Microinjection of galanin-like peptide into the medial preoptic area stimulates food intake in adult male rats

Galanin-like peptide (GALP) is a neuropeptide implicated in the regulation of feeding behaviour, metabolism and reproduction. GALP is an endogenous ligand of the galanin receptors, which are widely expressed in the hypothalamus. GALP is predominantly expressed in arcuate nucleus (ARC) neurones, which project to the paraventricular nucleus (PVN) and medial preoptic area (mPOA). Intracerebroventricular or intraparaventricular (iPVN) injection of GALP acutely increases food intake in rats. The effect of GALP injection into the mPOA on feeding behaviour has not previously been studied. In the present study, intra-mPOA (imPOA) injection of GALP potently increased 0-1-h food intake in rats. The dose-response effect of imPOA GALP administration on food intake was similar to that previously observed following iPVN administration. The effects of GALP (1 nmol) or galanin (1 nmol) on food intake were then compared following injection into the PVN, mPOA, ARC, dorsal medial nucleus (DMN), lateral hypothalamus and rostral preoptic area (rPOA). GALP (1 nmol) increased food intake to a similar degree when injected into the imPOA or iPVN, but produced no significant effect when injected into the ARC, DMN, lateral hypothalamus or rPOA. Similarly, galanin (1 nmol) significantly increased food intake following injection imPOA and iPVN. However, the effect was significantly smaller than that following administration of GALP (1 nmol). Galanin also had no significant effect on food intake when administered into the ARC, DMN, lateral hypothalamus and rPOA. These data suggest that the mPOA and the PVN may have specific roles in mediating the orexigenic effect of GALP and galanin.     

Preprothyrotropin-releasing hormone<Subscript>178–199</Subscript> affects tyrosine hydroxylase biosynthesis in hypothalamic neurons

ProThyrotropin-releasing hormone (proTRH) is a prohormone widely distributed in many areas of the brain. After biosynthesis, proTRH is subjected to post-translational processing to generate TRH and seven non-TRH peptides. Among these non-TRH sequences, we found previously that preproTRH_178–199 could regulate the secretion of prolactin in suckled rats by their pups. Dopamine (DA), the main regulator of prolactin secretion, is produced in dopaminergic tyrosine hydroxylase (TH)-positive neurons in the hypothalamic arcuate nucleus (ARC). In this study we investigated whether prolactin release during the estrous sexual cycle is regulated by prepro TRH_178–199 through its effecton DA neurons of the ARC. We observed that biotinylated prepro TRH_178–199 bound to neurons in the ARC; this was higher during proestrus than during diestrus. Binding of preproTRH_178–199 to DA neurons was seen only during proestrus in the ARC. Using primary neuronal hypothalamic cultures we found that preproTRH_178–199peptide decreased TH levels in a dose-responsive manner, whereas intra-ARC administration of preproTRH_178–199 induced a 20-fold increase in plasma prolactin levels. Together, these results suggest a potential role for preproTRH_178–199 in regulating dopaminergic neurons involved in the inhibition of pituitary prolactin release.     

Sexually dimorphic neuronal inputs to the neuroendocrine dopaminergic system governing prolactin release

Prolactin (PRL) is a pleiotropic hormone that was identified in the context of maternal care and its release from the anterior pituitary is primarily controlled by neuroendocrine dopaminergic (NEDA) neurones of the arcuate nucleus of the hypothalamus. The sexually dimorphic nature of PRL physiology and associated behaviours is evident in mammals, even though the number and density of NEDA neurones is reported as not being sexually dimorphic in rats. However, the underlying circuits controlling NEDA neuronal activity and subsequent PRL release are largely uncharacterised. Thus, we mapped whole‐brain monosynaptic NEDA inputs in male and female mice. Accordingly, we employed a rabies virus based monosynaptic tracing system capable of retrogradely mapping inputs into genetically defined neuronal populations. To gain genetic access to NEDA neurones, we used the dopamine transporter promoter. Here, we unravel 59 brain regions that synapse onto NEDA neurones and reveal that male and female mice, despite monomorphic distribution of NEDA neurones in the arcuate nucleus of the hypothalamus, receive sexually dimorphic amount of inputs from the anterior hypothalamic nucleus, anteroventral periventricular nucleus, medial preoptic nucleus, paraventricular hypothalamic nucleus, posterior periventricular nucleus, supraoptic nucleus, suprachiasmatic nucleus, lateral supramammillary nucleus, tuberal nucleus and periaqueductal grey. Beyond highlighting the importance of considering sex as a biological variable when evaluating connectivity in the brain, these results illustrate a case where a neuronal population with similar anatomical distribution has a subjacent sexually dimorphic connectivity pattern, potentially capable of contributing to the sexually dimorphic nature of PRL release and function.     

Amygdala enterostatin induces c-Fos expression in regions of hypothalamus that innervate the PVN

Enterostatin selectively inhibits the intake of the dietary fat after both central and peripheral administration. Our previous studies have shown that a central site of action is the central nucleus of amygdala. Serotonergic agonists administered into the paraventricular nucleus (PVN) inhibit fat intake and serotonergic antagonists block the feeding suppression induced by amygdala enterostatin, suggesting that there are functional connections between the PVN and amygdala that affect the feeding response to enterostatin. Our purpose was to identify the anatomic and functional projections from the amygdala to the PVN and hypothalamic area that are responsive to enterostatin, by using a retrograde tracer fluorogold (FG) and c-Fos expression. Rats were injected with fluorogold unilaterally into the PVN and a chronic amygdala cannula was implanted ipsilaterally. After 10 days recovery, rats were injected with either enterostatin (0.1 nmol) or saline vehicle (0.1 microl) into the amygdala and sacrificed 2 h later by cardiac perfusion under anesthesia. The brains were subjected to dual immunohistochemistry to visualize both FG and c-Fos-positive cells. FG/c-Fos double-labeled cells were found in forebrain regions including the PVN, amygdala, lateral hypothalamus (LH), ventral medial hypothalamus (VMH) and arcuate nucleus (ARC). The data provides the first anatomical evidence that enterostatin activates amygdala neurons that have functional and anatomic projections directly to the PVN and also activates neurons in the arcuate, LH and VMH, which innervate the PVN.     

Excitatory stimulation of neurons in the arcuate nucleus initiates central CRF-dependent stimulation of colonic propulsion in rats

It is well established that autonomic control of digestive function is modulated by central autonomic neurotransmission. In this context it has been shown that digestive function can be modulated by exogenous neuropeptides microinjected into specific brain sides. Furthermore, there is considerable evidence suggesting that neurons projecting from the arcuate nucleus (ARC) to the PVN may be the source of endogenous neuropeptide release in the PVN. Neuronal projections from the ARC have been proposed to target corticotropin-releasing factor (CRF)-positive neurons in the PVN. Exogenous CRF in the PVN has been shown to modulate digestive function like gastric acid secretion and GI motility. Recently we have demonstrated that activation of ARC neurons inhibits gastric acid secretion via central CRF receptor dependent mechanisms. This poses the question whether neuronal activation of the ARC alters digestive function beside gastric acid secretion. In the present study we investigated whether CRF pathways in the ARC-PVN axis are involved in the modulation of colonic motility. First we examined the effect of an excitatory amino acid, kainate, microinjected into the ARC on colonic motility in anesthetized rats. Colonic motility was measured with a non-absorbable radioactive marker using the geometric center method. Kainate (120 pmol/rat) bilaterally microinjected into the ARC induced a significant stimulation of colonic propulsion. To assess the contribution of hypothalamic CRF to the effects of neuronal stimulation in the ARC on colonic motility we performed consecutive bilateral microinjections of an antagonist to CRF receptors into the PVN and the excitatory amino acid kainate into the ARC. Microinjection of the non-selective CRF receptor antagonist, astressin (100 ng), into the PVN abolished the stimulatory effect of neuronal activation in the ARC by kainate on colonic motor function. The data indicate that activation of neurons in the ARC stimulates colonic motility via CRF-receptor-mediated mechanism in the PVN and underlines the important role of the ARC-PVN circuit for the integrative CNS regulation of GI function.     

Leptin Responsive and GABAergic Projections to the Rostral Preoptic Area in Mice

The adipocyte‐derived hormone leptin plays a critical role in the control of reproduction via signalling in hypothalamic neurones. The drivers of the hypothalamic‐pituitary‐gonadal axis, the gonadotrophin‐releasing hormone (GnRH) neurones, do not have the receptors for leptin. Therefore, intermediate leptin responsive neurones must provide leptin‐to‐GnRH signalling. We investigated the populations of leptin responsive neurones that provide input to the rostral preoptic area (rPOA) where GnRH cell bodies reside. Fluorescent retrograde tracer beads (RetroBeads; Lumafluor Inc., Naples, FL, USA) were injected into the rPOA of transgenic leptin receptor enhanced green fluorescent protein (Lepr‐eGFP) reporter mice. Uptake of the RetroBeads by Lepr‐eGFP neurones was assessed throughout the hypothalamus. RetroBead uptake was most evident in the medial arcuate nucleus (ARC), the dorsomedial nucleus (DMN) and the ventral premammillary nucleus (PMV) of the hypothalamus. The uptake of RetroBeads specifically by Lepr‐eGFP neurones was highest in the medial ARC (18% of tracer‐labelled neurones Lepr‐eGFP‐positive). Because neurones that are both leptin responsive and GABAergic play a critical role in the regulation of fertility by leptin, we next focussed on the location of these populations. To address whether GABAergic neurones in leptin‐responsive hypothalamic regions project to the rPOA, the experiment was repeated in GABA neurone reporter mice (Vgat‐tdTomato). Between 10% and 45% of RetroBead‐labelled neurones in the ARC were GABAergic, whereas uptake of tracer by GABAergic neurones in the DMN and PMV was very low (< 5%). These results show that both leptin responsive and GABAergic neurones from the ARC project to the region of the GnRH cell bodies. Our findings suggest that LEPR‐expressing GABA neurones from the ARC may be mediators of leptin‐to‐GnRH signalling.     

Increased feeding and neuropeptide Y (NPY) but not NPY mRNA levels in the hypothalamus of the rat following central administration of the serotonin synthesis inhibitorp-chlorophenylalanine

Neurons containing serotonin (5-HT), a potent anorexic agent, come into contact with neuropeptide Y-ergic neurons, that project from the arcuate nucleus (ARC) to the paraventricular nucleus (PVN). NPY powerfully stimulates feeding and induces obesity when injected repeatedly into the PVN. We hypothesize that 5-HT tonically inhibits the ARC-PVN neurons and that balance between the two systems determines feeding and energy homeostasis. This study aimed to determine whether central injection of the 5-HT synthesis inhibitor p-chlorophenylalanine (pCPA), which increases feeding, increased hypothalamic NPY and NPY mRNA levels. pCPA (10 mg/kg in 3 μl) was administered into the third ventricle either as a single injection (n = 8) or daily for 7 days (n = 8). Control rats received a similar injection of saline. pCPA significantly increased food intake compared with controls after both single and repeated injections (P < 0.05). NPY levels were measured by radioimmunoassay in microdissected hypothalamic extracts. NPY levels in the acutely treated group were significantly increased in the paraventricular nucleus (PVN; by 41%,P = 0.01), anterior hypothalamic area (AHA; by 34%,P < 0.01) and lateral hypothalamic area (LHA; by 41%,P < 0.02). In the 7-day-treated group, NPY levels were also increased in the same areas, i.e. PVN (by 24%,P < 0.01), AHA (by 30%,P < 0.01) and LHA (by 38%,P = 0.01). There were no significant changes in the ARC or any other region or in hypothalamic NPY mRNA levels. pCPA administration increased NPY levels in several regions notably the PVN. This is a major site of NPY release, where NPY injection induces feeding. We suggest that the hyperphagia induced by pCPA is mediated by increased NPY levels and secretion in the PVN. This is further evidence for interactions between NPY and 5-HT in the control of energy homeostasis.     

The dromedary camel displays annual variation in hypothalamic kisspeptin and Arg–Phe-amide-related peptide-3 according to sex,season, and breeding activity

The dromedary camel (Camelus dromedarius) is a desert mammal whose cycles in reproductive activity ensure that the offspring's birth and weaning coincide with periods of abundant food resources and favorable climate conditions. In this study, we assessed whether kisspeptin (Kp) and arginine–phenylalanine (RF)-amide related peptide-3 (RFRP-3), two hypothalamic peptides known to regulate the mammalian hypothalamo-pituitary gonadal axis, may be involved in the seasonal control of camel's reproduction. Using specific antibodies and riboprobes, we found that Kp neurons are present in the preoptic area (POA), suprachiasmatic (SCN), and arcuate (ARC) nuclei, and that RFRP-3 neurons are present in the paraventricular (PVN), dorsomedial (DMH), and ventromedial (VMH) hypothalamic nuclei. Kp fibers are found in various hypothalamic areas, notably the POA, SCN, PVN, DMH, VMH, supraoptic nucleus, and the ventral and dorsal premammillary nucleus. RFRP-3 fibers are found in the POA, SCN, PVN, DMH, VMH, and ARC. POA and ARC Kp neurons and DMH RFRP-3 neurons display sexual dimorphism with more neurons in female than in male. Both neuronal populations display opposed seasonal variations with more Kp neurons and less RFRP-3 neurons during the breeding (December–January) than the nonbreeding (July–August) season. This study is the first describing Kp and RFRP-3 in the camel's brain with, during the winter period lower RFRP-3 expression and higher Kp expression possibly responsible for the HPG axis activation. Altogether, our data indicate the involvement of both Kp and RFRP-3 in the seasonal control of the dromedary camel's breeding activity.     

Hypothalamic and brainstem sources of pituitary adenylate cyclase-activating polypeptide nerve fibers innervating the hypothalamic paraventricular nucleus in the rat

The hypothalamic paraventricular nucleus (PVN) coordinates major neuroendocrine and behavioral mechanisms, particularly responses to homeostatic challenges. Parvocellular and magnocellular PVN neurons are richly innervated by pituitary adenylate cyclase-activating polypeptide (PACAP) axons. Our recent functional observations have also suggested that PACAP may be an excitatory neuropeptide at the level of the PVN. Nevertheless, the exact localization of PACAP-producing neurons that project to the PVN is not understood. The present study examined the specific contribution of various brain areas sending PACAP innervation to the rat PVN by using iontophoretic microinjections of the retrograde neuroanatomical tracer cholera toxin B subunit (CTb). Retrograde transport was evaluated from hypothalamic and brainstem sections by using multiple labeling immunofluorescence for CTb and PACAP. PACAP-containing cell groups were found to be retrogradely labeled from the PVN in the median preoptic nucleus; preoptic and lateral hypothalamic areas; arcuate, dorsomedial, ventromedial, and supramammillary nuclei; ventrolateral midbrain periaqueductal gray; rostral and midlevel ventrolateral medulla, including the C1 catecholamine cell group; nucleus of the solitary tract; and dorsal motor nucleus of vagus. Minor PACAP projections with scattered double-labeled neurons originated from the parabrachial nucleus, pericoeruleus area, and caudal regions of the nucleus of the solitary tract and ventrolateral medulla. These observations indicate a multisite origin of PACAP innervation to the PVN and provide a strong chemical neuroanatomical foundation for interaction between PACAP and its potential target neurons in the PVN, such as parvocellular CRH neurons, controlling physiologic responses to stressful challenges and other neuroendocrine or preautonomic PVN neurons.     

Suckling-Induced Fos-Immunoreactivity in Subgroups of Hypothalamic POMC Neurons of the Lactating Rat: Investigation of a Role for Prolactin

Attention has recently been focused on lactation-induced modifications of activity of neuronal populations in the arcuate nucleus (ARC) of the mediobasal hypothalamus.The ARC hosts the tubero-infundibular dopaminergic (TIDA system) responsible for the neuroendocrine control of prolactin (PRL), and other non-neuroendocrine neuronal populations, such as neuropeptide Y (NPY)- and proopiomelanocortin (POMC)-containing systems that are important modulators of hypothalamic gonadoliberin (GnRH) secretion. Our longstanding interest in the functional anatomy of the ARC led us to investigate whether the suckling stimulus would trigger an expression of Fos-ir in specific arcuate neuronal populations and to possibly characterize responsive neurons by using double-labeling immunohistochemistry. Freely nursing lactating females expressed strong Fos-ir in neurons of the ARC compared to diestrous females. Fos-ir was encountered in neurons not belonging to the TIDA system and that was for a large proportion identical to the POMCergic neurons.We showed that, in lactating females submitted to suppression of the suckling stimulus by removal of the pups, the pattern of expression of Fos-ir is similar to that seen in diestrous females and that, a pattern of expression of Fos-ir indistinguishable from that observed during free lactation is reinstated a short time after the return of the pups and restoration of the suckling stimulus, suggesting that this expression of Fos-ir strictly depends upon the presence of the newborns and the suckling stimulus. By lowering circulating levels of the PRL with bromocryptine-or PRL antiserum-treatment, we noticed a decrease in the number of (β-endorphin+Fos)-ir neurons compared to non-injected freely nursing lactating females. By maintaining high levels of circulating PRL with haloperidol-treatment, we observed a number of colocalizations close to that observed in freely nursing lactating females. Our results suggest that during lactation a rostral subgroup of the arcuate POMCergic neuronal population is activated at least partially in response to the suckling-induced secretion of PRL and that this activation participates in maintaining the endocrine and/or metabolic demands of the lactational status.     

Plasticity in Expression of Immunoreactivity for Neuropeptide Y, Enkephalins and Neurotensin in the Hypothalamic Tubero-lnfundibular Dopaminergic System during Lactation in Mice

In lactating nursing vs lactating pup-deprived mice, single or multiple immunolabeling was performed to compare immunoreactivities (ir) for neuropeptide Y (NPY), enkephalins (ENK) and neurotensin (NT) in the tyrosine hydroxylase (TH)-ir hypothalamic tubero-infundibular dopaminergic (TIDA) system. NPY-, ENK- and NT-irs were intensely expressed and coexisted in virtually all TH-ir endings in the median eminence (ME) of nursing mice. Removal of the pups induced a marked depletion of the peptide-irs from the ME TH-ir endings. In the arcuate nucleus (ARC) of colchicine-treated nursing mice which received peripheral injections of Fluoro-Gold (FG) to retrogradely label neuroendocrine cells, virtually all dorsal A12 TH-ir perikarya simultaneously expressed, with individual variations, NPY-, ENK- and NT-irs, and all contained FG. These results suggest that the synthesis of NPY, ENK and NT is enhanced in TIDA neurons during lactation and that these neuromessengers may be co-released together with DA from the ME to regulate the suckling-induced prolactin secretion at the hypothalamic and/or pituitary levels.     

alpha-Melanocyte-stimulating hormone is contained in nerve terminals innervating thyrotropin-releasing hormone-synthesizing neurons in the hypothalamic paraventricular nucleus and prevents fasting-induced suppression of prothyrotropin-releasing hormone gene expression.

The hypothalamic arcuate nucleus has an essential role in mediating the homeostatic responses of the thyroid axis to fasting by altering the sensitivity of prothyrotropin-releasing hormone (pro-TRH) gene expression in the paraventricular nucleus (PVN) to feedback regulation by thyroid hormone. Because agouti-related protein (AGRP), a leptin-regulated, arcuate nucleus-derived peptide with alpha-MSH antagonist activity, is contained in axon terminals that terminate on TRH neurons in the PVN, we raised the possibility that alpha-MSH may also participate in the mechanism by which leptin influences pro-TRH gene expression. By double-labeling immunocytochemistry, alpha-MSH-IR axon varicosities were juxtaposed to approximately 70% of pro-TRH neurons in the anterior and periventricular parvocellular subdivisions of the PVN and to 34% of pro-TRH neurons in the medial parvocellular subdivision, establishing synaptic contacts both on the cell soma and dendrites. All pro-TRH neurons receiving contacts by alpha-MSH-containing fibers also were innervated by axons containing AGRP. The intracerebroventricular infusion of 300 ng of alpha-MSH every 6 hr for 3 d prevented fasting-induced suppression of pro-TRH in the PVN but had no effect on AGRP mRNA in the arcuate nucleus. alpha-MSH also increased circulating levels of free thyroxine (T4) 2.5-fold over the levels in fasted controls, but free T4 did not reach the levels in fed controls. These data suggest that alpha-MSH has an important role in the activation of pro-TRH gene expression in hypophysiotropic neurons via either a mono- and/or multisynaptic pathway to the PVN, but factors in addition to alpha-MSH also contribute to the mechanism by which leptin administration restores thyroid hormone levels to normal in fasted animals.     

GABA-ergic innervation of thyrotropin-releasing hormone-synthesizing neurons in the hypothalamic paraventricular nucleus of the rat

To determine whether GABA-ergic axons are anatomically situated to directly influence TRH neurons in the PVN, double-labeling light- and electronmicroscopic immunocytochemistry was performed using antisera against glutamic acid decarboxylase (GAD) and prothyrotropin-releasing hormone (proTRH). In the anterior, periventricular and medial parvocellular subdivisions of the PVN, GAD-immunoreactive (IR) axon varicosities were closely apposed to all proTRH containing cell bodies and proximal dendrites. Ultrastucturally, GAD-IR nerve terminals established symmetric type synapses with both perikarya and dendrites of proTRH-IR neurons, indicating the inhibitory nature of the contacts. Since a subpopulation of neuropeptide Y (NPY) neurons in the hypothalamic arcuate nucleus co-synthesize GABA, and NPY-containing neurons of arcuate nucleus origin densely innervate TRH neurons in the PVN, we performed triple labeling immunocytochemistry to elucidate the origin of the GAD-IR innervation of hypophysiotropic TRH neurons. While axons co-containing GAD and NPY were observed throughout the PVN, only approximately 10% of GAD-IR terminals in contact with TRH neurons were found to contain NPY-immunoreactivity. We conclude that GABA-ergic neurons are in position to act directly on hypophysiotropic TRH neurons and while this innervation arises partly from neurons in the arcuate nucleus that co-synthesize NPY, the majority of the GABA-ergic input arises from other neuronal groups.     

Activity of Hypothalamic Dopaminergic Neurones During the Day of Oestrus: Involvement in Prolactin Secretion

A secretory surge of prolactin occurs on the afternoon of oestrus in cycling rats. Pituitary prolactin is inhibited by dopamine. We evaluated the activity of the neuroendocrine dopaminergic neurones during oestrus and dioestrus, as determined by dopaminergic activity in the median eminence and neurointermediate lobe of the pituitary, as well as Fos‐related antigen expression in tyrosine hydroxylase (TH)‐immunoreactive (ir) neurones of the arcuate nucleus (ARC) and periventricular nucleus (Pe). During oestrus, the 4‐dihydroxyphenylacetic acid/dopamine ratio in the median eminence decreased at 16.00 h, coinciding with the increase in plasma prolactin levels. Similarly, the expression of Fos‐related antigen in TH‐ir neurones of Pe and rostral‐, dorsomedial‐ and caudal‐ARC also decreased at 16.00 h. On dioestrus, 4‐dihydroxyphenylacetic acid/dopamine ratio in the median eminence and Fos‐related antigen expression in TH‐ir neurones of Pe and rostral‐ARC decreased at 18.00 h, whereas prolactin levels were unaltered. No variation in dopaminergic activity was found in the neurointermediate lobe of the pituitary on either oestrus or dioestrus. The number of TH‐ir neurones in the ARC and parameters of dopaminergic activity were found to be generally lower on oestrus compared to dioestrus. The transitory decrease in the activity of neuroendocrine dopaminergic neurones temporally associated with the prolactin surge on the afternoon of oestrus suggests a role for dopamine in the generation of the oestrous prolactin surge.     

Effects of various neuroactive substances on single-unit activities of hypothalamic arcuate neurons in brain slices

Extracellular single-unit activities of 288 dorsomedial/ventrolateral hypothalamic arcuate (ARC) neurons were studied electrophysiologically in brain slices in vitro. We tested the effects of several neuroactive substances plus some of their analogs in this study. Among them, baclofen, a GABA_b-receptor agonist, inhibited 95.6% of ARC neurons tested, although GABA itself was much less effective (23.8%). About half of baclofen's effect was blocked by phaclofen, a GABA_b antagonist. Serotonin and dopamine also exhibited mostly inhibitory effects on responsive ARC neurons (80 and 78.4%, respectively), although more than half ARC neurons tested (53.3% of 169) were not responsive to dopamine. Neither ketanserin, a 5-HT₂, nor domperidone, a D₂ receptor antagonist, had much effect on blocking the actions of 5-HT or DA. Histamine and carbachol excited 67.4% and 52.2% of ARC neurons tested, respectively. Moreover, their effects were completely blocked by pyrilamine, a H₁ and atropine, a muscarinic receptor antagonist, respectively. Ranitidine, a H₂ receptor antagonist, however, was less effective. Norepinephrine had about equal number of excitation (33.3%) and inhibition (38.5%) on ARC neurons. Cholecystokinin-octapeptide sulphate (CCK-8S), a neuropeptide tested exerted potent excitatory effects on ARC neurons (62.8% of 137). In summary, ARC neurons in a more localized region aiming at the tuberoinfundibular dopaminergic neurons were selected in this study. Their responses to many agents and the implications on the regulation of prolactin secretion were discussed.