providing support for the observations discussed above demonstrating direct effects of OX on MNC neurones (Shirasaka et al. 2001).

As in other regions of the brain, most fast excitatory synaptic events in the PVN have been shown to be mediated by glutamate release leading to activation of ionotropic glutamate receptors (van den Pol et al. 1990). Anatomical (Csaki et al. 2000) and electrophysiological (Boudaba et al. 1997; Daftary et al. 1998) studies have also suggested that such excitatory glutamatergic inputs in the PVN may be derived from a presynaptic glutamate neurone located within the boundaries of the PVN. Our experiments demonstrating that the ionotropic glutamate receptor antagonist kynurenic acid blocked both EPSCs and changes in membrane potential evoked by bath application of OX-A supports a role for such a presynaptic glutamate neurone in mediating OX-A effects on MNC neurones. Further studies will, however, be necessary to identify both the specific location of this input as well as the specific glutamate receptors involved. It is also possible that one of the subpopulations of electrophysiologically described PARVO neurones described below may in fact provide this input. Our observations showing that OX-A enhances glutamate-evoked currents in MNCs also provide the first direct evidence in support of postsynaptic effects of OX-A in modulating glutamate receptor function.

Parvocellular neurones

Bath application of OX-A resulted in concentration-dependent depolarization of over 70 % of parvocellular neurones tested, effects which were again rapid, reversible and repeatable. In contrast to effects on MNC neurones these depolarizing effects on PARVO neurones were maintained in TTX suggesting them to be the result of direct actions of OX-A on these cells. OX-A administration also resulted in a significant decrease in input resistance that accompanied the membrane potential effects in PARVO neurones. Analysis of the I-V curves identified a reversal potential of -45.5 mV for PARVO neurones. This reversal potential is consistent with the activation of a non-selective cationic conductance as previously described in PVN (Powis et al. 1998), supraoptic nucleus (Bourque, 1989) and subfornical organ (Washburn et al. 1999) neurones.

Our voltage clamp experiments were undertaken to specifically test the hypothesis that OX-A depolarizes PARVO neurones as a consequence of the activation of this NSCC. Slow voltage ramps revealed a small voltage-independent OX-A-sensitive current (IOX-A) in PARVO neurones which displayed characteristics similar to the previously described leptin modulation of a NSCC (Powis et al. 1998) in PVN neurones. Importantly, these effects of OX-A on the NSCC in PARVO neurones would be expected to result in the 10-15 mV depolarizations observed in these neurones. In addition, our own recent work demonstrating similar OX-A effects on a NSCC in area postrema neurones (Yang & Ferguson, 2002) suggests that orexin receptor-mediated modulation of this conductance may represent a common mechanism through which OX peptides exert control over neuronal excitability. The signal transduction mechanisms underlying this modulation of the NSCC have not been examined in the present study, although previous work has shown that the OX2 receptor couples to a Gq protein (Sakurai et al. 1998), the activation of which results in activation of the phospholipase C system (Exton, 1994).

Physiological relevance

These studies clearly identify cellular actions of OX-A in the PVN, but do not provide direct evidence regarding the physiological relevance of these observations. Previous physiological studies do however provide important clues in the demonstration of effects of i.c.v. injection of OX-A on the regulation of neuroendocrine systems and blood pressure. The depolarizing effects of OX-A on both MNC and PARVO neurones reported here may underlie the previously reported hypertensive effects of i.c.v. administration of this peptide (Samson et al. 1999). These hypertensive effects could be the result of OX-A stimulation of either MNC or PARVO PVN neurones. Activation of the former would increase blood pressure through modulation of vasopressin release, while similar activation of descending projections to either the medullary NTS or the intemediolateral cell column of the spinal cord would be expected to result in similar hypertensive outcomes.

Recent studies showing effects of orexin on the HPA axis-A (Kuru et al. 2000; Al Barazanji et al. 2001; Samson & Taylor, 2001) suggest potentially important physiological roles for OX in control of stress. The largest population of PARVO PVN neurones has been characterized as CRH-synthesizing neurones projecting to the median eminence, the physiological role of which is the control of CRH secretion and thus maintenance of the HPA axis (Guillemin, 1967). Clearly our demonstration of direct effects of OX-A on PARVO neurones of the PVN identifies a mechanism which may underlie the effects of this peptide on the HPA axis.

Acknowledgments

This work was supported by the Heart and Stroke Foundation of Ontario.

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