Morphine withdrawal increases intrinsic ... - Wiley Online Library

European Journal of Neuroscience, Vol. 21, pp. 501–512, 2005

ª Federation of European Neuroscience Societies

Morphine withdrawal increases intrinsic excitability of oxytocin neurons in morphine-dependent rats Colin H. Brown,1 Javier E. Stern,2 Keshia L. M. Jackson,2 Philip M. Bull,1 Gareth Leng1 and John A. Russell1 1 2

School of Biomedical and Clinical Laboratory Sciences, University of Edinburgh, Edinburgh EH8 9XD, UK Department of Psychiatry, Genome Research Institute, Cincinnati, OH 45237, USA

Keywords: after-hyperpolarization, opioid dependence, supraoptic nucleus, transient outward rectification, vasopressin

Abstract To determine whether intrinsic mechanisms drive supraoptic nucleus oxytocin neuron excitation during morphine withdrawal, we calculated the probability of action potential (spike) firing with time after each spike for oxytocin neurons in morphine-naı¨ve and morphine-dependent rats in vivo and measured changes in intrinsic membrane properties in vitro. The opioid receptor antagonist, naloxone, increased oxytocin neuron post-spike excitability in morphine-dependent rats; this increase was greater for short interspike intervals (< 0.1 s). Naloxone had similar, but smaller (P ¼ 0.04), effects in oxytocin neurons in morphine-naı¨ve rats. The increased post-spike excitability for short interspike intervals was specific to naloxone, because osmotic stimulation increased excitability without potentiating excitability at short interspike intervals. By contrast to oxytocin neurons, neither morphine dependence nor morphine withdrawal increased post-spike excitability in neighbouring vasopressin neurons. To determine whether increased postspike excitability in oxytocin neurons during morphine withdrawal reflected altered intrinsic membrane properties, we measured the in vitro effects of naloxone on transient outward rectification (TOR) and after-hyperpolarization (AHP), properties mediated by K+ channels and that affect supraoptic nucleus neuron post-spike excitability. Naloxone reduced the TOR and AHP (by 20% and 60%, respectively) in supraoptic nucleus neurons from morphine-dependent, but not morphine-naı¨ve, rats. In vivo, spike frequency adaptation (caused by activity-dependent AHP activation) was reduced by naloxone (from 27% to 3%) in vasopressin neurons in morphine-dependent, but not morphine-naı¨ve, rats. Thus, multiple K+ channel inhibition increases post-spike excitability for short interspike intervals, contributing to the increased firing of oxytocin neurons during morphine withdrawal.

Introduction Opiate drugs are important analgesics that are abused for their euphoric actions. Chronic opiate use can result in dependence and addiction. Whilst the factors driving continued opiate use are complex, avoidance of withdrawal symptoms is of fundamental importance. At present, the cellular processes that induce dependence and underpin the withdrawal syndrome are poorly understood. Many central neurons express l-opioid receptors (Mansour et al., 1995) for which morphine is the classical agonist, but few types of neuron undergo withdrawal excitation upon removal from chronic morphine exposure, including neurons in the striatum, nucleus accumbens, locus coeruleus, ventral tegmental area and frontal cortex (Nye & Nestler, 1996) as well as the supraoptic nucleus and paraventricular nucleus (Jhamandas et al., 1996; Murphy et al., 1997; Johnstone et al., 2000). Whereas the phenotypes of most neurons that undergo withdrawal excitation have yet to be determined, supraoptic nucleus and paraventricular nucleus oxytocin magnocellular neurosecretory cells are the only identified peptidergic neurons shown to do so (Russell et al., 1995). Oxytocin cells are acutely inhibited by morphine and develop tolerance to chronic intracerebroventricular (i.c.v.) morphine (Bicknell et al., 1988), which is revealed by the requirement for progressively higher doses of morphine to cause the same magnitude of inhibition (Pumford et al., 1991). During

Correspondence: Dr C. H. Brown, as above. E-mail: [email protected] Received 5 August 2004, revised 18 November 2004, accepted 21 November 2004

doi:10.1111/j.1460-9568.2005.03885.x

chronic morphine exposure to induce dependence, there is a change in the physiology of oxytocin cells (that occurs in parallel with the changes that induce tolerance) such that they require continued morphine exposure to function apparently normally, and this is revealed by rebound hyperexcitation upon morphine withdrawal (Bicknell et al., 1988). Thus, oxytocin cells provide a robust model that is amenable to detailed analysis of the cellular mechanisms of morphine dependence (Brown & Russell, 2004). Here, we investigated the effects of morphine withdrawal on the organization of electrical activity in supraoptic nucleus cells in vivo and on their membrane properties in vitro. Given that morphine withdrawal induces a profound and sustained increase in oxytocin cell activity, without a marked change in the activation of their major afferent inputs (Murphy et al., 1997), we hypothesized that increased oxytocin cell intrinsic excitability might be involved in driving oxytocin cell firing at this time. We analysed changes in magnocellular neurosecretory cell post-spike excitability by constructing hazard functions before and after administration of the broad-spectrum opioid receptor antagonist, naloxone, to morphine-dependent rats to induce withdrawal excitation. Hazard functions reflect the changes in excitability of neurons following spontaneous action potentials (spikes) by displaying the probability of spike firing with time after the preceding spike, and are deduced to reveal the influence of nonsynaptic post-spike potentials on the activity of magnocellular neurosecretory cells (Leng et al., 1995). Immediately after a spike, oxytocin cells are unexcitable, reflecting the influence of a post-spike hyperpolarizing after-potential and an after-hyperpolarization (AHP).

502 C. H. Brown et al. A relatively long time after the last spike, oxytocin cell excitability stabilizes, reflecting the average rate of synaptic input and the steadystate resting potential. We show that morphine withdrawal increases post-spike excitability, particularly in the period < 0.1 s after each spike. Furthermore, we show that morphine withdrawal involves a reduction of multiple K+-mediated membrane properties in magnocellular neurosecretory cells in vitro that might induce the changes in post-spike excitability observed in vivo.

Materials and methods All procedures were carried out in accordance with the UK Animals (Scientific Procedures) Act, 1986 and associated guidelines (in vivo electrophysiology) or with the policy of Wright State University regarding the use and care of animals (in vitro electrophysiology).

Induction of morphine dependence Morphine dependence was induced as previously described (Rayner et al., 1988). Briefly, virgin female Sprague–Dawley rats (250–350 g) were anaesthetized with halothane (5% in an O2 ⁄ N2O mixture, both flow rates at c. 500 mL ⁄ min, in vivo experiments) or with ketamine ⁄ xylazine mixture (90 and 5 mg ⁄ kg, intraperitoneal (i.p.), respectively, in vitro experiments). An Alzet model 2001 miniosmotic pump (Charles River UK Ltd, Margate, Kent, UK or Durect Corporation, Cupertino, CA, USA) was placed subcutaneously (s.c.) and connected via polythene tubing to a 21-gauge stainless steel cannula implanted into the right lateral cerebral ventricle (3.0 mm caudal, 2.0 mm lateral to bregma and 4.5 mm below the surface of the skull). The pump and tubing were filled with morphine (in sterile pyrogen-free water) to deliver increasing doses (10 lg ⁄ h, 20 lg ⁄ h for 40 h each and 50 lg ⁄ h for the remainder at 1 lL ⁄ h) over 5 days. The cannula was secured using dental acrylic bonded to stainless steel screws inserted in the skull. Following surgery rats were housed individually with food and water available ad libitum. As we have previously shown that chronic i.c.v. infusion of vehicle does not alter the response of oxytocin cells to intravenous (i.v.) naloxone (Brown et al., 1996) or hypertonic saline (Bull et al., 2003), control rats were not subjected to surgery.

In vivo electrophysiology On the day of the experiment (the sixth day following minipump implantation for morphine-dependent rats), the rats were anaesthetized by i.p. injection of urethane (ethyl carbamate; 1.25 g ⁄ kg) and a catheter inserted into the left femoral vein for drug ⁄ hypertonic saline injection. The pituitary stalk and right supraoptic nucleus were exposed through the oral cavity (Leng & Dyball, 1991). Extracellular single-unit recordings were made using a glass recording microelectrode (15–40 MW) and conventional electrophysiological recording techniques. A side-by-side stimulating electrode (Clark Electromedical Instruments, Pangbourne, Reading, UK) was placed on the pituitary stalk to elicit antidromic spikes in supraoptic nucleus cells. Neurons were confirmed as magnocellular neurosecretory cells by collision of antidromic spikes with spontaneous orthodromic spikes (Lincoln & Wakerley, 1974). Magnocellular neurosecretory cells were characterized as oxytocin cells on the basis of a continuous firing pattern and by transient excitation following i.v. cholecystokinin injection (20 lg ⁄ kg, 0.5 mL ⁄ kg in 0.9% saline; Brown et al., 1996); or as vasopressin cells by virtue of spontaneous phasic activity or a transient inhibition of continuously active cells following cholecystokinin injection (Sabatier

et al., 2004). At the end of the experiments, the rats were killed by an i.v. anaesthetic overdose (60 mg ⁄ kg pentobarbitone).

In vivo data analysis Oxytocin and vasopressin cell activity was downloaded onto a personal computer using a 1401 interface and Spike2 software package (Cambridge Electronic Design, Cambridge, UK) and analysed offline. The probability of spike firing (hazard) was calculated from the interspike interval histogram of individual cells using the following formula: hazard½i1;i ¼ n½i1;1 =ðN n½0;i1 Þ where hazard[i)1, i] is the hazard at interval i, n[i)1, 1] is the number of spikes in interval i, n[0, i)1] is the total number of spikes preceding the current interval and N is the total number of spikes in all intervals. This gives the inferred probability (as a decimal) of a cell firing a subsequent spike in any interval after a spike (at time 0), given that another spike has not occurred earlier (Leng et al., 1995; Brown & Leng, 2000; Brown et al., 2004; Sabatier et al., 2004).

In vitro electrophysiology On the day of experiment, the rats were restrained in a soft plastic cone (5–10 s), anaesthetized with sodium pentobarbitone (50 mg ⁄ kg, i.p.) and perfused through the heart with cold medium in which NaCl was replaced by an equi-osmolar amount of sucrose (in mm): sucrose, 200; KCl, 2.5; NaH2PO4, 1.25; MgSO4, 2; CaCl2, 2; NaHCO3, 26; glucose, 20; and ascorbic acid, 0.4; pH 7.4 (297–300 mOsm). The rats were then decapitated, the brain rapidly removed and coronal hypothalamic slices (350 lm) containing the supraoptic nucleus were obtained as previously described (Stern et al., 1999). The perfusate contained (in mm): NaCl, 120; KCl, 2.5; NaH2PO4, 1.25; MgSO4, 1; CaCl2, 2; NaHCO3, 26; glucose, 20; and ascorbic acid, 0.4; pH 7.4 (297–300 mOsm). For slices from morphine-dependent rats, the perfusate also contained 1 lm morphine sulphate to prevent morphine withdrawal. Solutions bathing the slices (2 mL ⁄ min) were kept at room temperature (22–24 C) and bubbled continuously with a gas mixture of 95%O2)5% CO2. Patch pipettes (4–8 MX) were pulled from thin-walled (1.5 mm outer diameter, 1.17 mm inner diameter) borosilicate glass (GC150T7.5, Clark, Reading, UK) on a horizontal electrode puller (P-97, Sutter Instruments, Novato, CA, USA). The pipette internal solution contained (in mm) d-gluconic acid, 130; KCl, 20; HEPES, 10; EGTA, 0.5; and gramicidin, 0.26. The pH was slowly titrated to 7.25–7.3 and the tip of the pipette was filled with gramicidin-free solution. Gramicidin-based perforated patch recordings were obtained from supraoptic nucleus cells under visual control using an upright microscope (Axioscop, Zeiss, Germany) equipped with Nomarski IR-DIC optics and a water-immersion lens (· 40). Electrical recordings were obtained using an Axopatch 200B patch-clamp amplifier (Axon Instruments, Union City, CA, USA). The voltage output was digitized at 16-bit resolution using pClamp 8 software (Digidata 1320, Axon Instruments). Data were digitized at 10 kHz and transferred to disk. Only experiments for which series resistance was stable throughout the recording were included in the analyses.

In vitro data analysis All cells analysed had membrane potentials more negative than )50 mV and spike amplitudes of at least 60 mV. For transient outward

ª 2005 Federation of European Neuroscience Societies, European Journal of Neuroscience, 21, 501–512

Intrinsic excitability in morphine withdrawal 503 rectification (TOR), magnocellular neurosecretory cells were held at )80 mV and 180 ms depolarizing current pulses of increasing amplitude (10 pA, 10 pulses) were delivered through the recording electrode; changes in TOR were evaluated by measuring the time to the first spike during the first depolarizing pulse that was able to trigger a spike. Input resistance was calculated from the voltage response to 5 ms, 0.1 nA depolarizing current pulses. The afterhyperpolarization (AHP) area was measured following 400 ms depolarizing pulses that evoked trains of 7–10 spikes and was corrected for the number of spikes in each evoked train. Spike broadening was analysed by measuring the widths of the first nine spikes of the evoked spike trains used to elicit AHPs, at 50% spike height (measured from threshold). These parameters were measured in 6 magnocellular neurosecretory cells from morphine-dependent rats before and during bath application of naloxone (10 lm). A set of control experiments (n ¼ 6) was run in which the effects of 10 lm naloxone were tested in morphine-naı¨ve rats.

revealed that the excitability of oxytocin cells was markedly elevated following each spike during morphine withdrawal, and that this increase in post-spike excitability was more pronounced in the period < 0.1 s after each spike (Fig. 1F and I). Plotting the hazard functions (Fig. 1E) from the interspike interval histograms (Fig. 1C) of oxytocin cells recorded from morphine-naı¨ve rats showed no sustained steady-state change in post-spike excitability after naloxone administration but did reveal a small increase in the period < 0.1 s after each spike (Fig. 1E and G). To quantify these changes in the shape of the hazard functions, we calculated the ratio of the peak early hazard (< 0.07 s after each spike) to the mean late hazard (averaged between 0.2 and 0.3 s after each spike). While the early:late hazard ratio in oxytocin cells was increased by naloxone administration in both morphine-naı¨ve and morphine-dependent rats (P ¼ 0.003 and P ¼ 0.002, respectively, Fig. 1H), the increase was greater in oxytocin cells from morphinedependent rats than in morphine-naı¨ve rats (P ¼ 0.04).

Statistics

Correlation of early : late hazard ratio with firing rate after naloxone administration

All averaged data are expressed as the mean ± SEM. All differences within groups were evaluated on SigmaStat software (SPSS Science, Chicago, IL, USA) using paired t-tests or one-way repeated measures analysis of variance (anova), and between groups using two-way anova or two-way repeated measures anova. Where the F-ratio was significant, all-pairwise post hoc comparisons were completed using Student–Newman–Keuls tests. Correlations were analysed using Pearson Product Moment Correlations.

Drugs Morphine sulphate was supplied by The Royal Infirmary of Edinburgh, Edinburgh, UK or purchased from the Sigma Chemical Company Ltd (St. Louis, MO, USA). Naloxone hydrochloride was from Sigma (Poole, Dorset, UK, or St. Louis) and cholecystokinin from Bachem (UK) Ltd. (Saffron Walden, Essex, UK).

Results Effects of morphine withdrawal on post-spike excitability of oxytocin cells in vivo The firing rates of oxytocin cells in morphine-naı¨ve and morphinedependent rats were similar at 2.8 ± 0.4 (n ¼ 11) and 2.6 ± 0.4 spikes ⁄ s (n ¼ 12), respectively (P ¼ 0.83). As previously reported (Bicknell et al., 1988; Brown et al., 1996, 1997, 1998b; Ludwig et al., 1997), i.v. administration of the opioid receptor antagonist, naloxone (5 mg ⁄ kg), did not change the firing rate of oxytocin cells in morphinenaı¨ve rats (0.6 ± 0.3 spikes ⁄ s increase, P ¼ 0.23, n ¼ 11), but did increase the firing rate of oxytocin cells in morphine-dependent rats by 3.3 ± 0.6 spikes ⁄ s (P < 0.001, n ¼ 12, e.g. Fig. 1A and B). The interspike interval histograms (generated in 0.01 s bins) of spike firing for 10–20-min periods before and after naloxone injection (Fig. 1D) showed that more spikes fired during morphine withdrawal, simply reflecting the increase in firing rate that occurs during morphine withdrawal excitation of oxytocin cells. The shapes of these histograms differ before and during morphine withdrawal, indicating differences in post-spike excitability. These differences in post-spike excitability are more clearly exposed by the plots of hazard functions, which directly describe these differences. Plotting the hazard functions (Fig. 1F) from these interspike interval histograms

There was no correlation between the post-naloxone early:late hazard ratio and the post-naloxone firing rate for all 23 oxytocin cells in rats administered naloxone (Pearson product moment correlation coefficient, r ¼ )0.03, P ¼ 0.89), or for morphine-naı¨ve oxytocin cells (r ¼ 0.14, P ¼ 0.68, n ¼ 11) and morphine-dependent oxytocin cells (r ¼ –0.52, P ¼ 0.09, n ¼ 12) alone. Together, these findings indicate that the potentiated increase in the early hazard did not result simply from increases in firing rate.

Effects of osmotic stimulation on post-spike excitability in vivo To further determine whether the change in the shape of the hazard function induced by naloxone resulted from mechanisms induced by an increase in activity per se, we used hyperosmotic stimulation to increase the firing rate of oxytocin cells in morphine-naı¨ve rats; this stimulus increases oxytocin cell activity via activation of stretchinactivated cation channels on cells to generate a persistent depolarization (Oliet & Bourque, 1993), decreases taurine release from glia within the supraoptic nucleus to disinhibit magnocellular neurosecretory cells (Hussy et al., 2000) and increases synaptic input activity (Leng et al., 2001). Infusion of 2 m hypertonic saline (at 26 lL ⁄ min, i.v.) induced a ramp increase in the firing rate of oxytocin cells in morphine-naı¨ve rats (e.g. Fig. 2A). We selected portions of these recordings in which the firing rates were elevated to a similar extent to those seen during morphine withdrawal (by 4.1 ± 0.6 spikes ⁄ s; n ¼ 7, averaged over 10 min) to generate hazard functions. However, by contrast to morphine withdrawal excitation, hazard functions (Fig. 2B) showed that the shapes of these histograms did not differ before and during hyperosmotic stimulation (Fig. 2B and C), with no significant difference in the early:late hazard ratio before and during hyperosmotic stimulation (P ¼ 0.29, paired t-test, Fig. 2D). Thus, the pronounced increase of post-spike excitability induced by naloxone in the period < 0.1 s after each spike is not a non-specific effect of excitation in general.

Effects of morphine withdrawal on post-spike excitability of vasopressin cells in vivo By contrast to oxytocin cells (Brown et al., 1997, 1998b; Ludwig et al., 1997;), the firing rate of vasopressin cells was not affected by


504 C. H. Brown et al.

Fig. 1. Morphine withdrawal increases post-spike excitability in oxytocin cells in vivo. (A and B) Extracellular recordings of oxytocin cell firing rates (averaged in 1 s bins) recorded from the supraoptic nucleus of urethane-anaesthetized (1.25 g ⁄ kg, i.p.) morphine-naı¨ve (A) and morphine-dependent (B) female Sprague– Dawley rats. The cell from the morphine-dependent rat displays a morphine withdrawal-induced increase in firing rate upon administration of naloxone (NLX, 5 mg ⁄ kg), whereas the cell from the morphine-naı¨ve rat shows no obvious change in firing rate, typical of oxytocin cells. (C and D) Mean interspike intervals (± SEM, in 0.01 s bins) pre- (open circles) and post-naloxone (filled circles) in morphine-naı¨ve (C, n ¼ 11) and morphine-dependent (D, n ¼ 12) rats. (E and F) Mean hazard functions (± SEM) of the interspike intervals of the cells from C and D pre- (open circles) and post-naloxone (filled circles) in morphine-naı¨ve (E) and morphine-dependent (F) rats. Note that the probability of spike firing varies as a function of time following the preceding spike; this is inferred to reflect the influence of post-spike potentials on the excitability of oxytocin cells. (G) Mean differences in hazard functions pre- and post-naloxone (± SEM) of the cells from E and F in morphine-naı¨ve (open circles) and morphine-dependent (filled circles) rats. Note that the naloxone-induced difference in the probability of spike firing is greatest in the period < 0.1 s after each spike, changing the shape of the hazard function. (H) Ratios of peak early (< 0.07 s interspike interval) to mean late (0.2–0.3 s interspike intervals) hazards pre- and post-naloxone in morphine-naı¨ve (left-hand panel) and morphine-dependent (right-hand panel) rats. **P < 0.01 vs. pre-NLX data within each group, and P < 0.05 vs. matched data from morphine-naı¨ve rats, two-way repeated measures anova followed by Student–Newman– Keuls tests.

5 mg ⁄ kg naloxone in morphine-naı¨ve rats (4.3 ± 0.4 spikes ⁄ s, n ¼ 15, and 3.9 ± 0.7 spikes ⁄ s, n ¼ 8, before and after naloxone, respectively, P ¼ 0.57, e.g. Fig. 3A) or morphine-dependent rats (5.6 ± 0.7 spikes ⁄ s, n ¼ 18, and 6.4 ± 0.9 spikes ⁄ s, n ¼ 11, before and after naloxone, respectively, P ¼ 0.39; e.g. Fig. 3C). Nevertheless, we have previously shown that morphine withdrawal induces a reduction in the modal interspike interval of some vasopressin cells (Bicknell et al., 1988), so we generated plots of hazard functions for vasopressin cells in morphine-naı¨ve rats (Fig. 3B) and morphinedependent rats (Fig. 3D). For vasopressin cells, the ratio of the peak early hazard to the mean late hazard was not different between morphine-naı¨ve rats and morphine-dependent rats (P ¼ 0.80) and was

not altered by acute i.v. naloxone administration in morphine-naı¨ve rats and morphine-dependent rats (P ¼ 0.26, Fig. 3E), indicating that naloxone did not preferentially increase the post-spike excitability for short interspike intervals in vasopressin cells. Effects of morphine withdrawal on input resistance and membrane time constant in supraoptic nucleus cells in vitro Before naloxone administration, input resistance was significantly greater in supraoptic nucleus cells from morphine-naı¨ve rats than in cells from morphine-dependent rats (1032.6 ± 178.2 and 589.2 ± 83.6 MX, respectively; P ¼ 0.04; both n ¼ 6). Input resistance was not


Intrinsic excitability in morphine withdrawal 505

Fig. 2. Hyperosmotic stimulation increases post-spike excitability in oxytocin cells in vivo. (A) Extracellular recording of the firing rate of an oxytocin cell recorded from the supraoptic nucleus of a urethane-anaesthetized morphinenaı¨ve rat, showing a ramp increase in firing rate during hyperosmotic stimulation (2 m hypertonic saline at 26 lL ⁄ min, i.v.). (B) The mean hazard function of the interspike intervals (not shown) before (open circles) and during (filled circles) hyperosmotic stimulation (n ¼ 7). C, The mean difference in hazard (± SEM) of the cells from B before and during hyperosmotic stimulation. Note that, by contrast to naloxone (Fig. 1E–G), hyperosmotic stimulation did not change the shape of the hazard function. (D) Ratio of peak early-to-mean late hazards before (Pre) and during (Post) hyperosmotic stimulation (NaCl). P ¼ 0.29, paired t-test.

significantly affected by 10 lm naloxone in magnocellular neurosecretory cells from morphine-naı¨ve rats (at 798.6 ± 158.5 MX; P > 0.05). By contrast to cells from morphine-naı¨ve rats, 10 lm naloxone decreased the input resistance of cells from morphine-dependent rats to 450.5 ± 93.9 MX (P ¼ 0.02). The example voltage–current plots (Fig. 4B inset) show that the decreased input resistance occurred at all membrane potentials tested above )80 mV (holding potential). Before naloxone administration, the membrane time constant (s) was significantly greater in cells from morphine-naı¨ve rats than in cells from morphine-dependent rats (33.4 ± 4.6 ms and 18.4 ± 2.2, respectively; P ¼ 0.04; both n ¼ 6). 10 lm naloxone did not significantly alter the s of cells from morphine-naı¨ve rats (29.1 ± 3.5 ms; P > 0.05) or morphine-dependent rats (14.5 ± 1.1 ms; P > 0.05).

Effects of morphine withdrawal on transient outward rectification in supraoptic nucleus cells in vitro

Fig. 3. Effects of morphine withdrawal on post-spike excitability in vasopressin cells in vivo. (A and C) Extracellular recordings of vasopressin cell firing rates (averaged in 1-s bins) recorded from the supraoptic nucleus of urethaneanaesthetized (1.25 g ⁄ kg, i.p.) morphine-naı¨ve (A) and morphine-dependent (C) female Sprague–Dawley rats. Neither cell displays an increase in firing rate upon administration of naloxone (NLX, 5 mg ⁄ kg), typical of vasopressin cells. (B and D) Mean hazard functions (± SEM) of vasopressin cells in morphinenaı¨ve rats (B) pre-naloxone (open circles, n ¼ 15) and post-naloxone (filled circles, n ¼ 8) and morphine-dependent rats (D) pre-naloxone (open circles, n ¼ 18) and post-naloxone (filled circles, n ¼ 11). E, Ratios of peak early to mean late hazards pre- and post-naloxone in morphine-naı¨ve (left-hand panel) and morphine-dependent (right-hand panel) rats. Two-way anova showed that neither morphine (P ¼ 0.80) nor naloxone (P ¼ 0.26) altered the ratio of peak early to mean late hazards in vasopressin cells.

In response to membrane depolarization, and due to activation of a transient outward K+ current, magnocellular neurosecretory cells typically display a TOR (Bourque, 1988) that delays the occurrence

of the first spike during brief depolarizing current pulses. To determine whether the increase in post-spike excitability during


506 C. H. Brown et al.

Fig. 4. Morphine withdrawal decreases TOR in supraoptic nucleus cells in vitro. (A and B) Perforated patch recordings of the TOR from cells in hypothalamic slices from a morphine-dependent rat before (A) and during application of 10 lm naloxone (B) in the continued presence of 1 lm morphine. The inset displays a voltage–current plot (V–I plot) obtained from the traces in A (pre-naloxone, open symbols) and B (post-naloxone, closed symbols), taken at the beginning (squares, measured at the first arrow under voltage traces in Fig. 4B) and at steady state (circles, measured at the second arrow under voltage traces in Fig. 4B) of the voltage deflection. Note the decreased slope of the V–I plot in the presence of naloxone. (C) Overlays of the top traces from A and B, demonstrating that the time to the first spike (arrows) was reduced by naloxone (bold line) in the cell from the morphine-dependent rat (main panel), but not in a cell from a morphine-naı¨ve rat (inset). (D) Mean time to first spike during an evoked depolarization in morphine-naı¨ve supraoptic nucleus cells (left, n ¼ 5) and morphine-dependent cells (right, n ¼ 5) before and after naloxone. *P < 0.05 vs. pre-NLX, Student–Newman–Keuls test.

morphine withdrawal in vivo might be underpinned by a change in the transient outward K+ conductance, we investigated the TOR of magnocellular neurosecretory cells in response to depolarizing current injection before and during superfusion of 10 lm naloxone in hypothalamic slices from morphine-naı¨ve rats and morphinedependent rats. As shown in Fig. 4, depolarizing steps of increasing magnitude evoked a clear TOR in all recorded cells, which delayed the occurrence of a spike during the depolarizing step. In morphinedependent rats (in the continued presence of 1 lm morphine), bath application of naloxone (10 lm) significantly decreased the delay to the first evoked spike (Fig. 4B and C; P < 0.05), reflecting a reduced TOR during morphine withdrawal excitation. The reduced TOR can also be observed in the voltage-current plots displayed in the inset of Fig. 4. By contrast, naloxone failed to change the delay to the first evoked spike in morphine-naı¨ve rats (68.0 ± 6.8 ms and 63.9 ± 5.2 ms in the absence and presence of naloxone, respectively; P ¼ 0.25). Because the delay to the first spike during membrane depolarization could be influenced by input resistance and ⁄ or s, we determined whether a correlation among these variables existed. Overall, TOR did not significantly correlate with s (r ¼ 0.37, P ¼ 0.20) or input resistance (r ¼ 0.40, P ¼ 0.12) in magnocellular neurosecretory cells before application of naloxone. Furthermore, changes in TOR induced by naloxone in cells from morphinedependent rats did not correlate with changes in either input resistance (r ¼ 0.40, P ¼ 0.20) or s (r ¼ 0.45, P ¼ 0.11). Taken

together, these results suggest that changes in TOR induced by naloxone in morphine-dependent rats probably did not result from changes in input resistance and ⁄ or s. Effects of morphine withdrawal on the after-hyperpolarization in magnocellular neurosecretory cells in vitro Because the increase in post-spike excitability caused by naloxoneinduced withdrawal excitation in morphine-dependent oxytocin cells in vivo was more pronounced in the period < 0.1 s after each spike, we investigated the effects of naloxone-precipitated morphine withdrawal on the amplitude of the AHP in vitro in hypothalamic slices (Fig. 5). As shown in Fig. 5A, trains of evoked action potentials were followed by a pronounced AHP in all recorded cells. No differences in AHP area were observed between cells from morphine-naı¨ve rats and morphine-dependent rats prior to naloxone administration (P ¼ 0.14). In morphine-dependent rats (in the continued presence of 1 lm morphine), bath application of naloxone (10 lm) significantly decreased AHP area (Fig. 4A–C; P < 0.05). By contrast, naloxone did not alter the AHP area in morphine-naı¨ve rats (Fig. 5A inset, 0.7 ± 0.2 mV ⁄ spike and 0.6 ± 0.2 mV ⁄ spike, in the absence and presence of naloxone, respectively; P ¼ 0.17, Fig. 5B). As with the TOR, the evoked AHP area did not significantly correlate with s (r ¼ 0.42, P ¼ 0.18) or input resistance (r ¼ 0.51, P ¼ 0.1) in magnocellular neurosecretory cells before application of naloxone. Furthermore, changes in AHP area induced by naloxone in


Intrinsic excitability in morphine withdrawal 507

Fig. 5. Morphine withdrawal decreases AHP area in supraoptic nucleus cells in vitro. (A) Examples of the AHP (arrows) in cells from a morphine-naı¨ve rat (inset) and a morphine-dependent rat (main panel) before (Pre-NLX) and during (NLX) application of 10 lm naloxone. Note that the AHP was reduced by naloxone in the cell from the morphine-dependent rat, but not in the cell from the morphine-naı¨ve rat. (B and C) Mean AHP area in cells from morphine-naı¨ve rats (left, n ¼ 6) and morphine-dependent rats (right, n ¼ 6). *P < 0.05 vs. pre-NLX, Student–Newman–Keuls test.

cells from morphine-dependent rats did not correlate with changes in either input resistance (r ¼ 0.40, P ¼ 0.20) or s (r ¼ 0.43, P ¼ 0.20).

Fig. 6. Morphine withdrawal increases spike broadening in supraoptic nucleus cells in vitro. (A) Examples of the first and ninth (bold line) spike in the train used to elicit AHPs in a cell from a morphine-dependent rat before (Pre-NLX) and during (NLX) application of 10 lm naloxone. Spike broadening (arrows) was greater in the presence of naloxone (note that the two arrows in each panel are the same distance apart). (B) Percentage change in spike width in cells from morphine-naı¨ve rats (circles, n ¼ 5) and morphinedependent rats (squares, n ¼ 5) before (open symbols) and during (closed symbols) naloxone application. For the sake of clarity, the data from the morphine-naı¨ve rats and morphine-dependent rats are plotted offset from each other.

Effects of morphine withdrawal on spike frequency adaptation in vasopressin cells in vivo Effects of morphine withdrawal on spike broadening in magnocellular neurosecretory cells in vitro The AHP is generated by activation of a Ca2+-dependent K+ conductance (Bourque et al., 1985; Armstrong et al., 1994) and spike broadening with repeated firing in magnocellular neurosecretory cells is dependent upon Ca2+ entry (Bourque & Renaud, 1985), so we also measured spike broadening during the depolarizing pulses used to elicit AHPs. As shown in the examples of Fig. 5, spike duration progressively increased during repetitive firing in all tested cells. No differences in spike broadening were observed between cells from morphine-dependent and morphine-naı¨ve rats before naloxone application (P ¼ 0.12, Fig. 6B). Two-way repeated measures anova revealed that while spike broadening was observed before and after bath application of naloxone (P < 0.0001), the overall degree of broadening was significantly larger after naloxone in morphinedependent rats (P < 0.001, Fig. 6B) but not in morphine-naı¨ve rats (P ¼ 0.87).

Because the AHP was reduced by naloxone in all cells from morphinedependent rats tested in vitro and the supraoptic nucleus contains both oxytocin and vasopressin cells, evidently the AHP was reduced in both oxytocin and vasopressin cells. The AHP induces spike frequency adaptation over the course of bursts in phasic vasopressin cells in vitro (Kirkpatrick & Bourque, 1996) and treatments that block the AHP in vitro also prevent spike frequency adaptation in vivo (Johnstone et al., 2004), so we tested the effects of naloxone on spike frequency adaptation during bursts of activity in spontaneously phasic vasopressin cells in vivo (e.g. Fig. 7A and B). In morphine-dependent rats, there was a progressive reduction in firing rate by 26.6 ± 8.6% over the first 45 s of spontaneous phasic bursts (Fig. 7A and D) from an initial intraburst firing rate of 8.3 ± 1.5 spikes ⁄ s in the first 5 s (n ¼ 6). In morphine-withdrawn rats, firing rate decreased by only 3.4 ± 13.7% over the first 45 s of bursts (Fig. 7B and D) from 7.0 ± 2.2 spikes ⁄ s (n ¼ 4). Two-way repeated measures anova showed that the reduction of firing rate over the course of phasic bursts


508 C. H. Brown et al. reduction in firing rate over the first 45 s of bursts, from an initial firing rate of 8.4 ± 1.4 spikes ⁄ s in the first 5 s).

Discussion Here, we have shown that in vivo naloxone-precipitated morphine withdrawal preferentially increased oxytocin cell post-spike excitability < 0.1 s after each spike, an increase that was not seen in vasopressin cells and that was specific to naloxone because hyperosmotic stimulation did not induce such changes in oxytocin cells. Furthermore, we have also shown that morphine withdrawal reduced the TOR and AHP in supraoptic nucleus cells in vitro; these effects were specific to cells from morphine-dependent rats because naloxone did not affect the TOR or AHP in cells from morphine-naı¨ve rats. In oxytocin cells, such changes in intrinsic properties might underpin the increased post-spike excitability evident during morphine withdrawal excitation in vivo. In vitro, we used 10 lm naloxone to precipitously remove morphine from chronic occupancy of l-opioid receptors. Such a high dose of naloxone would be expected to act as an antagonist at l-, j- and d-opioid receptors. However, the supraoptic nucleus contains only l- and j-opioid receptors and there are very few opioid receptors in the region surrounding the supraoptic nucleus (Sumner et al., 1990). Furthermore, there is no cross-tolerance between l- and j-opioid receptors within the supraoptic nucleus of morphine-dependent rats (Pumford et al., 1993). Thus, the major in vitro effects of applied naloxone on the TOR and AHP in magnocellular neurosecretory cells from morphine-dependent rats probably result from removal of morphine from chronic occupancy of supraoptic nucleus l-opioid receptors.

Intrinsic neuronal excitability during morphine withdrawal

Fig. 7. Morphine withdrawal reduces spike frequency adaptation in vasopressin cells in vivo. (A and B) Examples of spontaneous phasic firing in different vasopressin supraoptic nucleus cells recorded from anaesthetized morphine-dependent rats: (A) without naloxone and (B) after injection of naloxone (5 mg ⁄ kg, i.v.). (C) Phasic burst parameters in cells from morphinedependent rats without naloxone (control, n ¼ 6), and in cells after naloxone injection (NLX, n ¼ 4). D, Spike frequency adaptation over the course of bursts in the cells from C. Phasic cells without naloxone (open circles) displayed spike frequency adaptation (decreased firing rate over the course of bursts), typical of cells from morphine-naı¨ve rats. By contrast, in cells recorded after injection of naloxone (closed circles), spike frequency adaptation was not evident. *P < 0.05 and **P < 0.01 vs. 5-s data (first point).

was significant in morphine-dependent rats, but not morphinewithdrawn rats (Fig. 7D), indicating that spike frequency adaptation was reduced by morphine withdrawal, consistent with reduced AHP amplitude in vasopressin cells following morphine withdrawal as seen in vitro. Similarly to morphine-dependent rats, firing rate was reduced by 25.5 ± 8.8% over the first 45 s of spontaneous phasic bursts in morphine-naı¨ve rats from an initial firing rate of 9.0 ± 0.9 spikes s)1 in the first 5 s (n ¼ 10). Six vasopressin cells displaying spontaneous phasic activity in morphine-naı¨ve rats were recorded after naloxone administration. By contrast to phasic cells in morphine-withdrawn rats, phasic cells in morphine-naı¨ve rats showed substantial spike frequency adaptation after naloxone administration (21.8% ± 3.7%

Hazard functions measure changes in neuronal excitability following spontaneous spikes (Leng et al., 1995). Immediately after a spike (typically < 0.01–0.02 s), magnocellular neurosecretory cells are unexcitable, indicating the influence of post-spike hyperpolarizing conductances. Vasopressin cells then enter a period of hyperexcitability reflecting the influence of a depolarizing after-potential (DAP) that sustains vasopressin cell activity (Andrew & Dudek, 1983). Usually about 0.2–0.3 s after the preceding spike, their excitability stabilizes at a level that reflects the average rate of synaptic input and the steady-state resting potential (Leng et al., 1995). j-Opioid inhibition of vasopressin cells reduces DAP amplitude in vitro (Brown et al., 1999; Brown & Bourque, 2004), and excitability < 0.1 s after each spike in vivo (Brown & Leng, 2000; Brown et al., 2004), indicating that changes in post-spike potentials measured in vitro are translated into changes in post-spike excitability in vivo. Oxytocin cells do not show post-spike hyperexcitability under basal conditions (Leng et al., 1995). However, small DAPs can be exposed in oxytocin cells by blockade of the slow AHP (Greffrath et al., 1998), and the incidence of DAPs in oxytocin cells is increased in pregnancy and lactation (Stern & Armstrong, 1996; Teruyama & Armstrong, 2002). Thus, enhanced DAP amplitude could increase oxytocin cell activity. The hazard function in oxytocin cells during morphine withdrawal is similar to that of vasopressin cells (Brown & Leng, 2000; Brown et al., 2004), so increased DAP amplitude in oxytocin cells might contribute to withdrawal excitation. However, naloxone also induced a similar, but smaller, increase in post-spike excitability < 0.1 s after each spike in morphine-naı¨ve rats without apparent effects on the TOR or AHP. Hence, whilst


Intrinsic excitability in morphine withdrawal 509 post-spike excitability might be enhanced by opioid receptor antagonism, morphine withdrawal does so more potently than opioid receptor antagonism alone. Because the DAP and AHP overlap temporally (Greffrath et al., 1998; Teruyama & Armstrong, 2002), increased post-spike excitability during withdrawal excitation in vivo might equally result from decreased AHP amplitude. Although we cannot eliminate potential effects on DAPs, the naloxone-induced decrease of AHP amplitude in cells from morphine-dependent rats, but not cells from morphine-naı¨ve rats, suggests that this might further increase oxytocin cell post-spike excitability < 0.1 s after each spike during morphine withdrawal. Post-spike potentials are not the only membrane properties that influence post-spike excitability; the TOR modulates spike duration, interspike interval and firing frequency (Bourque, 1988). Because the TOR in magnocellular neurosecretory cells is Ca2+-dependent (Bourque, 1988), depolarization and Ca2+ influx associated with spikes will activate the current underlying the TOR, to induce a transient membrane hyperpolarization. Hence TOR modulation will have a similar net effect to AHP modulation, albeit with a shorter timecourse. Morphine withdrawal reduced both the TOR and AHP, and so the reduced influence of these Ca2+-dependent K+ properties probably contributes to enhanced magnocellular neurosecretory cell post-spike excitability < 0.1 s after each spike during morphine withdrawal. Numerous studies have addressed the cellular adaptations that might mediate morphine dependence, identifying counteradaptations in several second messenger systems. However, previous work has failed to identify changes in K+ or Ca2+ conductances associated with morphine dependence (Williams et al., 2001). By contrast, we have shown that intrinsic properties that are mediated by K+ conductances are altered in magnocellular neurosecretory cells during morphine dependence. Several mechanisms could underlie inhibition of TOR and AHP during morphine withdrawal in magnocellular neurosecretory cells. First, morphine withdrawal could directly inhibit the K+ channels underlying the TOR and AHP. Second, the reduced TOR and AHP could result from reduced Ca2+ entry resulting from inhibition of voltage-gated Ca2+ channels, a common target of l-opioid receptors (Grudt & Williams, 1995). However, the fact that TOR and AHP were reduced concomitantly with increased spike broadening, which results from activity-dependent Ca2+ entry during repetitive firing (Bourque & Renaud, 1985), suggests that Ca2+ availability is increased rather than decreased. Instead, reduced TOR and AHP might reflect a morphine withdrawal-induced reduction of the Ca2+-sensitivity of the Ca2+-dependent K+ channels. Small conductance Ca2+-dependent K+ (SK) channels mediate the AHP in magnocellular neurosecretory cells (Greffrath et al., 2004) and changes in Ca2+ sensitivity could result from altered association between calmodulin and the alpha subunits of SK channels (Xia et al., 1998). Indeed, changes in Ca2+-calmodulin signalling mechanisms have been previously observed during chronic morphine treatment (Liang et al., 2004). Finally, the decreased input resistance and membrane s observed during morphine withdrawal might indirectly affect the TOR and AHP, and thus post-spike excitability. For example, changes in membrane s could influence the delay to the first spike during membrane depolarization. In addition, changes in input resistance could indirectly affect the AHP and TOR, by affecting Ca2+entry and ⁄ or the activation range of Ca2+-dependent K+ channels during membrane depolarization. However, there was no significant correlation between TOR or AHP area and input resistance or s, either under basal conditions, or following morphine withdrawal, suggesting the presence of such indirect interactions is unlikely. Nevertheless, a mechanistic link between changes in membrane s and ⁄ or input resistance, and changes in post-spike excitability, can not be ruled out by these studies.

Input resistance changed linearly throughout a wide range of membrane potentials suggesting that the change probably results from modulation of a background, K+ leak conductance. Indeed, such channels, including the tandem-pore K+ family, are highly expressed in magnocellular neurosecretory cells (Han et al., 2003). Alternatively, changes in input resistance could result from alterations in magnocellular neurosecretory cell morphology, such as have been observed during lactation in oxytocin cells (Stern & Armstrong, 1998). The effects of morphine withdrawal on K+ and Ca2+ currents have been most extensively investigated in locus coeruleus neurons. Similarly to supraoptic nucleus cells, local mechanisms contribute to morphine withdrawal excitation of locus coeruleus neurons (AstonJones et al., 1997). However, naloxone effects on the inwardly rectifying K+ conductance in locus coeruleus neurons are similar in morphinenaı¨ve rats and morphine-dependent rats (Christie et al., 1987). Similarly to the inwardly rectifying K+ conductance, naloxone-induced increases in the Ca2+ current, IBa, in locus coeruleus neurons are not different in morphine-naı¨ve rats and morphine-dependent rats (Connor et al., 1999). It appears likely that withdrawal-induced increases in firing rate in locus coeruleus neurons are driven by increased glutamate release during withdrawal (Akaoka & Aston-Jones, 1991). By contrast to locus coeruleus neurons, naloxone-induced reduction of the TOR and AHP in supraoptic nucleus cells was specific to morphine-dependent rats, suggesting these reductions are important contributors to oxytocin cell morphine withdrawal excitation. Whilst naloxone-precipitated morphine withdrawal in vivo preferentially increased oxytocin cell post-spike excitability < 0.1 s after each spike, steady-state excitability (> 0.1 s after each spike) was also increased during morphine withdrawal. Steady-state excitability is deduced to reflect the average rate of synaptic input and the steady-state resting potential. Because activity in oxytocin cells is entirely dependent upon synaptic input in vivo, it is possible that this increased steady-state excitability results from oxytocin cells passively following excitation in their afferent inputs during morphine withdrawal. Indeed, acute pharmacological blockade of any one of several excitatory inputs reduces withdrawal excitation of oxytocin cells (Russell et al., 1992b; Brown et al., 2000) and so full withdrawal excitation of oxytocin cells requires activity in their afferent inputs. However, the activity in these inputs is not necessarily increased during morphine withdrawal as there is little increase in Fos protein expression following withdrawal in the major forebrain and brainstem projections to the supraoptic nucleus (Murphy et al., 1997) and chronic neurotoxic lesion of brainstem inputs to the supraoptic nucleus does not affect withdrawal excitation of oxytocin cells (Brown et al., 1998b). Finally, direct intrasupraoptic nucleus naloxone administration to morphine-dependent rats evokes withdrawal excitation (Ludwig et al., 1997), as does systemic naloxone after chronic morphine infusion directly into the supraoptic nucleus (Johnstone et al., 2000). Thus, because the supraoptic nucleus contains only oxytocin and vasopressin cells and l-opioid receptors are scarce in the region surrounding the supraoptic nucleus (Sumner et al., 1990), the mechanisms underlying withdrawal excitation of oxytocin cells in morphine dependent rats appear to reside within the oxytocin cells themselves. Morphine-withdrawal induced changes in vasopressin cell activity The supraoptic nucleus contains both oxytocin and vasopressin cells. By contrast to oxytocin cells, vasopressin cells do not undergo morphine withdrawal excitation; naloxone administration to morphine-dependent rats does not consistently change vasopressin cell firing rate and only modestly increases vasopressin secretion (Bicknell


510 C. H. Brown et al. et al., 1988). Thus, it was unexpected that morphine withdrawal decreased the AHP and TOR in all supraoptic nucleus cells tested in vitro, and that it decreased spike frequency adaptation in VP neurons in vivo. However, the failure of the reduced AHP to induce functional morphine withdrawal excitation in vasopressin cells in vivo does not imply that this is not important in oxytocin cells. The DAP is much larger in vasopressin cells than in oxytocin cells, a fact that is reflected in the larger early:late hazard ratio in vasopressin cells (Fig. 3E) than oxytocin cells (Fig. 1H). The failure of the reduced AHP to consistently increase vasopressin cell firing rate during withdrawal is probably due to the greater influence of the larger DAP in these neurons. A further reason why vasopressin cells fail to exhibit morphinewithdrawal excitation might lie in the differential effects of peptides released from their dendrites (Ludwig, 1998). Dendritic oxytocin release is autoexcitatory, contributing to morphine withdrawal excitation (Brown et al., 1997). By contrast, vasopressin cells are subject to activity-dependent autocrine inhibition by coreleased dynorphin acting on j-opioid receptors (Brown & Bourque, 2004) contained in the same neurosecretory vesicles as dynorphin and vasopressin (Shuster et al., 2000). Thus, exocytosis will expose newly inserted j-opioid receptors to high local dynorphin concentrations and so even local j-opioid antagonist administration does not completely block endogenous dynorphin inhibition in opioidnaı¨ve rats (Brown et al., 1998a). Because there is no cross-tolerance between l- and j-opioid receptors in morphine-dependent rats (Pumford et al., 1993), activity-dependent j-opioid vasopressin cell inhibition will still occur, opposing the potential excitation induced by morphine withdrawal, even in the presence of naloxone. Furthermore, dendritically released vasopressin equalizes activity across the vasopressin cell population (Gouzenes et al., 1998; Brown et al., 2004). Thus, vasopressin cells possess intrinsic mechanisms that are expected to offset the potential excitation induced by morphine withdrawal.

Because some brain areas that express oxytocin receptors do not contain oxytocin fibres [e.g. the olfactory bulb (Yu et al., 1996; Breton & Zingg, 1997)], oxytocin released from magnocellular neurosecretory cell dendrites might diffuse through the parenchyma and ⁄ or cerebrospinal fluid to affect other brain areas by volume transmission (Yu et al., 1996). Thus withdrawal-induced oxytocin release from dendrites might be responsible for the central actions of oxytocin associated with opiate addiction.

Conclusions The increased post-spike excitability induced by naloxone in oxytocin cells of morphine-dependent rats probably involves inhibition of the K+ conductances that generate the TOR and AHP. These effects of morphine withdrawal on intrinsic membrane properties would allow the influence of ongoing (or increased) synaptic drive to be expressed as increased firing rate. Further studies will be required to determine the mechanisms of the altered regulation of the AHP and TOR in oxytocin cells from morphine-dependent rats.

Acknowledgements We are grateful to Drs N. P. Murphy, K. M. Pumford, A. C. Robson and S. Scullion for assistance in the completion of the in vivo experiments. Supported by the Wellcome Trust (C.H.B.), the Biotechnology and Biological Sciences Research Council (J.A.R.), the Human Frontier Science Program (J.E.S.) and the National Institutes of Health grant HL68725 (J.E.S.).

Abbreviations ANOVA, analysis of variance; AHP, after-hyperpolarization; DAP, depolarizing after-potential; TOR, transient outward rectification.

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