A neuromodulatory role for oxytocin within the supramammillary nucleus

Neuropeptides Neuropeptides 41 (2007) 217–226 www.elsevier.com/locate/npep

A neuromodulatory role for oxytocin within the supramammillary nucleus M.R. Cumbers, S.T. Chung, J.B. Wakerley

*

Department of Anatomy, University of Bristol, School of Medical Sciences, University Walk, Bristol BS8 1TD, UK Received 22 October 2006; accepted 10 April 2007 Available online 12 June 2007

Abstract Oxytocin functions as both a neurohypophysial hormone and central neuromodulatory peptide, and has been implicated in reproductive behaviours, anxiety and reward, as well as facilitation of the neuroendocrine milk-ejection reflex. A potential substrate for oxytocin is the supramammillary nucleus (SuM), a structure that contains oxytocin binding sites and serves as an important relay within the limbic system. Hence, this study investigated the neuromodulatory role of oxytocin within the SuM. Firstly, the effect of oxytocin on neuronal firing within the SuM was studied, using in vitro brain slices from virgin female rats. Oxytocin (106 M) excited approximately 50% of SuM neurones, and similar results were obtained with the selective oxytocin agonist, Thr4 Gly7 oxytocin (TGOT) (106 and 107 M). The remaining neurones were unaffected. The TGOT response was blocked by application of the oxytocin antagonist, [dðCH2 Þ5 1 ; TyrðMeÞ2 ; Thr4 ; Orn8 ; Tyr-NH2 9 ]-vasotocin. Repeat doses of TGOT caused diminution of the response, indicative of desensitisation. In the second series of experiments, immunocytochemical techniques were used to study the oxytocinergic innervation of the SuM. The supramammillary decussation was found to contain numerous oxytocinergic fibres, and some could be seen coursing ventrally to enter the SuM. Whereas, some were clearly ‘‘en passant’’ fibres innervating the neurohypophysis, others followed a more convoluted and branching course, and appeared to terminate within the nucleus. Finally, in vivo microinfusion studies investigated whether oxytocin injected into the SuM facilitated the milk-ejection reflex, a well known action of central oxytocin. Oxytocin microinfusion in the region of the SuM caused a pronounced facilitation of the reflex, contrasting with the much smaller effects of microinfusions made rostral or caudal to the nucleus. Collectively, these results strongly support a neuromodulatory role for oxytocin within the SuM. This could have important implications for understanding the diverse neuroendocrine and behavioural functions of central oxytocin, including its role in reward.  2007 Elsevier Ltd. All rights reserved. Keywords: Oxytocin; Supramammillary nucleus; Electrophysiological responses; Oxytocinergic fibres; Milk-ejection reflex; Facilitation

1. Introduction Oxytocin (OT) is neurohypophysial hormone that was first ascribed import peripheral actions on uterine contractility and milk-ejection, but has subsequently

*

Corresponding author. Tel.: +44 (0)117 9287406; fax: +44 (0)117 9291687. E-mail address: [email protected] (J.B. Wakerley). 0143-4179/$ - see front matter  2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.npep.2007.04.004

been recognised as an important neuromodulatory peptide (Landgraf and Neumann, 2004; Kiss and Mikkelsen, 2005; Leng et al., 2005). Within the limbic system, neuromodulatory effects of OT have been reported in the bed nucleus (Lambert et al., 1993; Wilson et al., 2005), hippocampus (Raggenbass et al., 1998) and amygdala (Condes-Lara et al., 1994; Terenzi and Ingram, 2005). These neuronal effects are thought to underlie a range of behavioural and neuroendocrine responses that are evoked by centrally-administered

218

M.R. Cumbers et al. / Neuropeptides 41 (2007) 217–226

OT, including facilitation of the milk-ejection reflex (Freund-Mercier et al., 1988; Richard et al., 1991), anxiolysis (Neumann et al., 2000; Amico et al., 2004), grooming (Stivers et al., 1988), penile erection, and yawning (Argiolas et al., 1989). Although, the central actions of OT have been extensively investigated, there still remain some important forebrain structures which have been ignored as potential targets for this peptide. One such structure is the supramammillary nucleus (SuM) of the posterior hypothalamus. The SuM is regarded as an integral part of the limbic system, and has extensive connections with the diagonal band, preoptic area, septum, amygdala, hippocampus, and raphe nuclei (Ottersen, 1980; Gonzalo-Ruiz et al., 1992; Hayakawa and Zyo, 1996; Kiss et al., 2000; Kiss et al., 2002). Functionally, the SuM has been implicated in the regulation of hippocampal theta activity (Kirk, 1998; Pan and McNaughton, 2002), memory (Shahidi et al., 2004; Aranda et al., 2006), anxiety (Aranda et al., 2006), and, most recently, in reward (Ikemoto et al., 2004). The SuM has been reported to contain OT binding sites (Kremarik et al., 1995), and mRNA for the OT receptor has also been detected in this nucleus (Yoshimura et al., 1993; Gould and Zingg, 2003). Moreover, several of the roles of the SuM overlap with functions that are modulated by OT, e.g. reward-related behaviour (Young et al., 2001; Insel, 2003), and memory (Kovacs et al., 1979; Ferguson et al., 2000). Thus, establishing a neuromodulatory action for OT within the SuM would help understand how OT brings about its various behavioural and neuroendocrine effects. The present series of experiments were therefore undertaken to investigate the neuromodulatory role of OT within the SuM. First, we investigated the electrophysiological effects of OT, and the selective OT agonist, Thr4 Gly7 OT (TGOT), on SuM neurones recorded from brain slices maintained in vitro. Second, we studied the detailed pattern of OTergic innervation of the SuM, using immunocytochemistry to localise OT-containing fibres within and surrounding this region. Finally, we investigated the effects of OT microinfusion made into the SuM on the neuroendocrine milk-ejection reflex. The milk-ejection reflex is facilitated by centrally-administered OT (Freund-Mercier et al., 1988; Richard et al., 1991) and microinfusion studies based on this facilitatory effect have previously helped to establish the neuromodulatory role of OT within the bed nucleus of the stria terminalis (Ingram et al., 1995). We reasoned that if OT acted as a neuromodulator within the SuM, a structure with a longestablished role in the milk-ejection reflex (Cowie and Tindal, 1971), then OT microinjections at this site should similarly facilitate the occurrence of milk-ejection responses.

2. Materials and methods 2.1. In vitro electrophysiological experiments Brain slices containing the SuM were prepared from 44 virgin female Wistar rats (150–225 g) from which a total of 59 neurones were recorded. Following decapitation under sodium pentobarbitone anaesthesia, the brain was rapidly removed and transverse brain slices (400 lm) prepared using a vibratome. Sections containing the SuM were trimmed to remove cortical tissue and transferred to the platform of an incubation chamber where they were maintained at 33 C with their under surface exposed to artificial cerebrospinal fluid (aCSF) (NaHCO3 26 mM, D-Glucose 10 mM, NaCl 124 mM, MgSO4 2.4 mM, KCl 3.25 mM, KH2PO4 1.25 mM, and CaCl2 1.0 mM). The upper surface of the slice was exposed to a 95% O2/5% CO2 humidified gas mixture. After a 90 min equilibration period, extracellular recordings were made from the SuM using glass microelectrodes filled with aCSF (impedance 5–10 MX). The nucleus was clearly visible as a translucent area lying between the principal mammillary tracts at the level of the caudal recess of the third ventricle (see Fig. 1). Action potentials were fed to a window discriminator and the resultant pulses recorded using Spike2 software (CED, Cambridge, UK). Following a minimum of 5 min of stable base-line recording, OT or the selective OT agonist, Thr4 Gly7 OT (TGOT) (Lowbridge et al., 1977) (Bachem, UK), was applied to the slice via the incubation medium. Drugs were applied as four-minute pulses, at concentrations of 107 or 106 M. Some neurones were also tested with simultaneous application of 107 M TGOT and the selective OT receptor antago2 nist, [dðCH2 Þ5 1 ; TyrðMeÞ ; Thr4 ; Orn8 ; Tyr-NH2 9 ]-vasotocin (Elands et al., 1988) (Bachem, UK). To reduce the problem of desensitisation, a recovery period of 15 min was allowed between each drug exposure and multiple drug applications to individual slices were avoided as far as possible. The electrophysiology data were displayed and analysed off-line using the Spike2 software to generate sequential histograms of neuronal firing rates. Counts in successive 30 s time epochs were converted to a Microsoft Excel file, enabling summation and averaging of data from multiple recordings within an Excel spreadsheet. 2.2. Immunocytochemical studies Material for histological analysis was collected from three adult male and five female Wistar rats weighing 250–325 g. The rats were deeply anaesthetised with sodium pentobarbital (45 mg/kg i.p.) and perfused transcardially with 100 ml of 0.2% (0.1 M) phosphate buffer (PB)(pH 7.4), followed by 100 ml of fixative solution

M.R. Cumbers et al. / Neuropeptides 41 (2007) 217–226

a

OT (10 -6 M)

219

TGOT (10 -6 M)

6

4

Spikes per sec

4 2 2 0

0 2

1

0 0

200

400

600

800

0 0

200

600

400

800 sec

b

SuM pm MMn

f

1 mm

1 mm

Fig. 1. (a) Shows individual examples of supramammillary neurones that were excited (upper traces) or unaffected (lower traces) by 106 M oxytocin (OT) and 106 M Thr4 Gly7 oxytocin (TGOT). (b) Indicates the position of the neurones recorded in these experiments. On the left is photograph of a brain slice, showing the position and appearance of the supramammillary nucleus (arrowed). On the right, the position of each recorded neurone is marked on two identical stereotaxic maps (Paxinos and Watson, 1986). The foreground map shows neurones that were responsive to 106 M OT (filled circles) or 106 M TGOT (filled squares), and the background map shows neurones unresponsive to these peptides (open circles and squares, respectively). Abbreviations: f, fornix; MMn, median mammillary nucleus; SuM, supramammillary nucleus; pm, principal mammillary tract.

containing 4% paraformaldehyde in 0.1 M PB. Brains were then removed, left in paraformaldehyde overnight and transferred to 25% sucrose dissolved in 0.1 M PB for 24–48 h. A tissue block was then prepared and mounted on a cryostat, and transverse sections (40 lm) were taken throughout the SuM region. Sections were collected in phosphate buffered saline (PBS, 0.9% sodium chloride in 0.01 M sodium phosphate buffer, pH 7.4) and incubated in PBS with 1% TritonX-100 (Sigma-Aldrich, Gillingham, UK). Sections were incubated in 0.3% H2O2 in PBS containing Triton X-100 for 10 min to block the endogenous peroxidase present in tissues, followed by incubation in normal goat serum (Vector Labs, Peterborough, UK). The sections were then washed in PB and incubated with the anti-OT primary antibody (Chemicon, Temecula, USA) (raised in rabbit) at a dilution of 1:5000 in PB at 4 C overnight. After further washing in PB, sections were incubated with secondary antibody (biotinylated goat anti-rabbit IgG, Vector Labs) at a dilution of 1:200 in PB for 2 h. Sections were further rinsed in PB and incubated for an hour with 1:200 avidin-conjugated peroxidase (ABC kit, Vector Labs), followed by washes with 0.05 M Tris buffer. The immunoperoxidase reaction was developed using 0.04% 3,3 0 -diaminobenzidine-4HCL (Sigma-Aldrich)

for 15–20 min at room temperature. The reaction was stopped with cold PB and the sections mounted in DPX. Localisation of immunoreactive OT fibres in the caudal hypothalamus was mapped using the rat brain atlas of Paxino and Watson (Paxinos and Watson, 1986). An indication of the density of OT fibres within the SuM was obtained by counting the number of immuno-stained fibres in the region in between the principal mammillary tracts (see Fig. 1b), an area of approximately 0.6 mm2. A mean count was obtained for each brain, based on the average of three sections, and values expressed as number of fibres per mm2 of section. 2.3. In vivo experiments on the milk-ejection reflex Experiments were undertaken on five lactating (day 8– 10) rats separated overnight from all but one of their pups, anaesthetised with urethane (1.1 g/Kg), and prepared for intramammary pressure recording during suckling, as previously described (Jiang and Wakerley, 1995). After stabilising the head within a stereotaxic frame, the scalp was incised, and a single bur hole (diameter 1.5 mm) was made on the mid-line at the level of the caudal hypothalamus. The superior sagittal sinus was then ligated, and the dura removed. The rat was then left for an hour before a litter of pups was applied to activate the milk-ejection reflex, and any milk-ejections were

220

M.R. Cumbers et al. / Neuropeptides 41 (2007) 217–226

recorded. Milk-ejections were recognised by an abrupt intramammary pressure increase, with an accompanying stretch response from the pups (Lincoln et al., 1973). After the pups had been applied, a 10 lL Hamilton micro-syringe containing 106 M OT (made up in physiological saline) was positioned with its needle located at the first microinjection site (see below). After one hour of suckling, regardless of the occurrence of reflex milk ejections, a slow microinfusion of 2 lL of 106 M OT (approx. 2 ng) was given over 10 min. Milk-ejection responses were then monitored for a 30 min period from the end of the infusion. The micro-syringe was then relocated to the second injection site and, after 30 min, a further OT microinfusion was undertaken. All microinfusions were made on the midline at a depth of 8.5 mm from the brain surface at different anterior–posterior co-ordinates estimated to be either rostral to the SuM (rostral sites, 4.9–5.7 mm anterior to interaural line), at the level of the SuM (middle sites, 4.0–4.8 mm anterior to interaural line), or caudal to the SuM (caudal sites, 3.1–3.9 mm anterior to interaural line). As anticipated from previous studies (Jiang and Wakerley, 1995), the rats displayed very few milkejection responses in the absence of OT-induced facilitation of the responses. Hence the effects of microinfusions were evaluated simply as the number milk-ejections observed in the 40 min period following onset of OT administration. The microinfusions were randomised with regard to their position, and where more than one microinfusion was made at a given site, the mean response was taken. Number of milk-ejections evoked in each animal at the different sites were compared using the Mann–Whitney U test. After the end of the experiment, the rats were deeply anaesthetised with 45 mg/Kg sodium pentobarbitone, perfused with fixative, and their brains prepared for histological analysis of the microinfusion sites. Microinfusion sites were identified by reconstructing the tracks of the micro-syringe needle through successive brain sections and marking these on a stereotaxic map (Paxinos and Watson, 1986). Rostral microinfusion sites were focused on the hypothalamic arcuate nucleus, middle sites on the SuM, and caudal sites on the linear nucleus of the raphe.

excited by OT, displaying an increase in firing lasting 7.9 ± 0.7 min, with a maximal change of 2.7 ± 0.5 spikes/s. The remaining seven neurones tested with OT were unresponsive to the peptide. Unresponsive cells were readily excited by 106 M NMDA, confirming that this lack of response was not due to a generalised loss of excitability. Similar results were obtained in a second series of 21 recordings using 106 M TGOT instead of OT (Fig. 1b). Thus, 10 out of 21 (48%) neurones responded to TGOT, displaying an increase in firing lasting 7.4 ± 0.9 min, with a maximal change of 3.9 ± 1.1 spikes/s. All of these recordings (i.e. both for TGOT and OT tests) were made in the rostral portion of the SuM, where the nucleus was easy to recognise in the slice as a translucent area lying between the principal mammillary tracts. Neurones responsive to OT or TGOT were located across the nucleus at this level, consistent with the diffuse distribution of OT receptor mRNA (Yoshimura et al., 1993), and there was no obvious difference in the distribution of responsive versus unresponsive neurones (Fig. 1b). Nineteen additional recordings were undertaken to further characterise the response to TGOT. Thus, SuM neurones were tested with 107 M TGOT, a concentration that was more comparable with studies in other brain regions. 107 M TGOT excited a similar proportion of neurones (9 out of 19 cells, 47%), but with a lower peak response (Fig. 2a). Interestingly, the decay following the response peak was noticeably slower with 107 M TGOT than observed with 106 M TGOT (Fig. 2a), perhaps indicating that the response to the higher concentration was attenuated by receptor desensitisation. The occurrence of desensitisation was also suggested from analysis of the reproducibility of the 106 M TGOT responses, which revealed a marked reduction in response amplitude when neurones were tested with a second exposure to TGOT (Fig. 2b). Finally, three recordings were undertaken to examine the effect of the OT receptor antagonist, [dðCH2 Þ5 1 ; TyrðMeÞ2 ; Thr4 ; Orn8 ; Tyr-NH2 9 ]-vasotocin (Elands et al., 1988) on the response to TGOT. This antagonist was found to completely abolish the TGOT excitation. (Fig. 2c). Only partial recovery of the response could be observed during the limited time course of these recordings, possibly due to receptor desensitisation or delayed washout of the antagonist.

3. Results 3.1. Effects of OT and TGOT on the firing of SuM neurones Spontaneously firing SuM neurones were readily encountered in vitro, with background firing ranging from 0.1 to 6.4 spikes/s. In the first series of experiments, 16 SuM neurones were tested with 106 M OT (see examples in Fig. 1a). Nine (56%) of the neurones were

3.2. Immunocytochemical analysis of the OTergic innervation of the SuM There have been several previous studies of the OTergic innervation of the rat brain (Buijs, 1980; Sofroniew, 1980), and this description will focus only on the SuM region. Immunoreactive OT fibres were readily identified in the sections by their dark-brown beaded appearance, and could often be followed for several hundred

M.R. Cumbers et al. / Neuropeptides 41 (2007) 217–226

Change in rate (spikes/s)

a

TGOT (10-7M)

221

TGOT (10-6M)

5

5

4

4

3

3

2

2

1

1 0

0 0

200

400

600

800

200

0

1000 sec

400

600

800

1000 sec

Change in rate (spikes/s)

b TGOT (10-6M)

TGOT (10-6M)

4 3 2 1 0 0

200

400

600

800

0

1000

200

400

600

800

1000 sec

Change in rat (spikes/s)

c TGOT (10-7M)

TGOT (10-7M)

TGOT (10-7M)

ANTAG (10-7M)

3 2 1 0 0

200

400

600

800

0

200

400

600

800

0

200

400

600

800

1000 sec

Fig. 2. (a) Shows the mean (±S.E.) change in firing rate of responsive supramammillary neurones during perfusion with 107 M (n = 9) and 106 M (n = 10) Thr4 Gly7 oxytocin (TGOT). Note the more rapid decay of the response to the higher concentration of TGOT. (b) Shows the mean response of neurones that were tested with two applications of 106 M TGOT (n = 8). Note the reduced response to the second test, indicative of desensitisation. (c) Shows blockade of the response to TGOT by a selective OT receptor antagonist (ANTAG), [dðCH2 Þ5 1 ; TyrðMeÞ2 ; Thr4 ; Orn8 ; Tyr-NH2 9 ]-vasotocin (n = 3). Note the partial recovery of the response (see text for explanation).

microns. At the level of the rostral portion of the SuM, many OT-containing fibres could be seen above the nucleus, passing laterally within the supramammillary decussation to arch over the principal mammillary tract and fornix before following their characteristic ventromedial course towards the base of the third ventricle (Fig. 3a). The supramammillary decussation also contained punctuate immuno-staining consistent with the presence of OT fibres orientated in a rostrocaudal direction at right angles to the plane of section. Along the dorsal border of the SuM, fibres could be seen leaving the supramammillary decussation to pass ventrally into the nucleus. Whereas, some of these fibres appeared to run in an uninterrupted ventral direction as if traversing the SuM, others followed a more convoluted path to become dispersed within the nucleus. Moreover, branching could be observed in some of these axons (Fig. 3b). The OTergic innervation of the SuM appeared to be denser in its rostral rather than caudal regions and where the nucleus becomes divided caudally into medial and lateral subdivisions, very few OT fibres were detected. However, the dense network of OT fibres located immediately above the nucleus was still present

at this level (Fig. 3a). The above description is based on studies in five female and three male rats and there were no obvious sex differences in the OTergic innervation of the SuM. Average density of OTergic fibres within sections of the SuM was estimated (see Section 2) to be 25.3 ± 3.1 fibres per mm2 for the female rats, compared to 33.9 ± 8.7 for the males, a difference that was non-significant (P < 0.3, Student t test). 3.3. Effect of microinfusion of OT within the SuM on facilitation of the milk-ejection reflex Results were obtained from five lactating rats in which microinfusions were made at rostral, middle and caudal sites (see Section 2). In all five rats, microinfusion of OT at the middle injection site (i.e. at the level of the SuM) was followed by the onset of a series of facilitated milk-ejection responses. An example is shown in Fig. 4a. The mean latency of this facilitation from the onset of the microinfusion was 8.84 ± 2.12 min. Termination of the facilitatory effect was accompanied by chewing and movements of the vibrissae, events that are characteristic of EEG arousal in the suckled urethane-anesthetised rat

222

M.R. Cumbers et al. / Neuropeptides 41 (2007) 217–226

Fig. 3. (a) Shows the distribution of oxytocin (OT) fibres in and surrounding different levels of the supramammillary nucleus. Presence of immunocytochemically-stained fibres is represented by freehand lines superimposed upon stereotaxic maps of the rat brain at planes 4.70, 4.48 and 4.20 anterior to interaural line. Maps are based on the rat brain atlas of Paxinos and Watson (Paxinos and Watson, 1986). (b) (lower) shows a high power (·200) photomicrograph of the dorsal supramammillary nucleus to illustrate the appearance of OT fibres (arrows) within the nucleus. The photomicrograph (uppper) indicates the position of the high power view (indicated by the white square). Abbreviations: f, fornix; fr, fasciculus retroflexus; ML, medial mammillary nucleus (lateral); MM medial mammillary nucleus (medial); MMn, median mammillary nucleus; mtg, mammillothalamic tract; PH, posterior hypothalamus; pm, principal mammillary tract; SuM, supramammillary nucleus; SuMM, supramammillary nucleus (medial division). See text for further explanation.

(Wakerley et al., 1989). Microinfusions made rostral or caudal to the SuM evoked significantly fewer milk-ejections than microinfusions at the level of the SuM (Fig. 4b). There was no evidence that mechanical stimulation at the microinfusion site, e.g. during repositioning of the micro-syringe needle, could facilitate of the milkejection reflex.

4. Discussion The present results showed that neurones within the SuM were modulated by OT, and this effect was mimicked by TGOT, an analogue with greatly enhanced selectivity for OT rather than vasopressin receptors (Lowbridge et al., 1977). Moreover, the TGOT responses were blocked by the specific OT receptor antagonist, 2 [dðCH2 Þ5 1 ; TyrðMeÞ ; Thr4 ; Orn8 ; Tyr-NH2 9 ]-vasotocin (Elands et al., 1988), indicating the involvement of ‘‘classical’’ OT receptors similar to those within the mammary

gland. These electrophysiological findings were consistent with previous reports that the SuM contained OT binding sites (Kremarik et al., 1995), and mRNA for the mammary-type of OT receptor (Yoshimura et al., 1993). In further experiments it was confirmed, using immunostaining, that OTergic fibres were present within the SuM. Finally, microinfusions of OT into the SuM in lactating rats were found to facilitate the neuroendocrine milk-ejection reflex, so providing a functional in vivo correlate for the in vitro electrophysiological results. Previous studies have established that OT exerts its central behavioural and neuroendocrine effects through multiple sites including the amygdala, bed nucleus and hippocampus (see Gimpl and Fahrenholz, 2001). Based on current findings, the SuM provides an additional target for OT, and most likely has a role in the central actions of this peptide. The electrophysiological results indicated that around 50% of SuM neurones responded to 106 OT or TGOT, and a similar result was obtained with a lower (107 M) dose of TGOT. This was comparable to the

M.R. Cumbers et al. / Neuropeptides 41 (2007) 217–226

223

OT microinfusion

a

SuM 1 mm 10 min

OT (i.v.)

b

5.4

4.7

Number of milk ejections

5

4

3

**

*

2

1

4.2 0 Rostral

Middle

Caudal

3.2

Fig. 4. (a) Individual experiment showing the effect of an oxytocin (OT) microinfusion into the supramammillary nucleus on reflex milk ejection responses evoked by suckling (see Section 2 for details). On the left is a low power photomicrograph showing the track of the microinfusion needle (arrowed) within the supramammillary nucleus. On the right is shown the sequence of facilitated milk-ejection responses that occurred following a slow (10 min) microinfusion of 2 lL of 106 M OT. Note that the response prior to the microinfusion (circle) was evoked by an intravenous injection of OT, given to confirm mammary OT sensitivity. (b) Shows summary data (n = 5 in each case) for microinfusions made at different locations relative to the position of the supramammillary nucleus (see Section 2). Location of the microinjection sites is shown on the left. Injections were made rostral to the supramammillary nucleus (cross hatched triangles), at the level of the supramammillary nucleus (filled circles), or caudal to the supramammillary nucleus (open squares) (see Section 2). On the right is shown the mean number of facilitated milk ejections evoked by OT microinfusion at the different sites (P < 0.05, P < 0.01, Mann–Whitney U test). Stereotaxic maps are based on Paxinos and Watson (1986) (see Fig. 3 for labels).

proportion of TGOT sensitive neurones in the bed nucleus (Ingram et al., 1990; Wilson et al., 2005), but higher than that reported for the amygdala (Terenzi and Ingram, 2005). In common with findings in these other regions, by far the most predominant effect of OT and TGOT in the SuM appears to be excitatory, and such excitations are thought to involve the opening of non-specific cation channels, or the closing of potassium channels (Raggenbass, 2001). Interestingly, the OT responses we encountered in the SuM were similar in magnitude to those previously found in the bed nucleus (Ingram et al., 1990; Wilson et al., 2005), despite a much lower level of OT binding in the SuM (Kremarik et al., 1995). Another characteristic of the SuM responses was their desensitisation with repeated doses of TGOT (Fig. 2b), and this has also been seen in the lateral dorsal bed nucleus (Wilson et al., 2005) and central nucleus of the amygdala (Terenzi and Ingram, 2005). A possible mechanism for this desensitisation could be inactivation and internalisation of OT receptors that is known to

occur after ligand binding (Plested and Bernal, 2001), and this might be particularly important in areas such as the SuM where there is a low level of OT receptor expression. However, against this suggestion, Wilson and co-workers found that occurrence of desensitisation in different regions of the bed nucleus was inversely related to the density of OT receptors (Wilson et al., 2005). Instead, these authors proposed that regional differences in susceptibility to desensitisation might reflect the presence of a particular type of OT receptor, or OT receptor coupling mechanism. Our electrophysiological evidence that OT acts as a neuromodulator within the SuM was further supported by immunocytochemical results showing the presence of OT fibres within this nucleus. Surprisingly, there has been no previous mention of OT fibres in the SuM, despite detailed studies of the OT innervation of the rat brain (Buijs, 1980; Sofroniew, 1980). Whilst many of these SuM OTergic fibres may have been en passant fibres, passing ventrally towards the neurohypophysis,

224

M.R. Cumbers et al. / Neuropeptides 41 (2007) 217–226

the presence of convoluted and branching axons is suggestive that some may have terminated locally. In any case, it has been demonstrated that exocytosis of OTcontaining granules may occur from multiple compartments of OTergic neurones, including axons and axonal swellings (Morris and Pow, 1991), so these axons may still provide a source of OT, even in the absence of specialised terminals. OT is also present in cerebrospinal fluid (Robinson and Coombes, 1993), so could reach the SuM by volume diffusion from the adjacent recess of the third ventricle. The present study did not identify the source of the OTergic fibres entering the SuM, but their close relationship with fibre bundles passing towards neurohypophysis would suggest that they were derived from the magnocellular paraventricular nucleus. Indeed, some of the OTergic fibres within the SuM might represent branches from axons innervating the neurohypophysis, although available evidence suggests that such axons tend not to have centrally projecting collaterals (Pittman et al., 1981). Additional evidence for a role for OT in the SuM was provided by functional studies in which microinfusions of OT into the SuM resulted in facilitation of the neuroendocrine milk-ejection reflex. The time course of the facilitated milk-ejection responses, and the chewing motions accompanying their termination, are comparable to observations made when OT is injected directly into the ventricular system (Wakerley et al., 1989). However, it would seem unlikely that this facilitation arose simply through diffusion of OT into the ventricular system since it was markedly diminished when infusions were made rostral to the SuM, in some cases impinging directly onto the caudal third ventricle. Involvement of the SuM in the milk-ejection reflex was first demonstrated by early electrical stimulation studies (Cowie and Tindal, 1971), and the present results confirm that the SuM contributes to the control of this reflex when activated by central OT. The SuM has widespread projections outside the hypothalamus, including to the septum and bed nucleus (Ottersen, 1980; Vertes, 1992), and this might argue against the idea that the SuM functions specifically as a relay to the paraventricular and supraoptic nuclei that mediate milk-ejection (see Wakerley, 2006). More probably, the ability of the SuM to alter the milk-ejection reflex arises through projections to other diverse structures that, in turn, impinge directly or indirectly upon these nuclei. Based on this interpretation, it is quite likely that facilitation of milk-ejection reflex would be one of a number of behavioural and neuroendocrine responses that, under other conditions, might be evoked by OT injections into the SuM. Our conclusion that OT acts as a neuromodulator within the SuM may help understand other central actions of this peptide. OT has been called a ‘‘neuropeptide of affiliation’’, on account of its actions in pro-

moting mating, pair bonding, and maternal behaviour (see Insel, 1992; McCarthy and Altemus, 1997). These behavioural effects may all stem from the ability of OT to reduce anxiety normally caused by social encounters with other individuals (McCarthy, 1995), and to increase their rewarding aspect (Young et al., 2005). Rewarding stimuli activate the mesolimbic dopamine system (Spanagel and Weiss, 1999) and, since many dopamine neurones in this system are located in the ventral tegmental area, it has been assumed that this region is involved in effects of OT on dopamine transmission and reward. This idea has been supported by evidence that, for example, dopamine-dependent grooming (Stivers et al., 1988), or maternal behaviour (Pedersen et al., 1994) can be evoked by OT microinjections into the ventral tegmentum. However, several detailed studies (Freund-Mercier et al., 1987; Tribollet et al., 1988) have failed to detect significant OT binding in the ventral tegmental area, and (Yoshimura et al., 1996) reported that oxytocin receptor mRNA was only expressed here during development. In preliminary experiments, we have been unable to detect electrophysiological responses to OT in this region. Furthermore, recent work by Ikemoto and co-workers cast doubt on the role of the ventral tegmental area as a principal relay for rewarding stimuli, but instead highlighted the important role of the SuM (Ikemoto et al., 2004). These same workers also found dopamine release in the nucleus accumbens, a major component of reward circuitry (Spanagel and Weiss, 1999), is regulated by the SuM rather than the ventral tegmental area (Ikemoto et al., 2004). Adding in the current results, one might reasonably conclude that the SuM may be a more likely candidate than the ventral tegmental area for participating in the effects of OT on dopamine and reward.

References Amico, J.A., Mantella, R.C., Vollmer, R.R., Li, X., 2004. Anxiety and stress responses in female oxytocin deficient mice. J. Neuroendocrinol. 16, 319–324. Aranda, L., Santin, L.J., Begega, A., Aguirre, J.A., Arias, J.L., 2006. Supramammillary and adjacent nuclei lesions impair spatial working memory and induce anxiolitic-like behavior. Behav. Brain Res. 167, 156–164. Argiolas, A., Melis, M.R., Stancampiano, R., Gessa, G.L., 1989. Penile erection and yawning induced by oxytocin and related peptides: structure-activity relationship. Peptides 10, 559–563. Buijs, R.M., 1980. Immunocytochemical demonstration of vasopressin and oxytocin in the rat brain by light and electron microscopy. J. Histochem. Cytochem. 28, 357–360. Condes-Lara, M., Veinante, P., Rabai, M., Freund-Mercier, M.J., 1994. Correlation between oxytocin neuronal sensitivity and oxytocin-binding sites in the amygdala of the rat: electrophysiological and histoautoradiographic study. Brain Res. 637, 277–286. Cowie, A.T., Tindal, J.S., 1971. The Physiology of Lactation. Edward Arnold.

M.R. Cumbers et al. / Neuropeptides 41 (2007) 217–226 Elands, J., Barberis, C., Jard, S., Tribollet, E., Dreifuss, J.J., Bankowski, K., Manning, M., Sawyer, W.H., 1988. 125I-labelled [dðCH2 Þ5 ; TyrðMeÞ2 ; Thr4 ; Tyr-NH2 9 ] OVT: a selective oxytocin receptor ligand. Eur. J. Pharmacol. 147, 197–207. Ferguson, J.N., Young, L.J., Hearn, E.F., Matzuk, M.M., Insel, T.R., Winslow, J.T., 2000. Social amnesia in mice lacking the oxytocin gene. Nat. Genet. 25, 284–288. Freund-Mercier, M.J., Moos, F., Poulain, D.A., Richard, P., Rodriguez, F., Theodosis, D.T., Vincent, J.D., 1988. Role of central oxytocin in the control of the milk ejection reflex. Brain Res. Bull. 20, 737–741. Freund-Mercier, M.J., Stoeckel, M.E., Palacios, J.M., Pazos, A., Reichhart, J.M., Porte, A., Richard, P., 1987. Pharmacological characteristics and anatomical distribution of [3H]oxytocin-binding sites in the Wistar rat brain studied by autoradiography. Neuroscience 20, 599–614. Gimpl, G., Fahrenholz, F., 2001. The oxytocin receptor system: structure, function, and regulation. Physiol. Rev. 81, 629–683. Gonzalo-Ruiz, A., Alonso, A., Sanz, J.M., Llinas, R.R., 1992. Afferent projections to the mammillary complex of the rat, with special reference to those from surrounding hypothalamic regions. J. Comp. Neurol. 321, 277–299. Gould, B.R., Zingg, H.H., 2003. Mapping oxytocin receptor gene expression in the mouse brain and mammary gland using an oxytocin receptor-LacZ reporter mouse. Neuroscience 122, 155– 167. Hayakawa, T., Zyo, K., 1996. Synaptic organization of afferent projections to the supramammillary nucleus of the rat. Anat. Embryol. (Berl.) 193, 249–257. Ikemoto, S., Witkin, B.M., Zangen, A., Wise, R.A., 2004. Rewarding effects of AMPA administration into the supramammillary or posterior hypothalamic nuclei but not the ventral tegmental area. J. Neurosci. 24, 5758–5765. Ingram, C.D., Adams, T.S., Jiang, Q.B., Terenzi, M.G., Lambert, R.C., Wakerley, J.B., Moos, F., 1995. Mortyn Jones Memorial Lecture. Limbic regions mediating central actions of oxytocin on the milk-ejection reflex in the rat. J. Neuroendocrinol. 7, 1–13. Ingram, C.D., Cutler, K.L., Wakerley, J.B., 1990. Oxytocin excites neurones in the bed nucleus of the stria terminalis of the lactating rat in vitro. Brain Res. 527, 167–170. Insel, T.R., 1992. Oxytocin–a neuropeptide for affiliation: evidence from behavioral, receptor autoradiographic, and comparative studies. Psychoneuroendocrinology 17, 3–35. Insel, T.R., 2003. Is social attachment an addictive disorder? Physiol. Behav. 79, 351–357. Jiang, Q.B., Wakerley, J.B., 1995. Analysis of bursting responses of oxytocin neurones in the rat in late pregnancy, lactation and after weaning. J. Physiol. 486 (Pt 1), 237–248. Kirk, I.J., 1998. Frequency modulation of hippocampal theta by the supramammillary nucleus, and other hypothalamo–hippocampal interactions: mechanisms and functional implications. Neurosci. Biobehav. Rev. 22, 291–302. Kiss, A., Mikkelsen, J.D., 2005. Oxytocin–anatomy and functional assignments: a minireview. Endocr. Regul. 39, 97–105. Kiss, J., Csaki, A., Bokor, H., Kocsis, K., Kocsis, B., 2002. Possible glutamatergic/aspartatergic projections to the supramammillary nucleus and their origins in the rat studied by selective [(3)H]D-aspartate labelling and immunocytochemistry. Neuroscience 111, 671–691. Kiss, J., Csaki, A., Bokor, H., Shanabrough, M., Leranth, C., 2000. The supramammillo–hippocampal and supramammillo–septal glutamatergic/aspartatergic projections in the rat: a combined [3H]D-aspartate autoradiographic and immunohistochemical study. Neuroscience 97, 657–669. Kovacs, G.L., Bohus, B., Versteeg, D.H., de Kloet, E.R., de Wied, D., 1979. Effect of oxytocin and vasopressin on memory

225

consolidation: sites of action and catecholaminergic correlates after local microinjection into limbic–midbrain structures. Brain Res. 175, 303–314. Kremarik, P., Freund-Mercier, M.J., Stoeckel, M.E., 1995. Oxytocin and vasopressin binding sites in the hypothalamus of the rat: histoautoradiographic detection. Brain Res. Bull. 36, 195–203. Lambert, R.C., Moos, F.C., Ingram, C.D., Wakerley, J.B., Kremarik, P., Guerne, Y., Richard, P., 1993. Electrical activity of neurons in the ventrolateral septum and bed nuclei of the stria terminalis in suckled rats: statistical analysis gives evidence for sensitivity to oxytocin and for relation to the milk-ejection reflex. Neuroscience 54, 361–376. Landgraf, R., Neumann, I.D., 2004. Vasopressin and oxytocin release within the brain: a dynamic concept of multiple and variable modes of neuropeptide communication. Front Neuroendocrinol. 25, 150– 176. Leng, G., Caquineau, C., Sabatier, N., 2005. Regulation of oxytocin secretion. Vitam. Horm. 71, 27–58. Lincoln, D.W., Hill, A., Wakerley, J.B., 1973. The milk-ejection reflex of the rat: an intermittent function not abolished by surgical levels of anaesthesia. J. Endocrinol. 57, 459–476. Lowbridge, J., Manning, M., Haldar, J., Sawyer, W.H., 1977. Synthesis and some pharmacological properties of [4-threonine, 7-glycine]oxytocin, [1-(L-2-hydroxy-3-mercaptopropanoic acid), 4-threonine, 7-glycine]oxytocin (hydroxy[Thr4, Gly7]oxytocin), and [7-Glycine]oxytocin, peptides with high oxytocic–antidiuretic selectivity. J. Med. Chem. 20, 120–123. McCarthy, M.M., 1995. Estrogen modulation of oxytocin and its relation to behavior. Adv. Exp. Med. Biol. 395, 235–245. McCarthy, M.M., Altemus, M., 1997. Central nervous system actions of oxytocin and modulation of behavior in humans. Mol. Med. Today 3, 269–275. Morris, J.F., Pow, D.V., 1991. Widespread release of peptides in the central nervous system: quantitation of tannic acid-captured exocytoses. Anat. Rec. 231, 437–445. Neumann, I.D., Torner, L., Wigger, A., 2000. Brain oxytocin: differential inhibition of neuroendocrine stress responses and anxiety-related behaviour in virgin, pregnant and lactating rats. Neuroscience 95, 567–575. Ottersen, O.P., 1980. Afferent connections to the amygdaloid complex of the rat and cat: II. Afferents from the hypothalamus and the basal telencephalon. J. Comp. Neurol. 194, 267–289. Pan, W.X., McNaughton, N., 2002. The role of the medial supramammillary nucleus in the control of hippocampal theta activity and behaviour in rats. Eur. J. Neurosci. 16, 1797–1809. Paxinos, G., Watson, C., 1986. The rat brain in stereotactic coordinates. Academic Press, San Diego. Pedersen, C.A., Caldwell, J.D., Walker, C., Ayers, G., Mason, G.A., 1994. Oxytocin activates the postpartum onset of rat maternal behavior in the ventral tegmental and medial preoptic areas. Behav. Neurosci. 108, 1163–1171. Pittman, Q.J., Blume, H.W., Renaud, L.P., 1981. Connections of the hypothalamic paraventricular nucleus with the neurohypophysis, median eminence, amygdala, lateral septum and midbrain periaqueductal gray: an electrophysiological study in the rat. Brain Res. 215, 15–28. Plested, C.P., Bernal, A.L., 2001. Desensitisation of the oxytocin receptor and other G-protein coupled receptors in the human myometrium. Exp. Physiol. 86, 303–312. Raggenbass, M., 2001. Vasopressin- and oxytocin-induced activity in the central nervous system: electrophysiological studies using in vitro systems. Prog. Neurobiol. 64, 307–326. Raggenbass, M., Alberi, S., Zaninetti, M., Pierson, P., Dreifuss, J.J., 1998. Vasopressin and oxytocin action in the brain: cellular neurophysiological studies. Prog. Brain Res. 119, 263–273. Richard, P., Moos, F., Freund-Mercier, M.J., 1991. Central effects of oxytocin. Physiol. Rev. 71, 331–370.

226

M.R. Cumbers et al. / Neuropeptides 41 (2007) 217–226

Robinson, I.C., Coombes, J.E., 1993. Neurohypophysial peptides in cerebrospinal fluid: an update. Ann. NY Acad. Sci. 689, 269–284. Shahidi, S., Motamedi, F., Naghdi, N., 2004. Effect of reversible inactivation of the supramammillary nucleus on spatial learning and memory in rats. Brain Res. 1026, 267–274. Sofroniew, M.V., 1980. Projections from vasopressin, oxytocin, and neurophysin neurons to neural targets in the rat and human. J. Histochem. Cytochem. 28, 475–478. Spanagel, R., Weiss, F., 1999. The dopamine hypothesis of reward: past and current status. Trends Neurosci. 22, 521–527. Stivers, J.A., Kaltwasser, M.T., Hill, P.S., Hruby, V.J., Crawley, J.N., 1988. Ventral tegmental oxytocin induces grooming. Peptides 9 (Suppl. 1), 223–231. Terenzi, M.G., Ingram, C.D., 2005. Oxytocin-induced excitation of neurones in the rat central and medial amygdaloid nuclei. Neuroscience 134, 345–354. Tribollet, E., Barberis, C., Jard, S., Dubois-Dauphin, M., Dreifuss, J.J., 1988. Localization and pharmacological characterization of high affinity binding sites for vasopressin and oxytocin in the rat brain by light microscopic autoradiography. Brain Res. 442, 105–118. Vertes, R.P., 1992. PHA-L analysis of projections from the supramammillary nucleus in the rat. J. Comp. Neurol. 326, 595–622.

Wakerley, J.B., 2006. Milk Ejection and its Control. In: Neill, J.D. (Ed.), Knobil and Neill’s Physiology of Reproduction, vol. 2. Elsevier, New York, pp. 3129–3190. Wakerley, J.B., Foreman, C.T., Ingram, C.D., 1989. Effect of centrally administered oxytocin on the association between cortical electroencephalogram and milk ejection in the rat. J. Neuroendocrinol. 1, 173–178. Wilson, B.C., Terenzi, M.G., Ingram, C.D., 2005. Differential excitatory responses to oxytocin in sub-divisions of the bed nuclei of the stria terminalis. Neuropeptides 39, 403–407. Yoshimura, R., Kimura, T., Watanabe, D., Kiyama, H., 1996. Differential expression of oxytocin receptor mRNA in the developing rat brain. Neurosci. Res. 24, 291–304. Yoshimura, R., Kiyama, H., Kimura, T., Araki, T., Maeno, H., Tanizawa, O., Tohyama, M., 1993. Localization of oxytocin receptor messenger ribonucleic acid in the rat brain. Endocrinology 133, 1239–1246. Young, L.J., Lim, M.M., Gingrich, B., Insel, T.R., 2001. Cellular mechanisms of social attachment. Horm. Behav. 40, 133–138. Young, L.J., Murphy Young, A.Z., Hammock, E.A., 2005. Anatomy and neurochemistry of the pair bond. J. Comp. Neurol. 493, 51–57.