Effects of neurotensin on the organization of activity in supraoptic nucleus cells in virgin and lactating rats

Neurotensin increases the firing rate of supraoptic nucleus oxytocin and vasopressin neurones in vitro and induces Fos protein expression in the supraoptic nucleus in vivo. Here, we used extracellular single-unit electrophysiological recording combined with local microdialysis administration of neurotensin (1 mM at 2 micro l/min) to investigate the effects of locally applied neurotensin on the firing of oxytocin and vasopressin neurones in urethane-anaesthetized virgin and lactating rats. Neurotensin decreased the mean firing rate of oxytocin cells in virgin, but not lactating, rats. In addition, neurotensin increased the index of dispersion (a measure of the variability of firing) in virgin, but not lactating, rats. By contrast to oxytocin cells, neurotensin increased the mean firing rate of vasopressin cells in both virgin and lactating rats, but did not alter the index of dispersion. The increase in firing of phasic vasopressin cells was achieved through an increase in intraburst frequency (rather than an increase in burst duration or decrease in interburst interval), which resulted from a reduction of the spike-frequency adaptation that develops over the course of phasic bursts. Thus, neurotensin has differential effects on activity patterning in oxytocin and vasopressin cells and the effects on oxytocin cells, but not vasopressin cells, depend upon the physiological status of the animal. The increase in the variability of firing of oxytocin cells induced by neurotensin in virgin rats, but not in lactating rats, suggests that neurotensin (or other neurotransmitters/neuromodulators with similar actions) might establish conditions that predispose oxytocin cells to fire in milk-ejection bursts in lactating rats.     

Electrical activity of dorsal horn neurons during the suckling-induced milk ejection reflex in the lactating rat

In lactating rats, suckling elicits the milk ejection reflex which consists of an intermittent synchronous activation of hypothalamic oxytocin neurons which releases oxytocin into the bloodstream. We here investigated the electrophysiological behaviour of spinal cord neurons linked to mammary innervation in relation to suckling and the suckling-induced milk ejection reflex. Experiments were carried out on 58 urethane-anaesthetized rats, paralysed with gallamine triethiodide and artificially ventilated. Fixation of the spinal cord and laminectomy significantly slowed down the reflex, which occurred in only 27% of the animals. In these rats, 31 dorsal horn neurons at the thoraco-lumbar level were found to be excited by nipple stimulation. During suckling by a litter of at least nine pups, they displayed an irregular pattern of brief bursts of activity (peak firing rate 22.0 ± 3.2 Hz, mean ± SD) correlated to the bouts of suckling of the pups. Seventeen out of 19 cells tested by stimulation of at least 2 adjacent nipples received convergent input from different ipsilateral nipples. Out of 11 cells tested, 8 were also activated by stimulation of a contralateral nipple. Fourteen out of 30 units were recorded through at least one reflex milk ejection. Their firing rate was significantly higher than the firing rate of cells recorded in animals which failed to milk eject (4.4 Hz ± 2.8 versus 1.5 Hz ± 0.7). At the moment of the high frequency discharge of action potentials, occurring in oxytocinergic cells 10 to 15 s before each milk ejection, spinal neurons showed no systematic change in electrical activity. In contrast, the stretch reaction of the pups, which corresponds to an intense period of suckling when milk ejection has started, induced, in 12 cells, a considerable increase in electrical activity. One unit was found to be inhibited by suckling and during the stretch reaction. Ten more units, which were not activated by stimulation of the nipples but responded to stimulation of excitatory receptive fields near the last three pairs of nipples, were recorded through reflex milk ejections: 8 remained silent during reflex milk ejections but 2 were activated when the pups stimulated their excitatory receptive field. We conclude that some dorsal horn neurons, able to respond readily to the suckling movements of pups, appear to receive an ungated input from the nipples. At the time of the activation of oxytocin neurons, they present no particular pattern of activation or inhibition which could account in a simple manner for the intermittence of the high frequency discharge in oxytocinergic cells. However, in so far as these dorsal horn neurons may be part of the milk ejection reflex pathway, their activity, showing convergence and summation of input, and being facilitated in milk ejecting animals, indicates that the reflex does undergo a certain degree of processing at the spinal cord level.     

Oxytocin in the Bed Nucleus of the Stria Terminalis and Lateral Septum Facilitates Bursting of Hypothalamic Oxytocin Neurons in Suckled Rats

Several regions of the forebrain possess high densities of oxytocin (OT)-binding sites including the bed nucleus of the stria terminalis (BST) and lateral septum (LS). In order to examine whether these regions participate in the central facilitation of the milk ejection reflex by OT, microinjections of OT (1 ng in 100 nl containing Janus Green dye) were made into the BST (13 tests) or LS (9 tests) of anaesthetized, suckled rats, while recording the electrical activity of OT neurons in the contralateral supraoptic nucleus. Histological localization of injection sites using Janus Green demonstrated that all BST injections were close to the anterior commissure, and LS injections were all located in the ventral division of the LS. Film autoradiographic visualization of OT-binding sites (in 7 tests using [¹²⁵I]OT antagonist) confirmed that the BST and LS injections were located within regions of high OT binding. Injections into both regions facilitated the milk ejection reflex by increasing either the frequency and/or amplitude of OT neuron bursts, or by triggering bursts in animals that previously had shown no milk ejection responses; the mean number of milk ejections in the 30 min before and after injection increasing from 1.6·0.5 to 3.6·0.5 for BST and from 1.5·0.6 to 3.9·0.4 for LS. The OT microinjections had a more variable effect on background activity of OT neurons, increasing firing in some cases and not in others. This facilitatory effect was similar to that induced by microinjections into the lateral ventricle, but was smaller and delayed compared to that induced by injection into the third ventricle (9 tests), possibly due to unilateral activation of target sites. The facilitatory effect was unlikely to have been due to diffusion of OT into the ventricle, since injections into control sites (striatum and thalamus) at similar distances from the ventricle (9 tests) had no facilitatory effect (number of bursts during 30 min before and after injection; 2.2·0.5 and 1.8·0.5, respectively). These data suggest that limbic structures (BST and LS) participate in the action of central OT on the pattern of milk ejections in the suckled rat.     

Peripheral insulin administration enhances the electrical activity of oxytocin and vasopressin neurones in vivo

Oxytocin neurones are involved in the regulation of energy balance through diverse central and peripheral actions and, in rats, they are potently activated by gavage of sweet substances. Here, we test the hypothesis that this activation is mediated by the central actions of insulin. We show that, in urethane‐anaesthetised rats, oxytocin cells in the supraoptic nucleus show prolonged activation after i.v. injections of insulin, and that this response is greater in fasted rats than in non‐fasted rats. Vasopressin cells are also activated, although less consistently. We also show that this activation of oxytocin cells is independent of changes in plasma glucose concentration, and is completely blocked by central (i.c.v.) administration of an insulin receptor antagonist. Finally, we replicate the previously published finding that oxytocin cells are activated by gavage of sweetened condensed milk, and show that this response too is completely blocked by central administration of an insulin receptor antagonist. We conclude that the response of oxytocin cells to gavage of sweetened condensed milk is mediated by the central actions of insulin.     

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.     

Stability of gene expression in postmortem brain revealed by cDNA gene array analysis

In the supraoptic nucleus (SON), the incidence of conducting gap junctions (gjs), as indicated by dye coupling, is low in cycling females, but dramatically elevated in nursing mothers. Functionally, this is consistent with the well-established presence of synchronous milk ejection bursts among oxytocin neurons only in the lactating rat. In situ hybridization data, however, revealed elevated gj mRNA expression on the last day of pregnancy, a time when burst firing by putative oxytocin neurons is absent. Using Lucifer Yellow dye coupling, we determined the incidence of high conductance gjs in SONs of proestrous, immediately prepartum, postpartum non-lactating, lactating day 1, and lactating day 9-10 rats. Results indicate that coupling incidence is high only at times when milk ejection bursts are known to occur, and that the elevated gj mRNA expression seen on the last day of pregnancy does not indicate conducting gjs. It is suggested that gj conductance states, but not gj expression, are modulated by plasma estradiol titers.     

Lesions of Hypothalamic Mammillary Body Desynchronise Milk‐Ejection Bursts of Rat Bilateral Supraoptic Oxytocin Neurones

Successful milk ejection depends on a bolus release of oxytocin, which results from the synchronised burst firing of magnocellular oxytocin neurones in several hypothalamic nuclei. Despite extensive studies of the mechanism underlying the burst synchrony of oxytocin neurones in the same nucleus, brain regions controlling burst synchronisation among different nuclei remain elusive. We hypothesised that some structures in the ventroposterior hypothalamus may function as the major component of neural circuits controlling burst synchronisation of bilateral oxytocin neurones. To test this hypothesis, we recorded burst firing of bilateral oxytocin neurones in the two supraoptic nuclei after microsurgical disconnection of different hypothalamic regions in anaesthetised lactating rats. The results obtained showed that the interhemispheric section of the caudal part of the hypothalamus but not the rostral hypothalamus resulted in burst desynchronisation. The difference in burst onset time between paired bursts of bilateral oxytocin neurones was 129.2 ± 34.7 s, which is significantly (P < 0.01) longer than that of sham‐lesioned controls (0.24 ± 0.02 s). Hypothalamic lesions leading to the desynchronisation involved the mammillary body, supramammillary nucleus and tuberomammillary nucleus in the ventroposterior hypothalamus. Consistently, electrolytic lesion of the median part of this mammillary body region also desynchronised the burst of bilateral oxytocin neurones and disrupted milk ejections. These results indicate that the mammillary body region is critically involved in the burst synchronisation of bilateral oxytocin neurones during suckling and possibly functions as the major component of a putative synchronisation centre.     

The magnocellular oxytocin system,the fount of maternity: adaptations in pregnancy

Oxytocin secretion from the posterior pituitary gland is increased during parturition, stimulated by the uterine contractions that forcefully expel the fetuses. Since oxytocin stimulates further contractions of the uterus, which is exquisitely sensitive to oxytocin at the end of pregnancy, a positive feedback loop is activated. The neural pathway that drives oxytocin neurons via a brainstem relay has been partially characterised, and involves A2 noradrenergic cells in the brainstem. Until close to term the responsiveness of oxytocin neurons is restrained by neuroactive steroid metabolites of progesterone that potentiate GABA inhibitory mechanisms. As parturition approaches, and this inhibition fades as progesterone secretion collapses, a central opioid inhibitory mechanism is activated that restrains the excitation of oxytocin cells by brainstem inputs. This opioid restraint is the predominant damper of oxytocin cells before and during parturition, limiting stimulation by extraneous stimuli, and perhaps facilitating optimal spacing of births and economical use of the store of oxytocin accumulated during pregnancy. During parturition, oxytocin cells increase their basal activity, and hence oxytocin secretion increases. In addition, the oxytocin cells discharge a burst of action potentials as each fetus passes through the birth canal. Each burst causes the secretion of a pulse of oxytocin, which sharply increases uterine tone; these bursts depend upon auto-stimulation by oxytocin released from the dendrites of the magnocellular neurons in the supraoptic and paraventricular nuclei. With the exception of the opioid mechanism that emerges to restrain oxytocin cell responsiveness, the behavior of oxytocin cells and their inputs in pregnancy and parturition is explicable from the effects of hormones of pregnancy (relaxin, estrogen, progesterone) on pre-existing mechanisms, leading through relative quiescence at term inter alia to net increase in oxytocin storage, and reduced auto-inhibition by nitric oxide generation. Cyto-architectonic changes in parturition, involving evident retraction of glial processes between oxytocin cells so they get closer together, are probably a response to oxytocin neuron activation rather than being essential for their patterns of firing in parturition.     

Inhibition of milk ejection by a visual stimulus in lactating rats: Implication of the pineal gland

The effect of visual and auditory stimuli on milk ejection during suckling was studied in normal and pinealectomized lactating rats. The photic and auditory stimuli were applied to each mother for 10s every 20s during the 30 min suckling period. Both stimuli inhibited milk ejection without altering the nursing behavior. In mothers kept in complete darkness or in which the visual stimulus shone continously during the suckling period, milk ejection was not affected. The inhibition of milk ejection is therefore produced by the light on-off sequence. In lactating rats exposed to the stimulus during 3 consecutive days, a significant inhibition of milk ejection was obtained each day. A normal milk-ejection response ocurred in both non-stimulated pinealectomized and in stimulated pinealectomized lactating rats. Pinealectomy did not prevent the inhibitory effect of the sound stimulus. Treatment with methysergide prevented the inhibition of milk ejection induced by the visual stimulus but did not prevent the inhibitory effect of the auditory stimulus. It seems that the pineal gland mediates an inhibitory visual reflex acting on oxytocin release and milk ejection.     

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.     

Comparison of firing patterns in oxytocin- and vasopressin-releasing neurones during progressive dehydration

The electrical activity of neurosecretory cells in the supraoptic nucleus of the urethane-anaesthetized lactating rat was examined after periods of water deprivation ranging from 0-24 h. Supraoptic units were identified by antidromic activation following stimulation of the neurohypophysis, and classified as oxytocin or vasopressin cells according to their response during reflex milk ejection. In 65 vasopressin cells, dehydration increased the mean firing rate from 2.1 spikes/sec at 0 h to 6.8 spikes/sec at 24 h and caused a change from a slow irregular to a phasic firing pattern. Thus, after 6 h or more of dehydration, 84-100% of the vasopressin cells fired phasically, compared to 12% under normal conditions. In phasic vasopressin cells , the intraburst firing rates were closely related to the stages of dehydration, rising from a mean of 6.3 spikes/sec at 6 h to 12.0 spikes/sec at 24 h. However, no systematic relationship was observed between the stages of dehydration and the mean burst or silence durations. In 77 identified oxytocin units, dehydration increased the firing rate from 0.9 spikes/sec to 2.8 spikes/sec after 24 h, but only 3 (4%) of the cells showed phasic firing. Instead, the oxytocin units changed from a slow irregular to a fast continuous discharge. In conclusion, both vasopressin and oxytocin neurones are activated during chronic dehydration, but there is a marked difference in the pattern of their response. The phasic firing of the vasopressin cells may be important in increasing the occurrence of short interspike intervals and thus facilitating hormone release.     

Oxytocin stimulates oxytocin release from isolated nerve terminals of rat neural lobes

Nerve endings from rat neural lobes isolated by homogenization were placed on a filter and constantly superfused. The effects of exogenous oxytocin and vasopressin (both added at 1 nM concentration) on basal and stimulated release of oxytocin and vasopressin were investigated. Stimulated release was evoked by 30 mM potassium and a simultaneous increase in osmolarity. A stimulatory effect of oxytocin on basal and evoked release of oxytocin was found while there was no effect on vasopressin release. The addition of vasopressin did not induce any change in the release of either hormone. The positive feedback mechanism observed with oxytocin might be active during the discharge of oxytocin which is known to occur in bursts.     

Further Evidence that the Septum is not part of the Main Pathway of the Milk Ejection Reflex in the Rat

The possible role of the medial septum in the control of oxytocin release and of the milk ejection reflex induced by suckling was investigated in lactating rats by using electrical stimulations and lesions. In anaesthetized animals, brief electrical stimulation of the medial septum at 5 to 50 Hz elicited a single brief milk ejection similar to natural reflex milk ejections, whereas prolonged low frequency stimulations (5 to 10 Hz) induced a prolonged inhibition of the reflex. In acute experiments under anaesthesia, lesions of the medial septum did not impair the amplitude and pattern of reflex milk ejections. In chronic experiments, lesions of the medial septum resulted first in a loss of body weight of the mothers and a parallel reduction in growth of the litters. After a few days, the litters gained weight normally, and the pattern of milk ejections was normal. Thus, the pathways which pass through or originate from the medial septum and which are excitatory for oxytocin release appear not to be involved in the regulation of the milk ejection reflex. In view of these results and those from our previous study on the lateral septum, we conclude that the whole septum is not essential to the milk ejection reflex. However, the effects of septal stimulation suggest that the medial and lateral septum may be involved in a secondary neural circuitry that can inhibit the reflex.     

Burst firing of oxytocin neurons in male rat hypothalamic slices

Burst firing and single spike activity play different roles in the modulation of local neuronal circuit activity and neurosecretion. In hypothalamic oxytocin (OT) neurons in vivo, burst firing is associated with pulsatile secretion of OT in the milk ejection reflex, and can be observed in slices from both immature and lactating rats in vitro. Whether OT neurons from male rats also possess burst firing capability is still an open question. To examine this possibility, whole-cell patch clamp recordings were made in supraoptic nucleus OT neurons in brain slices from male rats. In low Ca(2+) medium, the alpha(1)-adrenoceptor agonist, phenylephrine evoked bursts that were highly similar to those from lactating rats in vivo and in vitro: explosive onset, short-duration, quickly reaching peak firing rate and displaying an exponential decay in returning to the pre-burst rate. During bursts, spike durations increased, and spike amplitudes decreased, while riding on an arc of depolarization around peak rate. In comparison to those from lactating rats in vitro, the rising phase of male bursts was more rapid, the decay phase was slower, and the rising phase of the spike after hyperpolarization was faster. No significant differences, however, were seen in burst characteristics that are most important in determining the amount of peptide release: burst amplitudes (the number of spikes in a burst), firing frequency within bursts or peak firing rate. Thus, we conclude that OT neurons in males are capable of burst firing highly similar to that seen in lactating rats.     

Deficiency in Mouse Oxytocin Prevents Milk Ejection,but not Fertility or Parturition

Oxytocin is a nonapeptide hormone that participates in the regulation of parturition and lactation. It has also been implicated in various behaviors, such as mating and maternal, and memory. To investigate whether or not oxytocin (OT) is essential for any of these functions, we eliminated, by homologous recombination, most of the first intron and the last two exons of the OT gene in mice. Those exons encode the neurophysin portion of the oxytocin preprohormone which is hypothesized to help in the packaging and transport of OT. The homozygous mutant mice have no detectable neurophysin or processed oxytocin in the paraventricular nucleus, supraoptic nucleus or posterior pituitary. Interestingly, homozygous mutant males and females are fertile and the homozygous mutant females are able to deliver their litters. However, the pups do not successfully suckle and die within 24  h without milk in their stomachs. OT injection into the dams restores the milk ejection in response to suckling. These results indicate an absolute requirement for oxytocin for successful milk ejection, but not for mating, parturition and milk production, in mice.     

Excitatory effect of dopamine on oxytocin and vasopressin reflex releases in the rat

The involvement of dopamine in the release of oxytocin and vasopressin was investigated in lactating rats during suckling or after changes in plasma osmolality. The effects of intraventricular injections of dopamine, agonists and antagonists, were tested on electrical unit activity of oxytocinergic or vasopressinergic cells in the paraventricular nucleus, on intramammary pressure (index of oxytocin release) and diuresis (index of vasopressin release).In urethane-anaesthetized lactating suckled rats, dopamine (1 μg), apomorphine (2.5 and 5 μg) facilitated the established milk-ejection reflex, increasing the frequency and the amplitude of neurosecretory bursts of oxytocinergic cells. They also triggered the reflex in lactating rats without milk-ejections during suckling. The small doses injected were in no way such as to induce an acceleration in firing rate of oxytocinergic cells or an increase in mammary pressure.In alcohol-loaded rats, during water diuresis, dopamine (2 μg) and apomorphine (5 μg) activated the depressed vasopressinergic cells and inhibited diuresis. These facilitatory effects were progressive, reaching a maximum 10–15 min after injection.Haloperidol (5 μg) and α-flupentixol (10 μg) had an inhibitory effect on both types of neurosecretory cells in urethane-anaesthetized rats. They prevented the reflex activation of oxytocinergic cells induced by suckling and of vasopressinergic cells after a hyperosmotic stimulus (1 ml i.p 9% NaCl solution). These inhibitory effects were not of the ‘all-or-none’ type.So, we can postulate that dopamine regulates the reflex release of oxytocin and vasopressin in the hypothalamus. On the one hand, dopamine permits and controls the periodic activation of oxytocinergic cells as long as the mothers are being suckled. On the other hand, it modulates the activity of vasopressinergic cells whenever the plasma osmolality changes.     

Neuronal Activation of the Hypothalamic Magnocellular System in Response to Oropharyngeal Stimuli in the Rat

The present study was designed to delineate the neuronal site, the nature, and the gastrointestinal origin of the stimulation of the hypothalamic magnocellular system induced by the ingestion of sweetened condensed milk. Concomitant localization of the c- fos protein (Fos) with either arginine-vasopressin (AVP) mRNA or oxytocin (OT) mRNA in the paraventricular nucleus of the hypothalamus (PVH) and the supraoptic nucleus (SON) revealed that the hypothalamic neurons containing AVP and OT were activated following ingestion of sweetened condensed milk. Expression of c- fos mRNA was also determined in rats implanted with a gastric cannula that allowed for real, sham, and gastric feeding of sweetened condensed milk. The results provide evidence that the stimulation of the PVH and SON induced by sweetened condensed milk originate from oropharyngeal stimuli. Indeed, in real-and sham-fed rats, the postprandial levels of c- fos mRNA in the PVH and SON were significantly higher than the preprandial values, whereas there was no early postprandial rise in c- fos mRNA levels within the magnocellular division of the PVH and SON after gastric feeding. The results of this study also suggested that the stimulation of the PVH and SON induced by sweetened condensed milk was related to the hypertonicity of the milk. Indeed, ingestion of an hypertonic solution of sucrose with a carbohydrate content close to that of sweetened condensed milk led to a stimulation of the PVH and SON that was comparable to that induced by the milk, whereas ingestion of an isotonic solution of sucrose did not trigger any significant activation of the PVH and SON. Taken together, the present results indicate that magnocellular neurosecretory neurons are sensitive to oropharyngeal stimuli and further support the view of the existence of oropharyngeal osmoreceptors.     

Effect of Centrally Administered Oxytocin on the Association Between Cortical Electroencephalogram and Milk Ejection in the Rat

The milk-ejection reflex of the rat is closely associated with synchronized activity of the cortical electroencephalogram (EEG), and the frequency of milk ejections has been shown to be greatly facilitated by central oxytocin. The following experiments were undertaken to examine the changes in the EEG during facilitation of the reflex by central oxytocin. Intracerebroventricular injection of 1 mU (2.2 ng) oxytocin during suckling caused a rapid increase in the frequency of milk ejections but no change in the predominantly synchronized pattern of the EEG. However, after a delay (11.3 ± 0.8 min, mean ± SE) there appeared to be an increasing proportion of desynchronization, which correlated with cessation of the facilitated milk-ejection responses. Hence, the observed EEG desynchronization may signal activation of mechanisms inhibitory to the milk-ejection reflex. In the absence of the suckling stimulus oxytocin also caused a change to desynchronization. However, this effect was more pronounced and commenced after a much shorter latency (1.7 ± 0.4 min, mean ± SE; P < 1.001), suggesting that the desynchronizing effect of oxytocin on the EEG can be attenuated by the suckling stimulus. These results demonstrate two phases in the action of central oxytocin in the suckled rat. During the initial phase, the milk-ejection reflex is facilitated and although there may be a concomitant desynchronizing influence on the EEG this is prevented by the influence of the suckling stimulus. In the later phase, this desynchronizing influence predominates and is accompanied by cessation of milk-ejection responses. Although milk ejections were generally restricted to periods of a synchronized EEG as previously reported, during the oxytocin-induced change to desynchronization a number of milk ejections were observed to occur in the absence of a synchronized EEG. These results provide further evidence that the association between milk ejection and the EEG state is not an absolute causal relationship.