Milk Ejection Bursts of Supraoptic Oxytocin Neurones during Bilateral and Unilateral Suckling in the Rat

Extracellular recordings of the electrical activity of oxytocin neurones were made from the supraoptic nuclei (SON) of lactating rats, and the milk-ejection bursts and the background activity of oxytocin neurones were investigated during unilateral and bilateral suckling. When application of pups was limited to the nipples on either the same side (ipsilateral suckling) or the side opposite (contralateral suckling) to the oxytocin neurone recorded, the burst amplitude and background firing rate were significantly (P<0.05) lower and the inter-burst interval was significantly (P<0.05) longer than during bilateral suckling. Furthermore, the burst amplitude was significantly (P<0.05) lower during ipsilateral suckling than during contralateral suckling. The majority of the oxytocin neurones showed a gradual increase in the burst amplitude during bilateral (88.9%) and contralateral (77.3%) suckling, but during ipsilateral suckling only 40% of the neurones did. The inter-burst interval became shorter with the progress of the milk ejection reflex during any mode of suckling. Three pairs of oxytocin neurones recorded simultaneously from both SON were successfully tested for the effect of bilateral and unilateral suckling on the electrical activity, and the results showed the same direction of change in the burst amplitude, background activity and burst interval as shown in single side recordings. These findings indicate that the burst amplitude mainly depends on the amount of afferent suckling signals arising from the nipples on the side opposite to the recording side, and that there may exist bilateral summation centres coordinating with the synchronization mechanism of milk-ejection bursts of oxytocin neurones.     

Oxytocinase in the Female Rat Hypothalamus: A Novel Mechanism Controlling Oxytocin Neurones During Lactation

In addition to its peripheral actions, oxytocin released within the brain is important for birth and essential for milk ejection. The oxytocinase enzyme (placental leucine aminopeptidase; P‐LAP) is expressed both peripherally and centrally. P‐LAP controls oxytocin degradation in the uterus, placenta and plasma during pregnancy, although its role in the hypothalamus is unclear. We investigated P‐LAP expression and activity in the hypothalamus in virgin, pregnant and lactating rats, as well as its role in vivo during the milk‐ejection reflex. P‐LAP mRNA and protein were expressed in magnocellular neurones of the supraoptic (SON) and paraventricular (PVN) nuclei. Oxytocin neurones co‐expressed P‐LAP without strong subcellular co‐localisation of oxytocin and P‐LAP, indicating that they are packaged in separate vesicles. Examination of the intracellular distribution of oxytocin and P‐LAP showed a redistribution of P‐LAP to within 1 μm of the plasma membrane in the somata of oxytocin neurones during lactation. Both P‐LAP mRNA expression and hypothalamic leucyl/cystinyl aminopeptidase activity in the soluble fraction were higher during lactation than in late pregnant or virgin states. Inhibition of central enzyme activity by i.c.v. injection of amastatin in anaesthetised suckling mothers increased the frequency of reflex milk ejections. Because hypothalamic P‐LAP expression and activity increase in lactation, and the prevention of its action mimics central oxytocin administration, we conclude that P‐LAP regulates auto‐excitatory oxytocin actions during the suckling‐induced milk‐ejection reflex.     

Systemic Leptin Increases the Electrical Activity of Supraoptic Nucleus Oxytocin Neurones in Virgin and Late Pregnant Rats

In the rat hypothalamus, fasting attenuates the expression of oxytocin and this can be reversed by exogenous leptin administration. In the present study, we investigated the effects of systemically administered leptin on the electrical activity of magnocellular neurones in the supraoptic nucleus of urethane‐anaesthetised rats. In virgin female rats, systemic leptin significantly excited identified oxytocin neurones with no detected effects on the patterning of activity, as reflected by hazard function analyses. The lowest dose that was consistently effective was 100 μg/i.v., and this dose had no significant effect on vasopressin neurones. In virgin rats fasted overnight, the spontaneous firing rate of oxytocin neurones was significantly lower than in unfasted rats, although leptin had a similar excitatory effect as in unfasted rats. In late pregnant rats (days 19–21 of pregnancy), spontaneous firing rates of oxytocin neurones were higher than in virgins, and the initial response to leptin was similar to that in virgin rats, although the increase in activity was more persistent. In fasted pregnant rats, the mean spontaneous firing rate of oxytocin neurones was again lower than in unfasted rats, although leptin had no significant effect even at the higher dose of 1 mg/rat. Thus, fasting reduced the spontaneous firing rates of oxytocin neurones in nonpregnant rats, and this effect could be reversed by the excitatory effects of leptin. Pregnant rats showed some evidence of leptin resistance but only after an overnight fast.     

Role of central oxytocin in the control of the milk ejection reflex

The neuropeptide oxytocin, synthetized by magnocellular neurons in the hypothalamus, is well known for its peripheral action after it is released into the bloodstream from axons in the neurohypophysis. Less familiar is the notion that it is also released centrally to control the activity of oxytocinergic neurons themselves. When injected into the third ventricle of lactating rats during suckling, oxytocin increases the basal firing rate of oxytocinergic neurons as well as their activity at the time of each reflex milk ejection. On the other hand, centrally administered oxytocin engenders the neuronal-glial and synaptic plasticity characteristic of the oxytocin system when it is physiologically activated. From numerous in vivo and in vitro observations, it appears that central oxytocin is released in the hypothalamic nuclei themselves. For example, the use of push-pull cannulae inserted into one supraoptic nucleus of suckled rats shows that oxytocin is released inside the nucleus specifically during milk ejection. Moreover, ultrastructural immunocytochemistry reveals synaptic terminals in the supraoptic nucleus where both the pre- and postsynaptic elements are oxytocinergic. Nevertheless, the mechanism of the central release of the neuropeptide has still to be determined, especially in view of electrophysiological observations indicating that the release process in the hypothalamus is different from that within the neurohypophysis.     

Responses of paraventricular and supraoptic units to angiotensin II, SAR-ILE-angiotensin II and hypertonic NaCl administered into the cerebral ventricle

Extracellular recordings of action potentials were made from neurones antidromically identified as neurosecretory cells in the paraventricular and supraoptic nuclei of urethane-anesthetized female rats. Eighty-six neurones were examined for their responsiveness to 10 ng of angiotensin II (AII) injected into the third cerebral ventricle and 78 (91%) of them increased their firing rate following the AII injection. None of the neurosecretory cells tested showed a response to the intraventricular (IVT) injection of isotonic NaCl. Thalamic neurones and non-neurosecretory hypothalamic neurones did not respond to the AII given IVT. Firing activity of 13 neurosecretory neurones was recorded during reflex milk ejection induced by suckling pups in the lactating rats. Seven of them were classified as oxytocinergic cells because they showed a burst of activity before reflex milk ejections and the remaining 6 neurones which gave no burst of firing before milk ejections were classified as non-oxytocinergic neurones. The IVT application of AII resulted in activation of all the oxytocinergic neurones and 5 of the 6 non-oxytocinergic neurones. The effect of AII on the firing of the neurosecretory cell was inhibited by the simultaneous application of Sar¹-Ile⁸-AII (1 μg), a competitive AII antagonist. The IVT injection of the antagonist alone inhibited the spontaneous firing of the neurosecretory cells, but it did not affect the firing of thalamic or non-neurosecretory hypothalamic neurones. Hypertonic NaCl (0.85 M NaCl, 1 μ1 IVT) also activated 13 of 20 neurosecretory cells tested. Combined application of AII and hypertonic NaCl elicited a marked potentiation of the response of neurosecretory cells to each of the stimuli. These findings indicate that AII activates neurosecretory cells stimulating specific AII receptors in the brain and that AII has a synergistic action with hypertonic NaCl. Inhibition of spontaneous activity of neurosecretory cells by a competitive AII antagonist suggests that endogenous AII may participate in the maintenance of basal activity of neurosecretory cells.     

A dopaminergic projection to the rat mammillary nuclei demonstrated by retrograde transport of wheat germ agglutinin-horseradish peroxidase and tyrosine hydroxylase immunohistochemistry.

The presence and distribution of dopaminergic neurons and terminals in the hypothalamus of the rat were studied by tyrosine hydroxylase (TH) immunohistochemistry. Strongly labelled TH-immunoreactive neurons were seen in the dorsomedial hypothalamic nucleus, periventricular region, zona incerta, arcuate nucleus, and supramammillary nucleus. A few TH-positive neurons were also identified in the dorsal and ventral premammillary nucleus, as well as the lateral hypothalamic area. TH-immunoreactive fibres and terminals were unevenly distributed in the mammillary nuclei; small, weakly labelled terminals were scattered in the medial mammillary nucleus, while large, strongly labelled, varicose terminals were densely concentrated in the internal part of the lateral mammillary nucleus. A few dorsoventrally oriented TH-positive axon bundles were also identified in the lateral mammillary nucleus. A dopaminergic projection to the mammillary nuclei from the supramammillary nucleus and lateral hypothalamic area was identified by double labelling with retrograde transport of wheat germ agglutinin-horseradish peroxidase and TH-immunohistochemistry. The lateral mammillary nucleus receives a weak dopaminergic projection from the medial, and stronger projections from the lateral, caudal supramammillary nucleus. The double-labelled neurons in the lateral supramammillary nucleus appear to encapsulate the caudal end of the mammillary nuclei. The medial mammillary nucleus receives a very light dopaminergic projection from the caudal lateral hypothalamic area. These results suggest that the supramammillary nucleus is the principal source of the dopaminergic input to the mammillary nuclei, establishing a local TH-pathway in the mammillary complex. The supramammillary cell groups are able to modulate the limbic system through its dopaminergic input to the mammillary nuclei as well as through its extensive dopaminergic projection to the lateral septal nucleus.     

Neuronal connections between the cerebellar nuclei and hypothalamus in Macaca fascicularis: cerebello-visceral circuits.

The purpose of this study was to identify the basic pattern of interconnections between the cerebellar nuclei and hypothalamus in Macaca fascicularis. The distribution of retrogradely labeled cells and anterogradely filled cerebellofugal axons in the hypothalamus of M. fascicularis was investigated after pressure injections of a horseradish peroxidase mixture (HRP + WGA-HRP) in the cerebellar nuclei. Following injections in the lateral, anterior, and posterior interposed cerebellar nuclei retrogradely labeled cells were present in the following areas (greatest to least concentration): lateral and dorsal hypothalamic areas, dorsomedial nucleus, griseum periventriculare hypothalami, supramammillary and tuberomammillary nuclei, posterior hypothalamic area, ventromedial nucleus and periventricular hypothalamus, around the medial mammillary nucleus, lateral mammillary nucleus, and infundibular nucleus. Cell labeling was bilateral with an ipsilateral preponderance. In these same experiments anterogradely labeled cerebellar efferent fibers terminated in the contralateral posterior, dorsal and lateral hypothalamic areas, and the dorsomedial nucleus. In these regions retrogradely labeled hypothalamic cells were occasionally found in areas that also contained anterogradely filled cerebellar axons. This suggests a partial reciprocity in this system. In addition, sparse numbers of labeled cerebellar fibers recross in the hypothalamus to distribute to homologous areas ipsilateral to the injection site. Subsequent to an injection in the medial cerebellar nucleus (NM), cell labeling was present in more rostral hypothalamic levels including the lateral and dorsal hypothalamic areas, the dorsomedial nucleus, around or in fascicles of the column of the fornix, and in the periventricular hypothalamic area. Although no fastigiohypothalamic fibers were seen in this study, on the basis of information available from the literature it is likely that such a connection exists in primates. In summary, hypothalamic projections to NM originated mainly from rostral to midhypothalamic levels, whereas those projections to the lateral three cerebellar nuclei came from mid and more caudal levels. The existence of direct hypothalamic projections to cerebellar nuclei in M. fascicularis and of cerebellofugal projection to some hypothalamic centers indicates that circuitry is present through which the cerebellum may influence visceral functions. Furthermore, the fact that projections to NM versus the other cerebellar nuclei originate from somewhat different regions of the hypothalamus would suggest that the visceral functions modulated by each pathway is not the same.     

Effects of RFamide-related peptide (RFRP)-1 and RFRP-3 on oxytocin release and anxiety-related behaviour in rats

RFamide-related peptides (RFRP-1 and RFRP-3) are localised in neurones of the dorsomedial hypothalamus in rats. The dorsomedial hypothalamus plays an essential role in neuroendocrine and behavioural stress responses. In the present study, we examined the role of RFRP in the control of neuroendocrine and behavioural responses in rats. Stressful stimuli increased expression of Fos protein in RFRP-immunoreactive neurones of the dorsomedial hypothalamus, suggesting that stressful stimuli activate RFRP neurones. Intracerebroventricular injection of RFRPs increased the expression of Fos protein in oxytocin neurones in the hypothalamus and plasma concentrations of adrenocorticotrophic hormone and oxytocin. The hypothalamic paraventricular and supraoptic nuclei expressed mRNA of GPR147, the putative RFRP receptor, and application of RFRPs to isolated supraoptic nuclei facilitated oxytocin release, suggesting that RFRPs activate oxytocin neurones directly. Furthermore, the administration of RFRPs induced anxiety-related behaviour in rats in open-field tests. All these data taken together suggest that RFRPs play a role in the control of neuroendocrine and behavioural stress responses in rats.     

Responses of paraventricular and supraoptic units to angiotensin II,SAR1-ILE8-angiotensin II and hypertonic NaCl administered into the cerebral ventricle

Extracellular recordings of action potentials were made from neurones antidromically identified as neurosecretory cells in the paraventricular and supraoptic nuclei of urethane-anesthetized female rats. Eighty-six neurones were examined for their responsiveness to 10 ng of angiotensin II (AII) injected into the third cerebral ventricle and 78 (91%) of them increased their firing rate following the AII injection. None of the neurosecretory cells tested showed a response to the intraventricular (IVT) injection of isotonic NaCl. Thalamic neurones and non-neurosecretory hypothalamic neurones did not respond to the AII given IVT. Firing activity of 13 neurosecretory neurones was recorded during reflex milk ejection induced by suckling pups in the lactating rats. Seven of them were classified as oxytocinergic cells because they showed a burst of activity before reflex milk ejections and the remaining 6 neurones which gave no burst of firing before milk ejections were classified as non-oxytocinergic neurones. The IVT application of AII resulted in activation of all the oxytocinergic neurones and 5 of the 6 non-oxytocinergic neurones. The effect of AII on the firing of the neurosecretory cell was inhibited by the simultaneous application of Sar¹-Ile⁸-AII (1 μg), a competitive AII antagonist. The IVT injection of the antagonist alone inhibited the spontaneous firing of the neurosecretory cells, but it did not affect the firing of thalamic or non-neurosecretory hypothalamic neurones. Hypertonic NaCl (0.85 M NaCl, 1 μ1 IVT) also activated 13 of 20 neurosecretory cells tested. Combined application of AII and hypertonic NaCl elicited a marked potentiation of the response of neurosecretory cells to each of the stimuli. These findings indicate that AII activates neurosecretory cells stimulating specific AII receptors in the brain and that AII has a synergistic action with hypertonic NaCl. Inhibition of spontaneous activity of neurosecretory cells by a competitive AII antagonist suggests that endogenous AII may participate in the maintenance of basal activity of neurosecretory cells.     

Involvement of Prolactin‐Releasing Peptide in the Activation of Oxytocin Neurones in Response to Food Intake

Food intake activates neurones expressing prolactin‐releasing peptide (PrRP) in the medulla oblongata and oxytocin neurones in the hypothalamus. Both PrRP and oxytocin have been shown to have an anorexic action. In the present study, we investigated whether the activation of oxytocin neurones following food intake is mediated by PrRP. We first examined the expression of PrRP receptors (also known as GPR10) in rats. Immunoreactivity of PrRP receptors was observed in oxytocin neurones and in vasopressin neurones in the paraventricular and supraoptic nuclei of the hypothalamus and in the bed nucleus of the stria terminalis. Application of PrRP to isolated supraoptic nuclei facilitated the release of oxytocin and vasopressin. In mice, re‐feeding increased the expression of Fos protein in oxytocin neurones of the hypothalamus and bed nucleus of the stria terminalis. The increased expression of Fos protein in oxytocin neurones following re‐feeding or i.p. administration of cholecystokinin octapeptide (CCK), a peripheral satiety factor, was impaired in PrRP‐deficient mice. CCK‐induced oxytocin increase in plasma was also impaired in PrRP‐deficient mice. Furthermore, oxytocin receptor‐deficient mice showed an increased meal size, as reported in PrRP‐deficient mice and in CCK_A receptor‐deficient mice. These findings suggest that PrRP mediates, at least in part, the activation of oxytocin neurones in response to food intake, and that the CCK–PrRP–oxytocin pathway plays an important role in the control of the termination of each meal.     

Morphofunctional interrelationships between the hippocampus and hypothalamus in rats]

Evoked reactions of the hypothalamic arcuate and medial preoptic nuclei neurons were recorded when the hippocampus was stimulated by single stimuli in anaesthetized rats. In the arcuate nucleus phasic responses and primary inhibition were found to be dominant and in the medial preoptic nucleus--both phasic and nonspecifical responses. After injection of the horseradish peroxidase into the stimulated hippocampal region stained cells were found in the nuclei of the mammillary complex, mediobasal hypothalamus and in the medial preoptic nucleus. Groups of stained neurons were observed in the periphery of ventro- and dorsomedial, lateral and mammillary nuclei of the hypothalamus. In all the studied structures, except the medial mammillary nucleus, reticular-like cells were found alongside with spindle-like and triangle neurons. The data obtained are discussed in connection with the problem of hypothalamo-hippocampal interaction.     

A role of central oxytocin in autonomic functions: Its action in the motor nucleus of the vagus nerve

Neurones located in the dorsal motor nucleus of the vagus nerve were shown, in slices from the rat brainstem, to respond to oxytocin by a concentration-dependent increase in rate of firing. A newly available oxytocin antagonist suppressed the excitatory effect of oxytocin on single neurones, this antagonism was partially reversible. Further evidence that neurones located in the dorsal motor nucleus of the vagus nerve possess oxytocin receptors was obtained from in vitro light microscopical autoradiography using [¹²⁵I]-labelled oxytocin antagonist. In conjunction with data by others which showed that oxytocin antagonist microinjected into the dorsal motor nucleus of the vagus nerve blocks gastric and cardiac effects caused by stimulation of the hypothalamic paraventricular nucleus, our results suggest a role for central oxytocin in autonomic efferent activity.     

Projections of RFamide-related Peptide-3 Neurones in the Ovine Hypothalamus, with Special Reference to Regions Regulating Energy Balance and Reproduction

RFamide-related peptide-3 (RFRP-3) is a neuropeptide produced in cells of the paraventricular nucleus and dorsomedial nucleus of the ovine hypothalamus. In the present study, we show that these cells project to cells in regions of the hypothalamus involved in energy balance and reproduction. A retrograde tracer (FluoroGold) was injected into either the arcuate nucleus, the lateral hypothalamic area or the ventromedial nucleus. The distribution and number of retrogradely-labelled RFRP-3 neurones was determined. RFRP-3 neurones projected to the lateral hypothalamic area and, to a lesser degree, to the ventromedial nucleus and the arcuate nucleus. Double-label immunohistochemistry was employed to identify cells receiving putative RFRP-3 input to cells in these target regions. RFRP-3 cells were seen to project to neuropeptide Y and pro-opiomelanocortin neurones in the arcuate nucleus, orexin and melanin-concentrating hormone neurones in the lateral hypothalamic area, as well as orexin cells in the dorsomedial nucleus and corticotrophin-releasing hormone and oxytocin cells in the paraventricular nucleus. Neurones expressing gonadotrophin-releasing hormone in the preoptic area were also seen to receive input from RFRP-3 projections. We conclude that RFRP-3 neurones project to hypothalamic regions and cells involved in regulation of energy balance and reproduction in the ovine brain.     

Synchronization of oxytocin neurons in suckled rats: possible role of bilateral innervation of hypothalamic supraoptic nuclei by single medullary neurons

We have previously shown that oxytocin neurons located in the four hypothalamic magnocellular nuclei display synchronous bursts of action potentials before each milk ejection. The mechanisms involved in such a synchronization have, however, not yet been elucidated. In this study, we test the hypothesis of an extranuclear synchronization arising from a common extrahypothalamic input innervating bilateral magnocellular nuclei. First, two different retrograde tracers were injected into the right and left supraoptic nuclei of rats that were fixed 5-7 days later. Each tracer labelled numerous neurons in various brain regions ipsilateral or contralateral to the injection site, but colocalization of the two tracers within the same cell body could only be detected bilaterally in neurons in the ventromedial regions of the medulla oblongata. The axonal projections of these medullary neurons were then visualized by the unilateral microinjection of an anterograde tracer (BDA) within the ventromedial medulla oblongata. BDA-labelled axons afferent to the hypothalamus were found to branch towards both supraoptic nuclei through medial portions of the optic chiasma. Finally, in anaesthetized lactating rats, surgical lesions were placed medially through the optic chiasma and the electrical activity of oxytocin neurons in bilateral supraoptic nuclei was pair-recorded during suckling. The incidence of synchronous bursts in oxytocin neurons located within bilateral supraoptic nuclei were dramatically altered only when the medial portions of the optic chiasma were totally lesioned. Taken together, these data suggest that medullary neurons afferent to bilateral supraoptic nuclei are involved in the recruitment and synchronization of bursting in oxytocin neurons during suckling.     

Study of the connections of supposed diencephalic connections of the limbic system of the lizard using the technic of axonal transport of horseradish peroxidase

Connections of the anterior thalamic (n. dorsolateralis anterior, n. dorsomedialis) and habenular nuclei in lizards Ophisaurus apodus were studied by means of HRP administration into these nuclei. It was shown that all nuclei have overlapping locations of afferent sources (basotelencephalic structures, nuclei of anterior and hippocampal commissures, lateral area of the hypothalamus, superior raphe nucleus) and overlapping projectional zones (mammillary complex, ventral tegmental area). Besides common connections, specific ones for separate nuclei were revealed: for n. dorsolateralis anterior-reciprocal connection with dorsolateral hypothalamic nucleus, for habenular nuclei-projection to the interpeduncular nucleus, for n. dorsomedialis-projection to the dorsal hypothalamic area. No mammillary afferents were found for the anterior thalamic nuclei. All the nuclei studied are considered as diencephalic relay links of pathways which can be compared with dorsal (for habenular nuclei) and ventral (for anterior thalamic nuclei) pathways of the limbic system in mammals.     

Localization and developmental pattern of vasoactive intestinal polypeptide binding sites in the human hypothalamus

Using a quantitative in vitro autoradiographic approach, vasoactive intestinal polypeptide (VIP) binding site densities were compared in the post-mortem hypothalamus of human neonate/infant and adult. The densities were similar during development in most of the hypothalamic nuclei and areas examined underlying the stability of ¹²⁵I-VIP binding sites in the post-mortem hypothalamus of young and adult individuals. However, the ventral part of the medial preoptic area, the medial, lateral, and supramammillary nuclei were characterized by an increase of ¹²⁵I-VIP binding with age. In young and adult individuals, the highest densities of hypothalamic ¹²⁵1-VIP binding sites were detected in the supraoptic and infundibular nuclei; the ependyma; the organum vasculosum of the lamina terminalis; the horizontal limb of the diagonal band of Broca; the ventral part of the medial preoptic area (in adult); the suprachiasmatic, paraventricular, and periventricular nuclei; and the medial and lateral mammillary nuclei in adult. Moderate densities were found in the vertical limb of the diagonal band of Broca, the bed nucleus of the stria terminalis, the ventral part of the medial preoptic area in neonate/infant, the medial and lateral mammillary nuclei in neonate/infant, the supramammillary nucleus in adult, the dorsal hypothalamic area, and the ventromedial nucleus. Low to moderate binding site densities were observed in the other hypothalamic regions of young or adult individuals. The nonspecific binding ranged from 15% of the total binding in the anterior hypothalamus to 20% in the mediobasal and posterior hypothalamic levels. Taken together, these results provide evidence for a large distribution of VIP binding sites in neonate/infant and adult human hypothalamus suggesting the implication of VIP in the development of this brain structure and the maintenance of its various functions. © 1994 Wiley-Liss, Inc.     

Effect of anterior hypothalamic and mammillary area lesions on territorial aggressive behaviour in male rats

Adult male Wezob-rats were bilaterally lesioned in either the medial anterior hypothalamic area or the mammillary bodies. The behaviour of these animals when confronted with a male intruder within their own territory, was observed and recorded before and after lesioning and compared with the behaviour of sham-operated animals.Anterior hypothalamic lesions, including large parts of the anterior hypothalamus, the rostral part of the ventromedial hypothalamic nucleus and smaller caudal parts of the preoptic area, led to strong increases in defensive behaviour. This included a decreased tendency to investigate the intruder and an exaggerated defensive reaction when approached by the intruder. Ingestive behaviour and bodyweight were enhanced.Mammillary body lesions, including large parts of the ventral and dorsal premammillary nucleus, the caudal part of the arcuate nucleus, the medial mammillary nucleus, the posterior mammillary nucleus, the supramammillary peduncle and closely surrounding areas, led to a marked increase in offensive behaviour. This was characterized by high levels of initatives and aggression towards an intruder.It is suggested that two distinct neural substrates exist in the medial hypothalamus, which normally modulate defensive (anterior medial hypothalamus) or offensive (posterior medial hypothalamus) aspects of intermale aggression.