Efferent projections of Vglut2, Foxp2, and Pdyn parabrachial neurons in mice.

The parabrachial nucleus (PB) is a complex structure located at the junction of the midbrain and hindbrain. Its neurons have diverse genetic profiles and influence a variety of homeostatic functions. While its cytoarchitecture and overall efferent projections are known, we lack comprehensive information on the projection patterns of specific neuronal subtypes in the PB. In this study, we compared the projection patterns of glutamatergic neurons here with a subpopulation expressing the transcription factor Foxp2 and a further subpopulation expressing the neuropeptide Pdyn. To do this, we injected an AAV into the PB region to deliver a Cre-dependent anterograde tracer (synaptophysin-mCherry) in three different strains of Cre-driver mice. We then analyzed 147 neuroanatomical regions for labeled boutons in every brain (n = 11). Overall, glutamatergic neurons in the PB region project to a wide variety of sites in the cerebral cortex, basal forebrain, bed nucleus of the stria terminalis, amygdala, diencephalon, and brainstem. Foxp2 and Pdyn subpopulations project heavily to the hypothalamus, but not to the cortex, basal forebrain, or amygdala. Among the few differences between Foxp2 and Pdyn cases was a notable lack of Pdyn projections to the ventromedial hypothalamic nucleus. Our results indicate that genetic identity determines connectivity (and therefore, function), providing a framework for mapping all PB output projections based on the genetic identity of its neurons. Using genetic markers to systematically classify PB neurons and their efferent projections will enhance the translation of research findings from experimental animals to humans.

Neuropathic Pain Dysregulates Gene Expression of the Forebrain Opioid and Dopamine Systems.

Disturbances in the function of the mesostriatal dopamine system may contribute to the development and maintenance of chronic pain, including its sensory and emotional/cognitive aspects. In the present study, we assessed the influence of chronic constriction injury (CCI) of the sciatic nerve on the expression of genes coding for dopamine and opioid receptors as well as opioid propeptides in the mouse mesostriatal system, particularly in the nucleus accumbens. We demonstrated bilateral increases in mRNA levels of the dopamine D1 and D2 receptors (the latter accompanied by elevated protein level), opioid propeptides proenkephalin and prodynorphin, as well as delta and kappa (but not mu) opioid receptors in the nucleus accumbens at 7 to 14 days after CCI. These results show that CCI-induced neuropathic pain is accompanied by a major transcriptional dysregulation of molecules involved in dopaminergic and opioidergic signaling in the striatum/nucleus accumbens. Possible functional consequences of these changes include opposite effects of upregulated enkephalin/delta opioid receptor signaling vs. dynorphin/kappa opioid receptor signaling, with the former most likely having an analgesic effect and the latter exacerbating pain and contributing to pain-related negative emotional states.

Preproenkephalin-expressing ventral pallidal neurons control inhibitory avoidance learning.

The ventral pallidum (VP) is a critical component of the basal ganglia neurocircuitry regulating learning and decision making; however, its precise role in controlling associative learning of environmental stimuli conditioned to appetitive or aversive outcomes is still unclear. Here, we investigated the expression of preproenkephalin, a polypeptide hormone previously shown to be expressed in nucleus accumbens neurons controlling aversive learning, within GABAergic and glutamatergic VP neurons. Next, we explored the behavioral consequences of chemicogenetic inhibition or excitation of preproenkephalin-expressing VP neurons on associative learning of reward- or aversion-paired stimuli in autoshaping and inhibitory avoidance tasks, respectively. We reveal for the first time that preproenkephalin is expressed predominantly in GABAergic rather than glutamatergic VP neurons, and that excitation of these preproenkephalin-expressing VP neurons was sufficient to impair inhibitory avoidance learning. These findings indicate the necessity for inhibition of preproenkephalin-expressing VP neurons for avoidance learning, and suggest these neurons as a potential therapeutic target for psychiatric disorders associated with maladaptive aversive learning.

Neuronal Expression of Opioid Gene is Controlled by Dual Epigenetic and Transcriptional Mechanism in Human Brain.

Molecular mechanisms that define patterns of neuropeptide expression are essential for the formation and rewiring of neural circuits. The prodynorphin gene (PDYN) gives rise to dynorphin opioid peptides mediating depression and substance dependence. We here demonstrated that PDYN is expressed in neurons in human dorsolateral prefrontal cortex (dlPFC), and identified neuronal differentially methylated region in PDYN locus framed by CCCTC-binding factor binding sites. A short, nucleosome size human-specific promoter CpG island (CGI), a core of this region may serve as a regulatory module, which is hypomethylated in neurons, enriched in 5-hydroxymethylcytosine, and targeted by USF2, a methylation-sensitive E-box transcription factor (TF). USF2 activates PDYN transcription in model systems, and binds to nonmethylated CGI in dlPFC. USF2 and PDYN expression is correlated, and USF2 and PDYN proteins are co-localized in dlPFC. Segregation of activatory TF and repressive CGI methylation may ensure contrasting PDYN expression in neurons and glia in human brain.

Neocortical prodynorphin expression is transiently increased with learning: Implications for time- and learning-dependent neocortical kappa opioid receptor activation.

There are several lines of evidence that indicate a prominent role for the opioid system in the acquisition and consolidation of learned associations. Specifically, kappa opioid receptor (KOR) modulation has been demonstrated to alter various behavioral tasks including whisker trace eyeblink conditioning (WTEB). WTEB is an associative conditioning paradigm in which a neutral conditioned stimulus (CS; Whisker stimulation) is paired following a short stimulus free trace interval with a salient unconditioned stimulus that elicits a blink response (US; Eye shock). Work from our laboratory has shown that WTEB conditioning is dependent upon and induces plasticity in primary somatosensory cortex (S1), a likely site for memory storage. Our subsequent studies have shown that WTEB acquisition or consolidation are impaired when the initial or later phase of KOR activation in S1 is respectively blocked. Interestingly, this mechanism by which KOR is activated in S1 during learning remains unexplored. Dynorphin (DYN), KOR's endogenous ligand, is synthesized from the precursor prodynorphin (PD) that is synthesized from preprodynorphin (PPD). In S1, most PPD is found in inhibitory GABAergic somatostatin interneurons (SOM), suggesting that these SOM interneurons are upstream regulators of learning induced KOR activation. Using immunofluorescence to investigate the expression of PD and SOM, the current study found that PD/SOM expression was transiently increased in S1 during learning. Interestingly, these findings have direct implications towards a time- and learning-dependent role for KOR activation in neocortical mechanisms mediating learning.

Transcriptional repressor DREAM regulates trigeminal noxious perception.

Expression of the downstream regulatory element antagonist modulator (DREAM) protein in dorsal root ganglia and spinal cord is related to endogenous control mechanisms of acute and chronic pain. In primary sensory trigeminal neurons, high levels of endogenous DREAM protein are preferentially localized in the nucleus, suggesting a major transcriptional role. Here, we show that transgenic mice expressing a dominant active mutant of DREAM in trigeminal neurons show increased responses following orofacial sensory stimulation, which correlates with a decreased expression of prodynorphin and brain-derived neurotrophic factor in trigeminal ganglia. Genome-wide analysis of trigeminal neurons in daDREAM transgenic mice identified cathepsin L and the monoglyceride lipase as two new DREAM transcriptional targets related to pain. Our results suggest a role for DREAM in the regulation of trigeminal nociception. This article is part of the special article series "Pain".

Expression levels of OPRM1 and PDYN in human SH-SY5Y cells treated with morphine and methadone.

AIMS: The opioid receptor mu-1 (OPRM1, site of action for methadone and morphine, OMIM: 600018) and prodynorphin (PDYN, OMIM: 131340) genes are belonging to the endogenous opioid family. There is no data on alterations of mRNA levels of PDYN and OPRM1 in cells exposed to methadone. Therefore, the present study was carried out. MAIN METHODS: Here we have investigated the alterations of the expression levels of OPRM1 and PDYN genes in response to methadone (final concentrations 1, 2.5, 5, 7.5, 10 µM) and morphine (final concentrations 1, 5, 10 µM) in human SH-SY5Y cells (at 1h, 24h, 72 h, 18 days of exposure times). KEY FINDINGS: The most important findings are summaries as follow: 1) In the cells treated with morphine, the mRNA level of OPRM1 significantly decreased from 1h to 72 h in a dose dependent manner, but it is increased when the cells treated for 18 days by high concentrations of morphine; 2) Although the PDYN mRNA level is increased at 1 and 24h (for 5 and 10 µM morphine), it is decreased at 72 h and 18 days; 3) The mRNA level of OPRM1 negatively is associated with a methadone dose dependent and exposure time dependent manner; 4) In overall, the PDYN mRNA level is increased in the treated cells without any obvious trend by dose of methadone or exposure time. SIGNIFICANCE: Decreasing the PDYN mRNA levels in cells exposed to morphine for long period times, and increasing the level of PDYN mRNA in methadone treated cells, can interpret why heroine-dependent persons, easily accept methadone therapy.

Opioid-dependent regulation of high and low fear responses in two inbred mouse strains.

The molecular mechanisms underlying the susceptibility or resilience to trauma-related disorders remain incompletely understood. Opioids modulate emotional learning, but the roles of specific receptors are unclear. Here, we aimed to analyze the contribution of the opioid system to fear responses in two inbred mouse strains exhibiting distinct behavioral phenotypes. SWR/J and C57BL/6J mice were subjected to five consecutive electric footshocks (1mA each), and the contextual freezing time was measured. Stress-induced alterations in gene expression were analyzed in the amygdala and the hippocampus. In both strains, the fear response was modulated using pharmacological tools. SWR/J mice did not develop conditioned fear but exhibited increased transcriptional expression of Pdyn and Penk in the amygdala region. Blocking opioid receptors prior to the footshocks using naltrexone (2 mg/kg) or naltrindole (5 mg/kg) increased the freezing responses in these animals. The C57BL/6J strain displayed high conditioned fear, although no alteration in the mRNA abundance of genes encoding opioid precursors was observed. Double-injection of morphine (20 mg/kg) following stress and upon context re-exposure prevented the enhancement of freezing. Moreover, selective delta and kappa agonists caused a reduction in conditioned fear responses. To summarize, the increased expression of the Pdyn and Penk genes corresponded to reduced intensity of fear responses. Blockade of the endogenous opioid system restored freezing behavior in stress-resistant animals. The pharmacological stimulation of the kappa and delta opioid receptors in stress-susceptible individuals may alleviate fear. Thus, subtype-selective opioid receptor agonists may protect against the development of trauma-related disorders.

Constant light suppresses production of Met-enkephalin-containing peptides in cultured splenic macrophages and impairs primary immune response in rats.

The light-dark cycle is an environmental factor that influences immune physiology, and so, variations of the photoperiod length result in altered immune responsivity. Macrophage physiology comprises a spectrum of functions that goes from host defense to immune down-regulation, in addition to their homeostatic activities. Macrophages also play a key role in the transition from innate to adaptive immune responses. Met-enkephalin (MEnk) has been recognized as a modulator of macrophage physiology acting in an autocrine or paracrine fashion to influence macrophage activation, phenotype polarization and production of cytokines that would enhance lymphocyte activation at early stages of an immune response. Previously it was shown that splenic MEnk tissue content is reduced in rats exposed to constant light. In this work, we explored whether production of Met-enkephalin-containing peptides (MECPs) in cultured splenic macrophages is affected by exposure of rats to a constant light regime. In addition, we explored whether primary immune response was impaired under this condition. We found that in rats, 15 days in constant light was sufficient to disrupt their general activity rhythm. Splenic MEnk content oscillations and levels were also blunted throughout a 24-h period in animals subjected to constant light. In agreement, de novo synthesis of MECPs evaluated through incorporation of (35)S-methionine was reduced in splenic macrophages from rats exposed to constant light. Moreover, MECPs immunocytochemistry showed a decrease in the intracellular content and lack of granule-like deposits in this condition. Furthermore, we found that primary T-dependent antibody response was compromised in rats exposed to constant light. In those animals, pharmacologic treatment with MEnk increased IFN-γ-secreting cells. Also, IL-2 secretion from antigen-stimulated splenocytes was reduced after incubation with naloxone, suggesting that immune-derived opioid peptides and stimulation of opioid receptors are involved in this process. Thus, the immune impairment observed from early stages of the response in constant light-subjected rats, could be associated with reduced production of macrophage-derived enkephalins, leading to a sub-optimal interaction between macrophages and lymphocytes in the spleen and the subsequent deficiency in antibody production.

[Construction of recombinant over expression vector of PENK gene and the function study of PENK gene].

OBJECTIVE: To construct recombinant over expression vector of Homo sapiens proenkephalin (PENK) gene and explore the function of PENK gene. METHODS: Fragment containing PENK ORF gene was inserted into vector plasmid HIV, then the recombinant over was confirmed by enzyme digestion and sequencing. Lentivirus containing the recombinant over expression vector was produced by virus packaging with 293Ta cell,and then the lentivirus was transfected into HT1080 cell and the virus titer was estimated. The PC12 were tansfected with resulting lentivirus and un-transfected PC12 cells as control. The images of the PC12 cells were captured at 48 h post-transfection and the number of cells was also evaluated; the changes of PENK mRNA in transfection and control group were measured with RT-PCR. RESULTS: The constructed PENK ORF recombinant over expression vector was confirmed by enzyme digestion and sequencing. The number of PC12 cells in transfection and control group at 48 h post-transfection was 127.93 +/- 2.48 and 88. 60 +/- 2.55 respectively, and the statistical difference between them was observed (P < 0.01). CONCLUSION: Recombinant over expression vector of PENK gene was successfully constructed and the PENK gene can promote the growth of PC12.

Dynorphin/KOP and nociceptin/NOP gene expression and epigenetic changes by cocaine in rat striatum and nucleus accumbens.

Cocaine induces neurochemical changes of endogenous prodynorphin-kappa opioid receptor (pDYN-KOP) and pronociceptin/orphaninFQ-nociceptin receptor (pN/OFQ-NOP) systems. Both systems play an important role in rewarding mechanisms and addictive stimulus processing by modulating drug-induced dopaminergic activation in the mesocortico-limbic brain areas. They are also involved in regulating stress mechanisms related to addiction. The aim of this study was to investigate possible changes of gene expression of the dynorphinergic and nociceptinergic system components in the nucleus accumbens (NA) and in medial and lateral caudate putamen (mCPu and lCPu, respectively) of rats, following chronic subcutaneous infusion of cocaine. In addition, the epigenetic histone modifications H3K4me3 and H3K27me3 (an activating and a repressive marker, respectively) at the promoter level of the pDYN, KOP, pN/OFQ and NOP genes were investigated. Results showed that cocaine induced pDYN gene expression up-regulation in the NA and lCPu, and its down-regulation in the mCPu, whereas KOP mRNA levels were unchanged. Moreover, cocaine exposure decreased pN/OFQ gene expression in the NA and lCPu, while NOP mRNA levels appeared significantly increased in the NA and decreased in the lCPu. Specific changes of the H3K4me3 and H3K27me3 levels were found at pDYN, pN/OFQ, and NOP gene promoter, consistent with the observed gene expression alterations. The present findings contribute to better define the role of endogenous pDYN-KOP and pN/OFQ-NOP systems in neuroplasticity mechanisms following chronic cocaine treatment. The epigenetic histone modifications underlying the gene expression changes likely mediate the effects of cocaine on transcriptional regulation of specific gene promoters that result in long-lasting drug-induced plasticity.

Enkephalin downregulation in the nucleus accumbens underlies chronic stress-induced anhedonia.

Restraint and immobilization have been extensively used to study habituation of the neuroendocrine response to a repeated stressor, but behavioral consequences of this stress regimen remain largely uncharacterized. In this study, we used sucrose preference and the elevated-plus maze to probe behavioral alterations resulting from 14 days of restraint in rats. We observed a decrease in sucrose preference in stressed animals, particularly in a subgroup of individuals, but no alteration in anxiety behaviors (as measured in the elevated-plus maze) four days following the last restraint. In these low-sucrose preference animals, we observed a downregulation of the expression of preproenkephalin mRNA in the nucleus accumbens. Furthermore, we observed a strong correlation between enkephalin expression and sucrose preference in the shell part of the nucleus accumbens, with a lower level of enkephalin expression being associated with lower sucrose preference. Interestingly, quantification of the corticosterone response revealed a delayed habituation to restraint in the low-sucrose preference population, which suggests that vulnerability to stress-induced deficits might be associated with prolonged exposure to glucocorticoids. The induction of ΔFosB is also reduced in the nucleus accumbens shell of the low-sucrose preference population and this transcription factor is expressed in enkephalin neurons. Taken together, these results suggest that a ΔFosB-mediated downregulation of enkephalin in the nucleus accumbens might underlie the susceptibility to chronic stress. Further experiments will be needed to determine causality between these two phenomena.

Impaired periamygdaloid-cortex prodynorphin is characteristic of opiate addiction and depression.

Negative affect is critical for conferring vulnerability to opiate addiction as reflected by the high comorbidity of opiate abuse with major depressive disorder (MDD). Rodent models implicate amygdala prodynorphin (Pdyn) as a mediator of negative affect; however, evidence of PDYN involvement in human negative affect is limited. Here, we found reduced PDYN mRNA expression in the postmortem human amygdala nucleus of the periamygdaloid cortex (PAC) in both heroin abusers and MDD subjects. Similar to humans, rats that chronically self-administered heroin had reduced Pdyn mRNA expression in the PAC at a time point associated with a negative affective state. Using the in vivo functional imaging technology DREAMM (DREADD-assisted metabolic mapping, where DREADD indicates designer receptors exclusively activated by designer drugs), we found that selective inhibition of Pdyn-expressing neurons in the rat PAC increased metabolic activity in the extended amygdala, which is a key substrate of the extrahypothalamic brain stress system. In parallel, PAC-specific Pdyn inhibition provoked negative affect-related physiological and behavioral changes. Altogether, our translational study supports a functional role for impaired Pdyn in the PAC in opiate abuse through activation of the stress and negative affect neurocircuitry implicated in addiction vulnerability.

Early modulation by the dopamine D4 receptor of morphine-induced changes in the opioid peptide systems in the rat caudate putamen.

The peptides dynorphin and enkephalin modulate many physiological processes, such as motor activity and the control of mood and motivation. Their expression in the caudate putamen (CPu) is regulated by dopamine and opioid receptors. The current work was designed to explore the early effects of the acute activation of D4 and/or µ opioid receptors by the agonists PD168,077 and morphine, respectively, on the regulation of the expression of these opioid peptides in the rat CPu, on transcription factors linked to them, and on the expression of µ opioid receptors. In situ hybridization experiments showed that acute treatment with morphine (10 mg/kg) decreased both enkephalin and dynorphin mRNA levels in the CPu after 30 min, but PD168,077 (1 mg/kg) did not modify their expression. Coadministration of the two agonists demonstrated that PD168,077 counteracted the morphine-induced changes and even increased enkephalin mRNA levels. The immunohistochemistry studies showed that morphine administration also increased striatal µ opioid receptor immunoreactivity but reduced P-CREB expression, effects that were blocked by the PD168,077-induced activation of D4 receptors. The current results present evidence of functional D4 -µ opioid receptor interactions, with consequences for the opioid peptide mRNA levels in the rat CPu, contributing to the integration of DA and opioid peptide signaling.

Locomotor velocity and striatal adaptive gene expression changes of the direct and indirect pathways in Parkinsonian rats.

BACKGROUND: In Parkinson's disease (PD), bradykinesia, or slowness of movement, only appears after a large striatal dopamine depletion. Compensatory mechanisms probably play a role in this delayed appearance of symptoms. OBJECTIVE: Our hypothesis is that the striatal direct and indirect pathways participate in these compensatory mechanisms. METHODS: We used the unilateral 6-hydroxydopamine (6-OHDA) rat model of PD and control animals. Four weeks after the lesion, the spontaneous locomotor activity of the rats was measured and then the animals were killed and their brain extracted. We quantified the mRNA expression of markers of the striatal direct and indirect pathways as well as the nigral expression of dopamine transporter (DAT) and tyrosine hydroxylase (TH) mRNA. We also carried out an immunohistochemistry for the striatal TH protein expression. RESULTS: As expected, the unilateral 6-OHDA rats presented a tendency to an ipsilateral head turning and a low locomotor velocity. In 6-OHDA rats only, we observed a significant and positive correlation between locomotor velocity and both D1-class dopamine receptor (D1R) (direct pathway) and enkephalin (ENK) (indirect pathway) mRNA in the lesioned striatum, as well as between D1R and ENK mRNA. CONCLUSIONS: Our results demonstrate a strong relationship between both direct and indirect pathways and spontaneous locomotor activity in the parkinsonian rat model. We suggest a synergy between both pathways which could play a role in compensatory mechanisms and may contribute to the delayed appearance of bradykinesia in PD.

A single neonatal injury induces life-long deficits in response to stress.

Approximately 500,000 infants are born prematurely each year in the United States. These infants typically require an extensive stay in the neonatal intensive care unit (NICU), where they experience on average 14 painful and invasive procedures each day. These procedures, including repeated heel lance, insertion of intravenous lines, and respiratory and gastric suctioning, typically result in an inflammatory response, inducing pain and stress in the newborn. Remarkably, the majority of these procedures are performed in the complete absence of pre- or post-emptive analgesics. Recent clinical studies report that former NICU patients have increased thresholds for pain and stress later in life as compared with term-born infants. However, to date, the mechanisms whereby early-life inflammation alters later-life response to stress and pain are not known. The present studies were conducted to determine if neonatal injury impairs adult responses to anxiety- and stress-provoking stimuli. As we have previously reported that early-life pain results in a significant increase in opioid peptide expression within the midbrain periaqueductal gray, the role of endogenous opioids in our behavioral studies was also examined. Male and female rats received an intraplantar injection of the inflammatory agent carrageenan (1%) on the day of birth. In adulthood, animals were assessed for changes in response to anxiety- and stress-provoking stimuli using the open field and forced swim tests, respectively. Injury-induced changes in sucrose preference and stress-induced analgesia were also assessed. As adults, neonatally injured animals displayed a blunted response to both anxiety- and stress-provoking stimuli, as indicated by significantly more time spent in the inner area of the open field and a 2-fold increase in latency to immobility in the forced swim test as compared to controls. No change in sucrose preference was observed. Using in situ hybridization and immunohistochemistry, we observed a 2-fold increase in enkephalin mRNA and protein expression, respectively, in stress-related brain regions including the central amygdala and lateral septum. Administration of the opioid receptor antagonist naloxone reversed the attenuated responses to forced swim stress and stress-induced analgesia, suggesting the changes in stress-related behavior were opioid-dependent. Together, these data contribute to mounting evidence that neonatal injury in the absence of analgesics has adverse effects that are both long-term and polysystemic.

Region-specific diversity of striosomes in the mouse striatum revealed by the differential immunoreactivities for mu-opioid receptor, substance P, and enkephalin.

The complexity of the internal structure of the striatum is not completely understood, and the striosomes/matrix compartmentalization in particular has been one of the intriguing substructures of the striatum. Although various neurochemical markers have been used to visualize striosomes with sufficient clarity, it still remains obscure whether striosomes that are detectable by a single marker represent all of the striosomal compartments and to what extent the compartments are uniform across different intrastriatal positions. Triple immunohistochemical labeling for the three representative striosomes/matrix markers, µ-opioid receptor (MOR), substance P (SP), and enkephalin (Enk), was applied to serial sections covering the whole striatum of the mouse (n=8). The majority of MOR-positive striosomes were confined to the rostral quarter of the striatum. In contrast, SP-positive striosomes were distributed more broadly in the rostral two-thirds of the striatum. No striosomes were observable in the caudal third by the present method. In the rostral striatum, the majority of striosomes were labeled for both MOR and SP, but some at the most rostral positions were detectable only by MOR, while caudally located striosomes were identifiable only by SP. Thus MOR- and SP-immunoreactivities in striosomes exhibited contrasting patterns along the rostrocaudal axis. The Enk immunohistochemistry produced complicated profiles and was unsuitable for the detection of striosomes in mice. However, Enk immunoreactivity in MOR and/or SP-positive striosomes was higher in the ventral portion than in the dorsal portion in the rostral striatum. The present study revealed the region-specific diversity of striosomes, suggesting site-dependent differential regulation of striosomal neurons by MOR ligands and SP that are contained in indirect- and direct-pathway neurons, respectively. The results further suggest the necessity of viewing the striosomes as non-uniform compartments in addition to the traditional dichotomous view, which focuses on discrimination between the striosomes and the matrix.

Lmx1b controls peptide phenotypes in serotonergic and dopaminergic neurons.

Serotonin (5-HT) neurons synthesize a variety of peptides. How these peptides are controlled during development remains unclear. It has been reported that the co-localization of peptides and 5-HT varies by species. In contrast to the situations in the rostral 5-HT neurons of human and rat brains, several peptides do not coexist with 5-HT in the rostral 5-HT neurons of mouse brain. In this study, we found that the peptide substance P and peptide genes, including those encoding peptides thyrotropin-releasing hormone, enkephalin, and calcitonin gene-related peptide, were expressed in the caudal 5-HT neurons of mouse brain; these findings are in line with observations in rat and monkey 5-HT neurons. We also revealed that these peptides/peptide genes partially overlapped with the transcription factor Lmx1b that specifies the 5-HT cell fate. Furthermore, we found that the peptide cholecystokinin was expressed in developing dopaminergic neurons and greatly overlapped with Lmx1b that specifies the dopaminergic cell fate. By examining the phenotype of Lmx1b deletion mice, we found that Lmx1b was required for the expression of above peptides expressed in 5-HT or dopaminergic neurons. Together, our results indicate that Lmx1b, a key transcription factor for the specification of 5-HT and dopaminergic transmitter phenotypes during embryogenesis, determines some peptide phenotypes in these neurons as well.

Neurochemical heterogeneity of rats predicted by different measures to be high ethanol consumers.

BACKGROUND: Alcoholism is a heterogeneous disease, with subjects possibly differing both in the best measure that predicts their excess consumption and in their most effective pharmacotherapy. Two different measures, high novelty-induced activity and high-fat-induced triglycerides (TGs), are known to identify subgroups of animals prone to consuming higher amounts of ethanol (EtOH). The question investigated here is whether these subgroups are, in fact, similar in their neurochemical phenotype that may contribute to their overconsumption. METHODS: EtOH-naïve, Sprague-Dawley rats were subgrouped based on the 2 predictor measures of activity or TG levels, and then quantitative real-time polymerase chain reaction and digoxigenin-labeled in situ hybridization were used to measure their expression of hypothalamic peptides that affect EtOH intake. In additional subgroups subsequently trained to drink 9% EtOH, the opioid antagonist and alcoholism medication, naltrexone, was tested at a low dose (0.02 mg/kg, s.c.) to determine the rats' sensitivity to its effects. RESULTS: The 2 measures, while both effective in predicting amount of EtOH intake, were found to identify distinctive subgroups. Rats with high compared to low activity exhibited significantly greater expression of galanin and enkephalin in the paraventricular nucleus (PVN) and of orexin in the perifornical lateral hypothalamus (PFLH), but no difference in melanin-concentrating hormone in PFLH or neuropeptide Y in arcuate nucleus. This contrasts with rats having high TG, which exhibited greater expression only of PVN galanin, along with reduced PFLH orexin. The high-activity rats with elevated enkephalin, but not high-TG rats, were also unusually sensitive to naltrexone, which significantly reduced their alcohol intake. CONCLUSIONS: In addition to revealing differences in endogenous peptides and drug responsiveness in predicted high EtOH drinkers, this study demonstrates that these disturbances differ markedly between the 2 at-risk subgroups. This indicates that simple tests may be effective in identifying subjects most responsive to a specific pharmacotherapy.

Repetitive electroacupuncture causes prolonged increased met-enkephalin expression in the rVLM of conscious rats.

Enkephalinergic neurons in the rostral ventrolateral medulla (rVLM), an important presympathetic region in the brainstem, are activated by 30 min of low frequency (2 Hz) electroacupuncture (EA) at acupoints P5-P6, which overlie the median nerves. To more closely model the clinical application of acupuncture, we administered EA for 30 min twice over a 72 h period to unsedated conscious rats to examine its prolonged action. We hypothesized that repetitive EA would increase preproenkephalin mRNA and met-enkephalin in the rVLM of unsedated conscious rats. Rats received either EA (1-4 mA, 0.5 ms, 2 Hz) or sham stimulation (needle placement without electrical stimulation) twice at P5-P6 acupoints bilaterally. Preproenkephalin mRNA and its peptide met-enkephalin in the rVLM were measured 24 or 48 h after the final EA or sham procedure. Relative ratios of preproenkephalin mRNA levels (normalized with the 18S housekeeping gene) were almost doubled at 24h compared to sham (6.1 ± 0.79 vs. 3.1 ± 0.47). Met-enkephalin measured in rVLM tissue pooled from several rats exposed to the same treatment was increased by repeated EA by 68% after 24h and 51% after 48h, relative to sham. These findings suggest that repeated application of EA in the conscious rats enhances transcription and translation of enkephalin in rVLM for days. Since opioids in the rVLM contribute importantly to the action of EA on sympathetic outflow, this mechanism may contribute to the prolonged action of acupuncture on elevated blood pressure in patients.