Mammary duct luminal epithelium controls adipocyte thermogenic programme.

Sympathetic activation during cold exposure increases adipocyte thermogenesis via the expression of mitochondrial protein uncoupling protein 1 (UCP1)1. The propensity of adipocytes to express UCP1 is under a critical influence of the adipose microenvironment and varies between sexes and among various fat depots2-7. Here we report that mammary gland ductal epithelial cells in the adipose niche regulate cold-induced adipocyte UCP1 expression in female mouse subcutaneous white adipose tissue (scWAT). Single-cell RNA sequencing shows that glandular luminal epithelium subtypes express transcripts that encode secretory factors controlling adipocyte UCP1 expression under cold conditions. We term these luminal epithelium secretory factors 'mammokines'. Using 3D visualization of whole-tissue immunofluorescence, we reveal sympathetic nerve-ductal contact points. We show that mammary ducts activated by sympathetic nerves limit adipocyte UCP1 expression via the mammokine lipocalin 2. In vivo and ex vivo ablation of mammary duct epithelium enhance the cold-induced adipocyte thermogenic gene programme in scWAT. Since the mammary duct network extends throughout most of the scWAT in female mice, females show markedly less scWAT UCP1 expression, fat oxidation, energy expenditure and subcutaneous fat mass loss compared with male mice, implicating sex-specific roles of mammokines in adipose thermogenesis. These results reveal a role of sympathetic nerve-activated glandular epithelium in adipocyte UCP1 expression and suggest that mammary duct luminal epithelium has an important role in controlling glandular adiposity.

Homocysteine Hypothesis on the Impaired Peripheral but Not Central Nervous System Oxytocin Responses in Cystathionine γ-Lyase-Deficient Dam Mice.

An elevated plasma homocysteine level is an independent risk factor for cardiovascular diseases, neurological disorders, and pregnancy complications. We recently demonstrated partial lactation failure in cystathionine γ-lyase-deficient (Cth-/-) dam mice and their defective oxytocin responses in peripheral tissues: uterine (ex vivo) and mammary gland (in vivo). We reasoned that elevated levels of circulatory homocysteine in Cth-/- dam mice counteract with oxytocin-dependent milk ejection from the mammary gland. Based on our observation that those mice displayed normal maternal behaviors against their pups and adult Cth-/- male mice exhibited normal social behaviors against adult wild-type female mice, both of which are regulated by oxytocin in the central nervous system (CNS), we conducted the present study to investigate the amino acid profiles, including total homocysteine, in both blood and cerebrospinal fluid (CSF) of wild-type and Cth-/- female mice before pregnancy and at day 1 of lactation (L1). Serum levels of total homocysteine in wild-type and Cth-/- L1 dam mice were 9.44 and 188 µmol/L, respectively, whereas their CSF levels were below 0.21 (limit of quantification) and 3.62 µmol/L, respectively. Their CSF/serum level ratio was the lowest (1/51.9) among all 20 proteinogenic amino acids, sulfur-containing amino acids, and citrulline/ornithine in Cth-/- mice. Therefore, we hypothesize that the blood-brain barrier protects the CNS from high levels of circulatory homocysteine in Cth-/- dam mice, thereby conferring normal oxytocin-dependent maternal behaviors.

Sympathetic innervation, norepinephrine content, and norepinephrine turnover in orthotopic and spontaneous models of breast cancer.

Activation of the sympathetic nervous system (SNS) drives breast cancer progression in preclinical breast cancer models, but it has yet to be established if neoplastic and stromal cells residing in the tumor are directly targeted by locally released norepinephrine (NE). In murine orthotopic and spontaneous mammary tumors, tyrosine hydroxylase (TH)+ sympathetic nerves were limited to the periphery of the tumor. No TH+ staining was detected deeper within these tumors, even in regions with a high density of blood vessels. NE concentration was much lower in tumors compared to the more densely innervated spleen, reflecting the relative paucity of tumor TH+ innervation. Tumor and spleen NE concentration decreased with increased tissue mass. In mice treated with the neurotoxin 6-hydroxydopamine (6-OHDA) to selectively destroy sympathetic nerves, tumor NE concentration was reduced approximately 50%, suggesting that the majority of tumor NE is derived from local sympathetic nerves. To evaluate NE utilization, NE turnover in orthotopic 4T1 mammary tumors was compared to spleen under baseline and stress conditions. In non-stressed mice, NE turnover was equivalent between tumor and spleen. In mice exposed to a stressor, tumor NE turnover was increased compared to spleen NE turnover, and compared to non-stressed tumor NE turnover. Together, these results demonstrate that NE in mammary tumors is derived from local sympathetic nerves that synthesize and metabolize NE. However, differences between spleen and tumor NE turnover with stressor exposure suggest that sympathetic NE release is regulated differently within the tumor microenvironment compared to the spleen. Local mammary tumor sympathetic innervation, despite its limited distribution, is responsive to stressor exposure and therefore can contribute to stress-induced tumor progression.

Chronic neural activity recorded within breast tumors.

Nerve fibers are known to reside within malignant tumors and the greater the neuronal density the worse prognosis for the patient. Recent discoveries using tumor bearing animal models have eluded to the autonomic nervous system having a direct effect on tumor growth and metastasis. We report the first direct and chronic in vivo measurements of neural activity within tumors. Using a triple-negative mammary cancer mouse model and chronic neural interface techniques, we have recorded neural activity directly within the tumor mass while the tumor grows and metastasizes. The results indicate that there is a strong connection between the autonomic nervous system and the tumor and could help uncover the mechanisms of tumor growth and metastasis.

Coordination of mammary metabolism and blood flow after refeeding in rats.

The production of milk is closely linked to nutritional state in many mammalian species, but the mechanisms by which changes in nutritional state are signaled to the mammary glands are poorly understood. Simultaneous measurements of mammary blood flow and glucose arterio-venous difference were made across the inguinal mammary glands of anesthetized, lactating rats. Blood flow to the mammary glands of previously fed rats was 0.48 mL/min per gram of mammary tissue. Glucose supply was 1.7 mumol/min per gram and 28% was extracted by the mammary glands. After food deprivation for 18 h, mammary blood flow decreased 48%, glucose arterio-venous difference decreased 72%, and hematocrit increased 7%, resulting in a 60% decrease in glucose supply and an 88% decrease in glucose uptake. After 1 h of refeeding, glucose supply had returned to a similar level to that of normally fed animals, but glucose uptake was 60% higher than in the normally fed state. Mammary glucose uptake was not closely linked to either blood flow or glucose supply, suggesting that substrate supply was not the primary determinant of mammary metabolism. Denervation experiments showed that the mammary metabolic response to altered nutritional state was also unlikely to be closely controlled by neural pathways. Severance of the cutaneous branch of the posterior division of the femoral nerve innervating the inguinal mammary glands did not reduce the high glucose uptake by mammary glands of either fed or refed rats, nor did denervation change the low glucose uptake by mammary glands of food-deprived rats. Denervation reduced blood flow in the associated mammary gland, however, indicating that neural pathways may play a role in supporting mammary blood flow when food is available. In in vitro experiments, the rate of glucose uptake was 35% lower in mammary acini from food-deprived rats than in fed rats 2.5 h after tissue removal, indicating some persistence of the food deprivation-induced suppression of mammary metabolism. Administration of insulin increased glucose uptake in acini from both fed and food-deprived rats, indicating that insulin may be involved in signaling the mammary gland of the restoration of nutrient supply when food-deprived rats are refed. The effects of administration of a gut extract in vivo and in vitro are discussed.

Balance between BDNF and Semaphorins gates the innervation of the mammary gland.

The innervation of the mammary gland is controlled by brain-derived neurotrophic factor (BDNF), and sexually dimorphic sequestering of BDNF by the truncated form of TrkB (TrkB.T1) directs male-specific axonal pruning in mice. It is unknown whether other cues modulate these processes. We detected specific, non-dimorphic, expression of Semaphorin family members in the mouse mammary gland, which signal through PlexinA4. PlexinA4 deletion in both female and male embryos caused developmental hyperinnervation of the gland, which could be reduced by genetic co-reduction of BDNF. Moreover, in males, PlexinA4 ablation delayed axonal pruning, independently of the initial levels of innervation. In support of this, in vitro reduction of BDNF induced axonal hypersensitivity to PlexinA4 signaling. Overall, our study shows that precise sensory innervation of the mammary gland is regulated by the balance between trophic and repulsive signaling. Upon inhibition of trophic signaling, these repulsive factors may promote axonal pruning.

Immunohistochemical Characterization of Sympathetic Chain Ganglia (SChG) Neurons Supplying the Porcine mammary Gland.

The aim of this study was to investigate the chemical coding of mammary gland-projecting SChG neurons using double-labelling immunohistochemistry. Earlier observation showed that after injection of the retrograde tracer fast blue (FB) into the second, right thoracic mamma, FB+ mammary gland-projecting neurons were found in Th1-3, Th9-14 and L1-4 right SChG. The greatest number of FB+ nerve cell bodies was observed in Th10 (approx. 843) and Th11 (approx. 567). Neurons projecting to the last right abdominal mamma were found in L1-4 SChG. The greatest number of FB+ neurons was observed in L2 (approx. 1200). Immunohistochemistry revealed that the vast majority of FB+ mammary-projecting neurons contained immunoreactivities to TH (96.97%) and/or DßH (95.92%). Many TH/DßH-positive neurons stained for SOM (41.5%) or NPY (33.2%), and less numerous nerve cells expressed VIP (16.9%). This observation strongly corresponds to the results of previous studies concerning the immunohistochemical characterization of nerve fibres supplying the porcine mammary gland.

Perinatal exposure to androgen suppresses sexual dimorphism in nerve trunk diameter, axon number, and fiber size spectrum: a quantitative ultrastructural study of the adult rat mammary nerve.

The adult mammary nerves (MNs) from female, male, and testosterone-androgenized female rats were studied by light and electron microscopy. The female MNs trunk has twice the diameter of that of the male. Morphometry showed a significantly more myelinated (307 +/- 6) and unmyelinated axons (1654 +/- 10) in the female MN than the male MN (278 +/- 6 and 1373 +/- 28, respectively). Perinatal exposure of the female to testosterone significantly reduced the number of both axon types in the MN in adulthood (244 +/- 6 myelinated and 1300 +/- 32, unmyelinated). Another sexual dimorphism is a distinct group of large (>7.0 microm in diameter) myelinated axons known to conduct sensory information (i.e., touch and vibration). Because the male and the perinatally-androgenized female MNs lack these fibers, it is concluded that gonadal sex hormones may promote the differentiation of specific sets of axons committed to transmission of sensory cues relevant to reproduction.

Release of catecholamines follows suckling or electrical stimulation of mammary nerve in lactating rats.

The hypothesis that catecholamines may be released by mammary gland stimulation during lactation was tested by measuring, with an HPLC electrochemical method, plasma epinephrine (E) and norepinephrine (NE) concentrations during suckling in conscious rats and during electrical stimulation (pulses: 1 msec duration, 10/sec at 5-30 V) of the central end of a cut abdominal mammary nerve in urethane-anesthetized rats. Plasma E and NE concentrations were significantly elevated in two different strains of rats (Wistar and Holtzman) within 5 min of suckling. The concentration of E and NE did not change in control unsuckled rats during the same time period. As a complementary indication of sympathetic activation, it was observed that piloerection occurred during suckling. Plasma E levels (but not NE levels) increased significantly within 30 sec of a 2-min period of nerve stimulation in lactating rats on either day 7 or day 21 of lactation, as well as in nonlactating rats. The effect was significantly greater in nonlactating rats. The levels of E and NE were not altered after sham stimulation, whereas adrenalectomy abolished the rise in plasma E after mammary nerve stimulation. Blockade of the rise in plasma E also occurred after rapid injection of 100 microliters milk intraductally into each of two thoracic mammary glands, 15 sec before the onset of mammary nerve stimulation. These results show that E and NE can be released in response to suckling, and that activation of ductal mechanoreceptors may inhibit such release. These mechanisms may operate to regulate the rate of milk removal during suckling in the rat.

Effect of electrical stimulation of mammary nerve upon pituitary and plasma prolactin concentrations in anesthetized lactating rats.

A 50-70% depletion in the anterior pituitary concentration of PRL occurred within 5 min of electrical stimulation of a single abdominal mammary nerve of urethane-anesthetized lactating rats previously nonsuckled for 6-8 h. The stimulus parameters were: 1-msec pulses, 10-20/sec at 5-30 V applied 5 sec on and 10 sec off. The anterior pituitary concentration of PRL remained low for another 15-30 min after depletion, then repleted to prestimulus levels by the 90th min. The same pattern of depletion-repletion occurred when mammary nerve stimulation was applied for 5 min as when it was applied for 180 min. In other experiments, plasma PRL concentration rose swiftly and attained a maximal level, 4- to 5-fold in Wistar rats and 8- to 10-fold in Holtzman rats, above basal concentrations within 10-15 min of mammary nerve stimulation. The maximal level was sustained throughout the time the nerve was stimulated and for 45 min after the stimulation was stopped, i.e. the sustained secretion of PRL into the plasma occurred at the same time that depleted PRL was repleting. Ligation of both adrenals or iv injection of the beta-adrenergic blocker, propranolol, before stimulation of the nerve had no effect upon the plasma PRL profile in response to mammary nerve stimulation. These data indicate that mammary nerve stimulation mimics that of suckling upon depletion, repletion, and the release of PRL into the circulation and add further support to the hypothesis that these phases are independent processes.

The decrease in hypothalamic dopamine secretion induced by suckling: comparison of voltammetric and radioisotopic methods of measurement.

Previous in situ voltammetric microelectrode measurements of median eminence dopamine release during mammary nerve stimulation of anesthetized lactating rats revealed a transient (1-3 min) 70% decline of dopamine concentrations. This dopamine was believed to be destined for secretion into the hypophysial portal circulation, but direct experimental support for this supposition was lacking. Thus, in the present study, [3H]dopamine release into brief sequential samples of hypophysial portal blood was compared with dopamine release in the median eminence measured by voltammetry. Lactating female rats were urethane anesthetized, and the median eminence pituitary region was exposed. [3H]Tyrosine was injected into a jugular cannula (100 microCi) followed by continuous infusion (5 microCi/min). In a preliminary experiment, this regimen produced a steady state level of [3H]dopamine in the portal blood within 45 min. In subsequent experiments, portal blood was collected as sequential 3-min samples, and electrochemical sampling from a microelectrode placed in the median eminence occurred at 1-min intervals. Electrochemical current resulting from the oxidation of dopamine in the medial median eminence was unvarying throughout the 75-min experiment in control rats (n = 4) and during the 30-min control period preceding mammary nerve stimulation in the other group (n = 4). These results were parallel by [3H] dopamine levels in portal blood during the same periods of time. All animals showed simultaneous decreases in oxidation current and [3H]dopamine levels within 1-4 min after initiation of mammary nerve stimulation (respectively, 35 +/- 7% and 62.5 +/- 5.9%, mean +/- SEM). Significant increases in oxidation current, taking the form of brief 2- to 6-min pulses began within an average of 18.5 min after initiation of stimulation. Similar increases in [3H]dopamine levels in portal blood were also observed. These and earlier results demonstrate that mammary nerve stimulation (and by extension, suckling) induces a momentary, but profound, decrease in hypothalamic dopamine secretion which precedes or accompanies the rise in PRL secretion evoked by the same stimulus.

Anatomic organization of the ascending branch of the milk-ejection reflex in sheep: primary afferent neurons.

The present study examines the anatomic characteristics of the primary afferent neurons that innervate the nipples and pseudonipples of ewes and the nipples of lambs. For this purpose, horseradish-peroxidase coupled to wheat germ agglutinin (WGA-HRP) was injected intradermally into the whole extent, the tip, or the base of the nipples and pseudonipples, as well as into a region of the posterior surface of the udder. After survival periods of 72-96 hours, dorsal root ganglia (DRG), segments of the spinal cord and medulla oblongata were sectioned and reacted histochemically with tetramethylbenzidine to reveal the transganglionically transported tracer. Injections of WGA-HRP in the nipples and pseudonipples of the ewe resulted in labeled cells in the second to fifth ipsilateral lumbar spinal ganglia (L(2)-L(5)) and third and fourth (L(3) and L(4)) lumbar spinal ganglia, respectively. Labeled cells after WGA-HRP injections in the nipples of the lamb were found in the ipsilateral L(3)-L(5) spinal ganglia. Central projections of the DRG-labeled cells were found in the medial part of laminae I-III of the ipsilateral L(3) and L(4) spinal segments (ewe and lamb) and in the ipsilateral dorsal column nuclei (ewe). Central projections of the DRG-labeled cells after injections in the pseudonipples of the ewe were located in the medial part of laminae I-III of the ipsilateral L(3) spinal segment. The results of this study demonstrate that, whereas the innervation of the nipples of the ewe originates from four successive lumbar spinal ganglia (L(2)-L(5)), the innervation of the nipples of the lamb and the pseudonipples of the ewe originates from three (L(3)-L(5)) and two (L(3) and L(4)) successive ganglia, respectively.

Afferent projections from the mammary glands to the spinal cord in the lactating rat--I. A neuroanatomical study using the transganglionic transport of horseradish peroxidase-wheatgerm agglutinin.

Horseradish peroxidase-wheatgerm agglutinin was injected subcutaneously into one or more nipples of lactating rats to determine the spinal organization of sensory afferents emanating from the mammary glands. After survival periods of 45-96 h, dorsal root ganglia and segments of the spinal cord and/or medulla oblongata were sectioned and reacted histochemically with tetramethylbenzidine to reveal the transganglionically transported tracer. For each nipple injected, the peroxidase reaction product was found in somata, ranging in diameter from 15 to 60 microns, and fibres in 5-11 contiguous dorsal root ganglia. The number of labelled profiles was highest in the 2-4 central-most ganglia of the series and generally decreased progressively rostrally and caudally. After separate injections into each of the six ipsilateral nipples, labelling occurred in all ipsilateral dorsal root ganglia between the 5th cervical and 6th lumbar spinal segments. Substantial overlap of the spinal projections from adjacent mammary glands was seen, a given dorsal root ganglion innervating 2-3 different glands. Label in the spinal cord was restricted to the medial portion of the superficial dorsal horn. It occurred in what appeared to be terminal fields and fibres essentially in the substantia gelatinosa, but was also seen to extend into the marginal zone and sometimes into deeper regions of the dorsal horn. Label was found in both the gracile and cuneate nuclei of the medulla oblongata, though only occasionally and then only very sparsely. The substantial spread and segmental overlap of labelled mammary afferents, and the fact that most labelled afferents terminated in the dorsal horn, suggest that this spinal region may be an important site for the integration of sensory input from the mammary glands that may play a role in the sensory induction of reflex milk ejection.

Transneuronal labelling of nerve cells in the CNS of female rat from the mammary gland by viral tracing technique.

Using the viral transneuronal tracing technique, the cell groups in the CNS transneuronally connected with the female mammary gland were detected. Lactating and non-lactating female rats were infected with pseudorabies virus injected into the mammary gland. The other group of animals was subjected to virus injection into the skin of the back. Four days after virus injection, infected neurons detected by immunocytochemistry, were present in the dorsal root ganglia ipsilateral to inoculation and in the intermediolateral cell column of the spinal cord. In addition, a few labelled cells could be detected in the dorsal horn and in the central autonomic nucleus (lamina X) of the spinal cord. At this survival time several brain stem nuclei including the A5 noradrenergic cell group, the caudal raphe nuclei (raphe obscurus, raphe pallidus, raphe magnus), the A1/C1 noradrenergic and adrenergic cell group, the nucleus of the solitary tract, the area postrema, the gigantocellular reticular nucleus, and the locus coeruleus contained virus-infected neurons. In some animals, additional cell groups, among others the periaqueductal gray and the red nucleus displayed labelling. In the diencephalon, a significant number of virus-infected neurons could be detected in the hypothalamic paraventricular nucleus. In most cases, virus-labelled neurons were present also in the lateral hypothalamus, in the retrochiasmatic area, and in the anterior hypothalamus. In the telencephalon, in some animals a few virus-infected neurons could be found in the preoptic area, in the bed nucleus of the stria terminalis, in the central amygdala, and in the somatosensory cortex. At the longer (5 days) survival time each cell group mentioned displayed immunopositive neurons, and the number of infected cells increased. The pattern of labelling was similar in animals subjected to virus inoculation into the mammary gland and into the skin. The distribution and density of labelling was similar in lactating and non-lactating rats. The present findings provide the first morphological data on the localization of CNS structures connected with the preganglionic neurons of the sympathetic motor system innervating the mammary gland. It may be assumed that the structures found virus-infected belong to the neuronal circuitry involved in the control of the sympathetic motor innervation of the mammary gland.

Electronmicroscopic and electrophysiological studies of the teat branch of the XIII thoracic nerve: relationship with lactation in the rat.

Electrical stimulation of the XIII thoracic nerve (the 'mammary nerve') causes milk ejection and the release of prolactin and other hormones. We have analysed the route of the suckling stimulus at the level of different subgroups of fibres of the teat branch of the XIII thoracic nerve (TBTN), which innervates the nipple and surrounding skin, and assessed the micromorphology of the TBTN in relation to lactation. There were 844 +/- 63 and 868 +/- 141 (S.E.M.) nerve fibres in the TBTN (85% non-myelinated) in virgin and lactating rats respectively. Non-myelinated fibres were enlarged in lactating rats; the modal value being 0.3-0.4 micron 2 for virgin and 0.4-0.5 micron 2 for lactating rats (P greater than 0.001; Kolmogorov-Smirnov test). The modal value for myelinated fibres was 3-6 micron 2 in both groups. The compound action potential of the TBTN in response to electrical stimulation showed two early volleys produced by the A alpha- and A delta-subgroups of myelinated fibres (conduction velocity rate of 60 and 14 m/s respectively), and a late third volley originated in non-myelinated fibres ('C') group; conduction velocity rate 1.4 m/s). Before milk ejection the suckling pups caused 'double bursts' of fibre activity in the A delta fibres of the TBTN. Each 'double burst' consisted of low amplitude action potentials and comprised two multiple discharges (33-37 ms each) separated by a silent period of around 35 ms. The 'double bursts' occurred at a frequency of 3-4/s, were triggered by the stimulation of the nipple and were related to fast cheek movements visible only by watching the pups closely. In contrast, the A alpha fibres of the TBTN showed brief bursts of high amplitude potentials before milk ejection. These were triggered by the stimulation of cutaneous receptors during gross slow sucking motions of the pup (jaw movements). Immediately before the triggering of milk ejection the mother was always asleep and a low nerve activity was recorded in the TBTN at this time. When reflex milk ejection occurred, the mother woke and a brisk increase in nerve activity was detected; this decreased when milk ejection was accomplished. In conscious rats the double-burst type of discharges in A delta fibres was not observed, possibly because this activity cannot be detected by the recording methods currently employed in conscious animals.(ABSTRACT TRUNCATED AT 400 WORDS)

Food intake, aggression, and fear behavior in the mother rat: control by neural systems concerned with milk ejection and maternal behavior.

Mother rats eat more, are more aggressive, and show less fear behavior (freezing) than during other stages of the reproductive cycle. Electrolytic lesions in the peripeduncular area of the lateral midbrain made nursing mother rats eat less and interact peacefully with male intruders. This midbrain area forms part of the ascending milk-ejection pathway, so it seems plausible that the suckling stimulus maintains hyperphagia and aggression in mother rats. Because no alteration in fear behavior was observed in mothers with lesions, it was predicted that the reduction in freezing was related primarily to maternal responsiveness to pup cues other than suckling. In line with this hypothesis, it was found that the experimental induction of maternal behavior in ovariectomized, hormone-treated females was associated with a significant decrease in fear behavior, with no concomitant changes in food intake or aggression.

In situ voltammetric microelectrodes: application to the measurement of median eminence catecholamine release during simulated suckling.

Catechol-sensitive microelectrodes (10-30 microns) were developed and then used to study the dynamic regulatory role of the prolactin inhibiting factor, dopamine, under conditions of simulated suckling. Current flow resulting from the electrochemical oxidation of catecholamines at the microelectrode surface was linearly related to the concentration of catecholamines present in solution over the range of 5-100 microM. Endogenous catecholamine levels in the rat median eminence were readily detectable and the electrochemical signal corresponding to dopamine release responded in an appropriate manner to various pharmacologic manipulations. We then implanted carbon microelectrodes into the medial median eminence region among capillaries of the primary portal plexus of urethane anesthetized lactating rats. Catecholamine release into the extracellular fluid was electrochemically measured once each minute before, during and after electrical stimulation (15 Hz, 5-30 V, 15 min) of a surgically isolated mammary nerve trunk. This simulated suckling paradigm reliably evoked prolactin secretory episodes qualitatively similar to those observed during suckling of the nipples by the young. During the period of nerve stimulation, a transient (3-5 min) 65% decline in electrochemically detectable catecholamine release was observed. Following cessation of nerve stimulation an oscillatory pattern of catecholamine release was observed with an overall trend toward an increased level of release. This latter observation corresponds with previous reports of increased hypothalamic dopamine turnover during or following suckling and with the increased levels of dopamine measured in hypophysial portal blood following mammary nerve stimulation. The transient nature of the decline of catecholamine release during the nerve stimulation period may explain why a similar observation has not been forth-coming from experiments utilizing the stalk blood collection technique (unless the standard collection periods are considerably shortened). These observations lead us to reject the hypothesis of a mirror image relationship between stalk blood dopamine and peripheral prolactin levels. Instead, we suggest that a transient decline in dopamine secretion coincident with the onset of suckling acts to prepare the pituitary lactotrophs to respond to a prolactin releasing factor which then facilitates prolactin secretion.

Suckling-induced activation of neural c-fos expression at lower thoracic rat spinal cord segments.

Suckling stimulation is essential for neuroendocrine and sympathetic reflex activation during lactation. In the present study, the induction of c-fos gene expression was used to identify neuronal populations in the spinal cord activated by acute 5 min suckling or by electrical stimulation of the central stump of the first abdominal mammary nerve in lactating rats previously separated from their litters for 6 or 18 h. In addition, to investigate whether spinal sympathetic preganglionic neurons are activated by suckling, dual immunostaining (Fos and choline acetyltransferase) was performed. Fos was expressed at low levels in continuously suckled and 6 h nonsuckled mothers, but no expression was found after 18 h of nonsuckling. On the other hand, in 6 h nonsuckled rats, significant increments in Fos expression occurred in several regions after acute suckling and after electrical stimulation. Also, the pattern of Fos expression in each spinal laminae was different for the two stimuli, i.e. more intense effects of suckling in deep laminae V-X and more intense effects in laminae I-IV with electrical stimulation. Double-labeling after suckling was found only in sympathetic preganglionic neurons from the intermedio-medial cell column, whereas after electrical stimulation, double label was observed only in neurons from the intermedio-lateral cell column. On the other hand, no effect upon Fos protein expression was observed after suckling and only a minor effect after electrical stimulation of mammary nerve in 18 h nonsuckled rats. These results are consistent with previous findings on the sympathetic reflex regulation of the mammary gland, as well as on the importance of the nonsuckling interval for optimal functioning of lactation.

The milk ejection pathway in brain studied with the 2-deoxyglucose method.

The neural pathways involved in the milk ejection reflex have been studied with the aid of the 2-deoxyglucose (2DG) method. All the experiments were carried out on Wistar female rats, 9-11 days post-partum, which had been separated from their pups (except for one) overnight. The effect of suckling on the relative metabolic activity (RMA) of the brain was studied in conscious rats and in rats anaesthetized with urethane. Control animals were similarly treated but were not suckled. In addition, the effect of mammary nerve stimulation on RMA was studied in animals anaesthetized with urethane; sham-operated animals served as controls for this group. Suckling (minimum of 10 pups) in conscious animals had no apparent effect on the RMA of any of the brain areas measured. However, in anaesthetized rats, suckling produced a significant increase in the RMA of the paraventricular and supraoptic nuclei (PVN and SON), but had no effect on the RMA of any other brain area or the pituitary gland. Stimulation of the mammary nerve, with a stimulus that causes milk ejection and an increase in prolactin release, produced a significant increase in the RMA of the PVN, SON, the pars distalis and pars nervosa and the spinothalamic tract, and a significant decrease in the ventromedial and mediodorsal nuclei of the thalamus, the zona incerta, the red nucleus and the ventral nucleus of the lateral lemniscus. These results show that suckling significantly increases the metabolic activity of afferent terminals in the PVN and SON. Activation of the cell bodies of the PVN and SON, as assessed by increased RMA of the pituitary gland, could be evoked by the more intense stimulus of mammary nerve stimulation. The ascending pathway from the mammary nerve involves the spinothalamic tract but could not be traced beyond the midbrain. The lack of effect of suckling in conscious animals may have been due to the inhibitory influence of stress mediated by forebrain structures.

Facilitatory effect of oxytocin on oxytocin cell background activity in the rat is suckling-dependent.

The injection of oxytocin into the third ventricle during suckling in lactating rats not only enhances neurosecretory bursts but also the background activity of oxytocin cells. However, removing the young rats 10 min after the oxytocin injection is immediately followed by a decrease in background activity, while injecting oxytocin alone (i.e. without suckling) or suckling alone (i.e. without oxytocin injection) has no effect. These results, which show that the facilitatory effect of oxytocin on oxytocin cell background activity is suckling-dependent, suggest that oxytocin could act either on the afferent pathways for milk ejection or on oxytocin cells themselves, but only if they received inputs from mammary glands. Various hypotheses on the site and mode of action of oxytocin are discussed.