Liver-specific decrease in Tff3 gene expression in infant mice perinatally exposed to 2,3,7,8-tetrabromodibenzofuran or 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Polybrominated dibenzo-p-dioxins and dibenzofurans (PBDD/DFs) are byproducts of brominated flame retardants and can cause adverse health effects. Although exposure to polychlorinated (PC) DD/DFs induces toxic effects, including liver injury and neurobehavioral disorder, little is known about toxicities associated with PBDD/DF exposure. Thus, we examined effects of perinatal exposure to brominated congener on the infant mouse. Gene expression in several organs, such as the liver and brain, was analyzed in mouse offspring born to dams administered 2,3,7,8-tetrabromodibenzofuran (TBDF; 9 or 45 µg/kg body weight) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 3 µg/kg body weight) on gestational day 12.5. An increase in liver size was observed in TBDF- or TCDD-exposed offspring in infancy. Gene microarray analysis revealed that 163 and 36 genes were markedly upregulated and downregulated, respectively, in the liver of TBDF-exposed mice compared with those in vehicle-treated mice on postnatal day (PND) 5. Significant increases in Cyp1a1, Cyp1a2, Fmo3, and Pnliprp1 and decreases in Tff3, Ocstamp, Kcnk16, and Lgals2 mRNA levels in TBDF-exposed offspring on PNDs 5 and 12 were confirmed by quantitative PCR. In particular, a significant reduction in Tff3 mRNA in the liver, but not in the brain, small intestine, colon, and kidney, was observed in offspring perinatally exposed to TBDF or TCDD. Ultrasonic calls of TBDF- or TCDD-exposed offspring on PNDs 3-5 were impaired. Taken together, perinatal exposure to polyhalogenated dioxin/furan congeners disrupts gene expression patterns in the liver and ultrasonic calling during infancy. These results suggest that liver injury may contribute to neurobehavioral disorder.

Neurons expressing the aryl hydrocarbon receptor in the locus coeruleus and island of Calleja major are novel targets of dioxin in the mouse brain.

The aryl hydrocarbon receptor (AhR) acts as a receptor that responds to ligands, including dioxin. The AhR-ligand complex translocates from the cytoplasm into the nucleus to induce gene expression. Because dioxin exposure impairs cognitive and neurobehavioral functions, AhR-expressing neurons need to be identified for elucidation of the dioxin neurotoxicity mechanism. Immunohistochemistry was performed to detect AhR-expressing neurons in the mouse brain and confirm the specificity of the anti-AhR antibody using Ahr-/- mice. Intracellular distribution of AhR and expression level of AhR-target genes, Cyp1a1, Cyp1b1, and Ahr repressor (Ahrr), were analyzed by immunohistochemistry and quantitative RT-PCR, respectively, using mice exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The mouse brains were shown to harbor AhR in neurons of the locus coeruleus (LC) and island of Calleja major (ICjM) during developmental period in Ahr+/+ mice but not in Ahr-/- mice. A significant increase in nuclear AhR of ICjM neurons but not LC neurons was found in 14-day-old mice compared to 5- and 7-day-old mice. AhR was significantly translocated into the nucleus in LC and ICjM neurons of TCDD-exposed adult mice. Additionally, the expression levels of Cyp1a1, Cyp1b1, and Ahrr genes in the brain, a surrogate of TCDD in the tissue, were significantly increased by dioxin exposure, suggesting that dioxin-activated AhR induces gene expression in LC and ICjM neurons. This histochemical study shows the ligand-induced nuclear translocation of AhR at the single-neuron level in vivo. Thus, the neurotoxicological significance of the dioxin-activated AhR in the LC and ICjM warrants further studies.

Strain differences in intermale aggression and possible factors regulating increased aggression in Japanese quail.

The National Institute for Environmental Studies (NIES) of Japan established a strain of Japanese quail (Coturnix japonica) known as NIES-L by rotation breeding in a closed colony for over 35years; accordingly, the strain has highly inbred-like characteristics. Another strain called NIES-Brn has been maintained by randomized breeding in a closed colony to produce outbred-like characteristics. The current study aimed to characterize intermale aggressive behaviors in both strains and to identify possible factors regulating higher aggression in the hypothalamus, such as sex hormone and neuropeptide expression. Both strains displayed a common set of intermale aggressive behaviors that included pecking, grabbing, mounting, and cloacal contact behavior, although NIES-Brn quail showed significantly more grabbing, mounting, and cloacal contact behavior than did NIES-L quail. We examined sex hormone levels in the blood and diencephalon in both strains. Testosterone concentrations were significantly higher in the blood and diencephalon of NIES-Brn quail compared to NIES-L quail. We next examined gene expression in the hypothalamus of both strains using an Agilent gene expression microarray and real-time RT-PCR and found that gene expression of mesotocin (an oxytocin homologue) was significantly higher in the hypothalamus of NIES-Brn quail compared to NIES-L quail. Immunohistochemistry of the hypothalamus revealed that numbers of large cells (cell area>500µm2) expressing mesotocin were significantly higher in the NIES-Brn strain compared to the NIES-L strain. Taken together, our findings suggest that higher testosterone and mesotocin levels in the hypothalamus may be responsible for higher aggression in the NIES-Brn quail strain.

Analyzing the effects of co-expression of chick (Gallus gallus) melanocortin receptors with either chick MRAP1 or MRAP2 in CHO cells on sensitivity to ACTH(1-24) or ACTH(1-13)NH2: Implications for the avian HPA axis and avian melanocortin circuits in the hypothalamus.

In order to better understand the roles that melanocortin receptors (cMCRs) and melanocortin-2 receptor accessory proteins (cMRAP1 and cMRAP2) play in the HPA axis and hypothalamus, adrenal gland and hypothalamus mRNA from 1day-old white leghorn chicks (Gallus gallus), were analyzed by real-time PCR. mRNA was also made for kidney, ovary, and liver. Mrap1 mRNA could be detected in adrenal tissue, but not in any of the other tissues, and mrap2 mRNA was also detected in the adrenal gland. Finally, all five melanocortin receptors mRNAs could be detected in the adrenal gland; mc2r and mc5r mRNAs were the most abundant. To evaluate any potential interactions between MRAP1 and the MCRs that may occur in adrenal cells, individual chick mcr cDNA constructs were transiently expressed in CHO cells either in the presence or absence of a chick mrap1 cDNA, and the transfected cells were stimulated with hACTH(1-24) at concentrations ranging from 10-13M to 10-6M. As expected, MC2R required co-expression with MRAP1 for functional expression; whereas, co-expression of cMC3R with cMRAP1 had no statistically significant effect on sensitivity to hACTH(1-24). However, co-expression of MC4R and MC5R with MRAP1, increased sensitivity for ACTH(1-24) by approximately 35 fold and 365 fold, respectively. However, co-expressing of cMRAP2 with these melanocortin receptors had no effect on sensitivity to hACTH(1-24). Since the real-time PCR analysis detected mrap2 mRNA and mc4r mRNA in the hypothalamus, the interaction between cMC4R and cMRAP2 with respect to sensitivity to ACTH(1-13)NH2 stimulation was also evaluated. However, no effect, either positive or negative, was observed. Finally, the highest levels of mc5r mRNA were detected in liver cells. This observation raises the possibility that in one-day old chicks, activation of the HPA axis may also involve a physiological response from liver cells.

Role of Environmental Chemical Insult in Neuronal Cell Death and Cytoskeleton Damage.

Environmental influences, such as chemical exposure, have long been considered potential risk factors for neurodegenerative disorders, including neuromuscular diseases. However, no definitive links between environmental chemical exposure and a pathogenic mechanism of neurodegenerative disease has yet been established. In this study, we describe that exposure to arsenic, an environmental pollutant naturally found in drinking water, induces neuronal cell death and alteration of morphology, particularly neurite outgrowth and in the cytoskeleton of neurons. Since progressive cell loss accompanied by the alteration of neuronal structures and cytoskeleton is considered the major pathologic feature of neurodegenerative disorders, arsenic-induced neurotoxicity might contribute to an etiologic mechanism of some neurodegenerative diseases. Further, we discuss the importance of in vitro assay, particularly an embryonic toxicity test, for assessing the neurotoxicity of chemicals, because most of chemicals found in our environment remain to be evaluated regarding their neurotoxicity risk for neurodegenerative diseases.

Neural basis of imprinting behavior in chicks.

Newly hatched chicks memorize the characteristics of the first moving object they encounter, and subsequently show a preference for it. This "imprinting" behavior is an example of infant learning and is elicited by visual and/or auditory cues. Visual information of imprinting stimuli in chicks is first processed in the visual Wulst (VW), a telencephalic area corresponding to the mammalian visual cortex, congregates in the core region of the hyperpallium densocellulare (HDCo) cells, and transmitted to the intermediate medial mesopallium (IMM), a region similar to the mammalian association cortex. The imprinting memory is stored in the IMM, and activities of IMM neurons are altered by imprinting. Imprinting also induces functional and structural plastic changes of neurons in the circuit that links the VW and the IMM. Of these neurons, the activity of the HDCo cells is strongly influenced by imprinting. Expression and modulation of NR2B subunit-containing N-methyl-D-aspartate (NMDA) receptors in the HDCo cells are crucial for plastic changes in this circuit as well as the process of visual imprinting. Thus, elucidation of cellular and molecular mechanisms underlying the plastic changes that occurred in the HDCo cells may provide useful knowledge about infant learning.

2-Chloro-3,7,8-tribromodibenzofuran as a new environmental pollutant inducing atypical ultrasonic vocalization in infant mice.

Epidemiological and experimental studies indicate that maternal exposure to environmental pollutants impairs the cognitive and motor functions of offspring in humans and laboratory animals. Infant ultrasonic vocalizations (USVs), the communicative behavior of pups toward caregivers, are impaired in rodent models of neurodevelopmental disorders, suggesting a useful method to evaluate the developmental neurotoxicity of environmental pollutants. Therefore, we investigated USVs emitted by mouse pups of dams exposed to 2-chloro-3,7,8-tribromodibenzofuran (TeXDF) and 1,2,3,7,8-pentabromodibenzofuran (PeBDF), which are detected in the actual environment. The USV duration and number in the pups born to dams administered with TeXDF 40 µg/kg body weight (b.w.), but not 8 µg/kg b.w., on gestational day (GD) 12.5, were significantly lower than those in the corresponding pups on postnatal days 3-9. Conversely, there was no statistical change in the USVs emitted by the pups of dams administered with PeBDF 35 or 175 µg/kg b.w. on GD 12.5. To examine whether maternal exposure leads to behavioral impairments in adulthood, we analyzed exploratory behaviors in a novel environment using IntelliCage, a fully automated testing apparatus for group-housed mice. Neither TeXDF nor PeBDF exposure induced significant differences in offspring exploration. Considered together, our findings revealed that TeXDF induces atypical USV emission in infant mice, suggesting the importance of further studies on the risk assessment of mixed brominated/chlorinated dibenzo-p-dioxins and dibenzofurans.

Presence of sibling during the learning phase of imprinting affects escape behavior from a new object in chicks.

Imprinting is a crucial learning behavior by the hatchlings of precocious birds. In nature, hatchlings in a group environment imprint on a hen, but the effect of siblings on the imprinting process remains largely unknown. To investigate this issue, we examined how the social context modulated visual imprinting in domestic chicks. One-day-old postnatal chicks in isolation (RS chicks) or with siblings (RD chicks), were first exposed to an imprinting stimulus, and subsequently the responses to the imprinting stimulus as well as a new stimulus were examined and compared. The experiment constituted three types of siblings: a 20-min pre-trained tutor, a 60-min pre-trained tutor, and a naïve chick. A multiple comparison test revealed that the preference score (PS) to the new stimulus of RD chicks trained with a 60-min pre-trained tutor was significantly lower than that of RS chicks. Multiple linear regression analysis revealed that the length of the tutor's pre-training significantly correlated negatively with the PS to the new stimulus, but this variable did not correlate with the PS to the imprinting stimulus. These results revealed that the presence of highly imprinted siblings could enhance the escape response to the new stimulus. We discussed the possible involvement of the chick's medial amygdala in the social aspect of imprinting.

Possible molecular mechanism for acute encephalopathy by angel-wing mushroom ingestion - Involvement of three constituents in onset.

In Japan in 2004, 59 people who had consumed angel-wing mushroom, Pleurocybella porrigens, experienced acute encephalopathy, and of these 17 died. We purified a lethal protein to mice, pleurocybelline (PC), from P. porrigens. Although PC caused no damage to the brain, PC formed a complex with a lectin (PPL) and showed exo-protease activity, degrading substrates from both N- and C-termini. In addition, the presence of an unstable toxic compound, pleurocybellaziridine (PA), in the mushroom was demonstrated. We hypothesized that the complex and PA are involved in disease development and verified that apoptotic cells in the hippocampus were significantly increased by injection of the mixture of PC, PPL, and PA, indicating that these substances might be involved in acute encephalopathy.

Elevated expression of brain-derived neurotrophic factor facilitates visual imprinting in chicks.

With the aim of elucidating the neural mechanisms of early learning, we studied the role of brain-derived neurotrophic factor (BDNF) in visual imprinting in birds. The telencephalic neural circuit connecting the visual Wulst and intermediate medial mesopallium is critical for imprinting, and the core region of the hyperpallium densocellulare (HDCo), situated at the center of this circuit, has a key role in regulating the activity of the circuit. We found that the number of BDNF mRNA-positive cells in the HDCo was elevated during the critical period, particularly at its onset, on the day of hatching (P0). After imprinting training on P1, BDNF mRNA-positive cells in the HDCo increased in number, and tyrosine phosphorylation of TrkB was observed. BDNF infusion into the HDCo at P1 induced imprinting, even with a weak training protocol that does not normally induce imprinting. In contrast, K252a, an antagonist of Trk, inhibited imprinting. Injection of BDNF at P7, after the critical period, did not elicit imprinting. These results suggest that BDNF promotes the induction of imprinting through TrkB exclusively during the critical period.

Behavioral impairments in infant and adult mouse offspring exposed to 2,3,7,8-tetrabromodibenzofuran in utero and via lactation.

Polybrominated dibenzo-p-dioxins and dibenzofurans (PBDD/DFs) have been unintentionally produced and emitted from the lifecycle of products containing brominated flame retardants, such as polybrominated diphenyl ether, which is suspected to cause developmental neurotoxicity (DNT). Although it is plausible that PBDD/DFs can also induce DNT, information regarding their neurotoxic potential is currently limited. Hence, in the present study, we examined the effects of in utero and lactational exposure to brominated dibenzofurans on infant and adult offspring behavior to understand the mechanism of PBDD/DFs toxicity and detect effective behavioral endpoints in DNT assessment. We analyzed the behavior of mouse offspring born to dams administered 2,3,7,8-tetrabromodibenzofuran (2,3,7,8-TeBDF; dose of 0, 9, or 45 µg/kg) or 2,3,8-tribromodibenzofuran (2,3,8-TrBDF; dose of 0, 75.6, or 378 µg/kg) on gestational day 12.5. In mouse offspring born to dams exposed to 2,3,7,8-TeBDF, the exploratory behavior in a novel environment in adulthood and ultrasonic vocalization (USV) during infancy were significantly reduced. Additionally, AhR-target genes, such as Cyp1a1, were induced in the liver of 2,3,7,8-TeBDF-exposed offspring in a dose-dependent manner. Conversely, no significant changes in the infant and adult behaviors and expression level of AhR-target genes were observed in the 2,3,8-TrBDF-exposed offspring. These results suggest that 2,3,7,8-TeBDF can induce DNT and that the analysis of exploratory behavior in a novel environment and USV may be useful endpoints to assess DNT of dioxin-related substances.

Estrogenic action by tris(2,6-dimethylphenyl) phosphate impairs the development of female reproductive functions.

Developmental exposure to environmental chemicals with estrogen-like activity is suspected to permanently impair women's health. In this study, a mouse model was used to evaluate whether tris(2,6-dimethylphenyl) phosphate (TDMPP), a chemical with a putative estrogen-like action, impairs sexual differentiation of the brain. Either TDMPP and 17ß-estradiol (E2) as positive controls or sesame oil as a negative control were administered subcutaneously to dams from gestational day (GD) 14 to parturition, and to pups from postnatal day (PND) 0 to 9. Precocious puberty, irregular estrous cycles, and a lowered lordosis response were found in the TDMPP- and E2-treated groups. A certain amount of TDMPP and its metabolites in the perinatal brain and the masculinization of sexual dimorphic nuclei in the hypothalamus of female mice after treatment were also detected. The experimental evidence demonstrates that TDMPP directly enters the fetal and neonatal brain, thereby inducing changes of sex-related brain structures and impairing female reproductive functions.

Regulation of the intracellular distribution, cell surface expression, and protein levels of AMPA receptor GluR2 subunits by the monocarboxylate transporter MCT2 in neuronal cells.

The neuronal monocarboxylate transporter, MCT2, is not only an energy substrate carrier but it is also purported to be a binding partner for the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluR2 subunit. To unravel a putative role of MCT2 in the regulation of GluR2 subcellular distribution, Neuro2A cells and primary cultures of mouse cortical neurons were co-transfected with plasmids containing sequences to express the fluorescent proteins mStrawberry (mStb)-fused MCT2 and Venus-fused GluR2. Subsequently, their subcellular distribution was visualized by fluorescence microscopy. GluR2 was led to form perinuclear and dendritic clusters together with MCT2 when co-transfected in Neuro2A cells or in neurons, following the original distribution of MCT2. MCT2 co-transfection had no effect on the intracellular distribution of several other post-synaptic proteins, although it partially affected the intracellular distribution of GluR1 similarly to GluR2. Both cell surface and total protein expression levels of GluR2 were significantly reduced by co-expression with MCT2. Finally, partial perinuclear and dendritic co-localization between MCT2 and Rab8, a member of the small GTPase family involved in membrane trafficking of AMPA receptors, was also observed in co-transfected neurons. These results suggest that MCT2 could influence AMPA receptor trafficking within neurons by modulating GluR2 sorting between different subcellular compartments.

Basal and stimulated lactate fluxes in primary cultures of astrocytes are differentially controlled by distinct proteins.

Lactate release by astrocytes is postulated to be of importance for neuroenergetics but its regulation is poorly understood. Basigin, a chaperone protein for specific monocarboxylate transporters (MCTs), represents a putatively important regulatory element for lactate fluxes. Indeed, basigin knockdown by RNA interference in primary cultures of astrocytes partially reduced both proton-driven lactate influx and efflux. But more strikingly, enhancement of lactate efflux induced by glutamate was prevented while the effect of sodium azide was significantly reduced by treatment of cultured astrocytes with anti-basigin small interfering RNA. Enhancement of glucose utilization was unaffected under the same conditions. Basal lactate uptake and release were significantly reduced by MCT1 knockdown, even more so than with basigin knockdown, whereas glutamate-driven or sodium azide-induced enhancement of lactate release was not inhibited by either MCT1, 2, or 4 small interfering RNAs. In conclusion, MCT1 plays a pivotal role in the control of basal proton-driven lactate flux in astrocytes while basigin is only partly involved, most likely via its interaction with MCT1. In contrast, basigin appears to critically regulate the enhancement of lactate release caused by glutamate (or sodium azide) but via an effect on another unidentified transporter at least present in astrocytes in vitro.

Oleic acid glucose-independently stimulates glucagon secretion by increasing cytoplasmic Ca2+ via endoplasmic reticulum Ca2+ release and Ca2+ influx in the rat islet alpha-cells.

The effect of long-chain free fatty acids on glucagon secretion from islet alpha-cells has been a controversial issue. This study examined direct effects of oleic acid (OA) on glucagon release from rat pancreatic islets and on cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) in single alpha-cells by fura-2 fluorescence imaging. OA at 30 microM increased glucagon release from isolated islets in the presence of low (2.8 mM) and elevated (8.3 mM) glucose concentrations. OA at 6-10 microm concentration-dependently increased [Ca(2+)](i) in alpha-cells, irrespective of glucose concentrations (1.4, 2.8, and 8.3 mM). OA at 10 mum increased [Ca(2+)](i) in 90% of alpha-cells. OA-induced [Ca(2+)](i) increases were strongly inhibited by the endoplasmic reticulum Ca(2+)-pump inhibitors cyclopiazonic acid and thapsigargin and by 2-aminoethoxydiphenyl borate, the blocker of both inositol 1,4,5-trisphosphate receptors and store-operated Ca(2+) channels. Furthermore, the amplitude, but not incidence, of OA-induced [Ca(2+)](i) increases was reduced substantially by Ca(2+)-free conditions and mildly by an L-type Ca(2+) channel blocker, nitrendipine, and an ATP-sensitive K(+) channel activator, diazoxide. OA-induced glucagon release was also inhibited mildly by nitrendipine and strongly by 2-aminoethoxydiphenyl borate. These results indicate that OA glucose-independently stimulates glucagon release by increasing [Ca(2+)](i) in rat pancreatic alpha-cells and that the [Ca(2+)](i) increase is triggered by Ca(2+) release from endoplasmic reticulum and amplified by Ca(2+) influx possibly via store-operated channels and via voltage-dependent L-type Ca(2+) channels. The glucose-independent action of OA to stimulate glucagon release from alpha-cells may operate under hypoglycemic conditions when plasma free fatty acids levels are elevated, possibly playing a role in maintaining glucose metabolism.

Leptin suppresses ghrelin-induced activation of neuropeptide Y neurons in the arcuate nucleus via phosphatidylinositol 3-kinase- and phosphodiesterase 3-mediated pathway.

Neuropeptide Y (NPY) neurons in the hypothalamic arcuate nucleus (ARC) play a central role in stimulation of feeding. They sense and integrate peripheral and central signals, including ghrelin and leptin. However, the mechanisms of interaction of these hormones in NPY neurons are largely unknown. This study explored the interaction and underlying signaling cross talk between ghrelin and leptin in NPY neurons. Cytosolic Ca(2+) concentration ([Ca(2+)](i)) in single neurons isolated from ARC of adult rats was measured by fura-2 microfluorometry. Ghrelin increased [Ca(2+)](i) in 31% of ARC neurons. The [Ca(2+)](i) increases were inhibited by blockers of phospholipase C, adenylate cyclase, and protein kinase A. Ghrelin-induced [Ca(2+)](i) increases were suppressed by subsequent administration of leptin. Fifteen of 18 ghrelin-activated, leptin-suppressed neurons (83%) contained NPY. Leptin suppression of ghrelin responses was prevented by pretreatment with inhibitors of phosphatidylinositol 3-kinase and phosphodiesterase 3 (PDE3) but not MAPK. ATP-sensitive potassium channel inhibitors and activators did not prevent and mimic leptin suppression, respectively. Although leptin phosphorylated signal-transducer and activator of transcription 3 (STAT3) in NPY neurons, neither STAT3 inhibitor nor genetic STAT3 deletion altered leptin suppression of ghrelin responses. Furthermore, orexigenic effect of intracerebroventricular ghrelin in rats was counteracted by leptin in a PDE3-dependent manner. These findings indicate that ghrelin increases [Ca(2+)](i) via mechanisms depending on phospholipase C and adenylate cyclase-PKA pathways in ARC NPY neurons and that leptin counteracts ghrelin responses via a phosphatidylinositol 3-kinase-PDE3 pathway. This interaction may play an important role in regulating ARC NPY neuron activity and, thereby, feeding.

Imprinting modulates processing of visual information in the visual wulst of chicks.

BACKGROUND: Imprinting behavior is one form of learning and memory in precocial birds. With the aim of elucidating of the neural basis for visual imprinting, we focused on visual information processing. RESULTS: A lesion in the visual wulst, which is similar functionally to the mammalian visual cortex, caused anterograde amnesia in visual imprinting behavior. Since the color of an object was one of the important cues for imprinting, we investigated color information processing in the visual wulst. Intrinsic optical signals from the visual wulst were detected in the early posthatch period and the peak regions of responses to red, green, and blue were spatially organized from the caudal to the nasal regions in dark-reared chicks. This spatial representation of color recognition showed plastic changes, and the response pattern along the antero-posterior axis of the visual wulst altered according to the color the chick was imprinted to. CONCLUSION: These results indicate that the thalamofugal pathway is critical for learning the imprinting stimulus and that the visual wulst shows learning-related plasticity and may relay processed visual information to indicate the color of the imprint stimulus to the memory storage region, e.g., the intermediate medial mesopallium.

Pituitary adenylate cyclase-activating polypeptide neurons of the ventromedial hypothalamus project to the midbrain central gray.

The pituitary adenylate cyclase-activating polypeptide (PACAP) and brain-derived neurotrophic factor (BDNF) neurons have recently been established as markers of the ventromedial hypothalamic nucleus (VMH). However, their neural projections from the VMH remained unknown. We examined whether PACAP and BDNF neurons in the VMH connected to the mesencephalic central gray (MCG), using the combination of in-situ hybridization and immunohistochemical tracing of Fluorogold (FG) injected into the MCG. Approximately 49% of PACAP neurons and 19% of BDNF neurons in the VMH contained the retrograde-transported FG, and 52% of FG-positive cells were PACAP neurons. These results indicate that a large number of PACAP neurons and a small number of BDNF neurons of the VMH project to the MCG.