Dynorphin Cells Research Articles

Sex-specific negative affect-like behaviour and parabrachial nucleus activation induced by BNST stimulation in adult mice with adolescent alcohol history.

Adolescent alcohol use is a strong predictor for the subsequent development of alcohol use disorders later in life. Additionally, adolescence is a critical period for the onset of affective disorders, which can contribute to problematic drinking behaviours and relapse, particularly in females. Previous studies from our laboratory have shown that exposure to adolescent intermittent ethanol (AIE) vapour alters glutamatergic transmission in the bed nucleus of the stria terminalis (BNST) and, when combined with adult stress, elicits sex-specific changes in glutamatergic plasticity and negative affect-like behaviours in mice. Building on these findings, the current work investigated whether BNST stimulation could substitute for stress exposure to increase the latency to consume a palatable food in a novel context (hyponeophagia) and promote social avoidance in adult mice with AIE history. Given the dense connections between the BNST and the parabrachial nucleus (PBN), a region involved in mediating threat assessment and feeding behaviours, we hypothesized that increased negative affect-like behaviours would be associated with PBN activation. Our results revealed that the chemogenetic stimulation of the dorsolateral BNST induced hyponeophagia in females with AIE history, but not in female controls or males of either group. Social interaction remained unaffected in both sexes. Notably, this behavioural phenotype was associated with higher activation of calcitonin gene-related peptide and dynorphin cells in the PBN. These findings provide new insights into the neurobiological mechanisms underlying the development of negative affect in females and highlight the potential involvement of the BNST-PBN circuitry in regulating emotional responses to alcohol-related stimuli.

242 Divergent Selection for Early Puberty Impacts KNDy Neuron Gene Expression in Gilts

Abstract Advancing gilt puberty onset is financially desirable for swine production. Neurons in the hypothalamic arcuate nucleus (ARC) that co-express kisspeptin, neurokinin B (NKB), and dynorphin (i.e. KNDy cells) are believed to control gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion, but their role in gilt pubertal development is unknown. We hypothesized that puberty onset in gilts would coincide with greater expression of mRNA for kisspeptin and NKB, and less expression of dynorphin. Using fluorescent in situ hybridization (RNAscope), we examined expression of kisspeptin, NKB, and dynorphin in pre- and postpubertal gilts from two genetic lines divergently selected for age at puberty. Prepubertal (n = 6/line) and postpubertal (n = 6/line) gilts were used, and postpubertal animals all received Matrix (0.22% altrenogest) orally for 14 days with tissue collection two days after the final dose. Gilts were euthanized and heads were perfused with 8 L of 4% paraformaldehyde (PFA). Hypothalamic brain tissue was removed, placed in 4% PFA for 24 hrs, and then in 20% sucrose until sectioning (50 µm). Sectioned tissue was stored in cryopreservative at -20°C until RNAscope. Data were analyzed using SAS software (Version 9.4, SAS Institute, Cary NC) with significance declared at P < 0.05. We determined mRNA expression for kisspeptin was not different between groups (P > 0.05). In addition, we found that mRNA expression for NKB was higher in prepubertal gilts compared to postpubertal gilts (P < 0.05) but was not different between lines; mRNA expression was lowest in postpubertal late puberty gilts. Furthermore, total number of dynorphin cells were higher in prepubertal gilts compared to postpubertal gilts (P < 0.05), while individual cell mRNA expression for dynorphin was greatest in postpubertal early puberty gilts (P < 0.05). Taken together, we suggest puberty onset in gilts is more dependent on NKB and dynorphin than kisspeptin.

Expression of Calretinin Among Different Neurochemical Classes of Interneuron in the Superficial Dorsal Horn of the Mouse Spinal Cord

Around 75% of neurons in laminae I-II of the mouse dorsal horn are excitatory interneurons, and these are required for normal pain perception. We have shown that four largely non-overlapping excitatory interneuron populations can be defined by expression of the neuropeptides neurotensin, neurokinin B (NKB), gastrin-releasing peptide (GRP) and substance P. In addition, we recently identified a population of excitatory interneurons in glabrous skin territory that express dynorphin. The calcium-binding protein calretinin is present in many excitatory neurons in this region, but we know little about its relation to these neuropeptide markers. Here we show that calretinin is differentially expressed, being present in the majority of substance P-, GRP- and NKB-expressing cells, but not in the neurotensin or dynorphin cells. Calretinin-positive cells have been implicated in detection of noxious mechanical stimuli, but are not required for tactile allodynia after neuropathic pain. Our findings are therefore consistent with the suggestion that neuropathic allodynia involves the neurotensin and/or dynorphin excitatory interneuron populations. Around a quarter of inhibitory interneurons in lamina I-II contain calretinin, and recent transcriptomic studies suggest that these co-express substance P. We confirm this, by showing that inhibitory Cre-expressing cells in a Tac1Cre knock-in mouse are calretinin-immunoreactive. Interestingly, there is evidence that these cells express low levels of peptidylglycine alpha-amidating monooxygenase, an enzyme required for maturation of neuropeptides. This may explain our previous finding that although the substance P precursor preprotachykinin A can be detected in some inhibitory interneurons, very few inhibitory axonal boutons are immunoreactive for substance P.

Does Dynorphin Play a Role in the Onset of Puberty in Female Sheep?

Puberty onset involves increased gonadotrophin-release (GnRH) release as a result of decreased sensitivity to oestrogen (E2 )-negative feedback. Because GnRH neurones lack E2 receptor α, this pathway must contain interneurones. One likely candidate is KNDy neurones (kisspeptin, neurokinin B, dynorphin). The overarching hypothesis of the present study was that the prepubertal hiatus in luteinising hormone (LH) release involves reduced kisspeptin and/or heightened dynorphin input. We first tested the specific hypothesis that E2 would reduce kisspeptin-immunopositive cell numbers and increase dynorphin-immunopositive cell numbers. We found that kisspeptin cell numbers were higher in ovariectomised (OVX) lambs than OVX lambs treated with E2 (OVX+E2 ) or those left ovary-intact. Very few arcuate dynorphin cells were identified in any group. Next, we hypothesised that central blockade of κ-opioid receptor (KOR) would increase LH secretion at a prepubertal (6months) but not postpubertal (10months) age. Luteinising hormone pulse frequency and mean LH increased during infusion of a KOR antagonist, norbinaltorphimine, in OVX+E2 lambs at the prepubertal age but not in the same lambs at the postpubertal age. We next hypothesised that E2 would increase KOR expression in GnRH neurones or alter synaptic input to KNDy neurones in prepubertal ewes. Oestrogen treatment decreased the percentage of GnRH neurones coexpressing KOR (approximately 68%) compared to OVX alone (approximately 78%). No significant differences in synaptic contacts per cell between OVX and OVX+E2 groups were observed. Although these initial data are consistent with dynorphin inhibiting pulsatile LH release prepubertally, additional work will be necessary to define the source and mechanisms of this inhibition.

Distinct Subpopulations of Nucleus Accumbens Dynorphin Neurons Drive Aversion and Reward

The nucleus accumbens (NAc) and the dynorphinergic system are widely implicated in motivated behaviors. Prior studies have shown that activation of the dynorphin-kappa opioid receptor (KOR) system leads to aversive, dysphoria-like behavior. However, the endogenous sources of dynorphin in these circuits remain unknown. We investigated whether dynorphinergic neuronal firing in the NAc is sufficient to induce aversive behaviors. We found that photostimulation of dynorphinergic cells in the ventral NAc shell elicits robust conditioned and real-time aversive behavior via KOR activation, and in contrast, photostimulation of dorsal NAc shell dynorphin cells induced a KOR-mediated place preference and was positively reinforcing. These results show previously unknown discrete subregions of dynorphin-containing cells in the NAc shell that selectively drive opposing behaviors. Understanding the discrete regional specificity by which NAc dynorphinerigic cells regulate preference and aversion provides insight into motivated behaviors that are dysregulated in stress, reward, and psychiatric disease.

Colocalization of CART peptide with prodynorphin and dopamine D1 receptors in the rat nucleus accumbens

CART peptide is a peptidergic neurotransmitter that is expressed in brain regions involved in critical biological processes such as feeding and stress, and in areas associated with drug reward and abuse including the dopamine-rich nucleus accumbens (NAcc), which can be considered part of the basal ganglia. Because CART has been shown to colocalize with substance P, a marker of the basal ganglia direct pathway, we now test for colocalization with other markers of the direct pathway to determine if CART colocalizes with dynorphin and dopamine D1 receptors. In the NAcc, CART peptide immunoreactivity (IR) was colocalized with prodynorphin-IR in neurons. Approximately 80.1% of CART-IR cells colocalized with prodynorphin-IR, while only 27.6% of prodynorphin-IR neurons contained CART-IR, suggesting that CART cells are a subset of dynorphin cells. In contrast, only about 25% of CART-IR cell bodies demonstrated dopamine D1 receptor-IR. Because dynorphin and D1 receptors are markers for the basal ganglia direct pathway, from the NAcc to the basal ganglia output nuclei, and because CART significantly colocalizes with these markers, some CART neurons are part of the direct pathway or some comparable pathway in the accumbens. The presence of CART in NAcc neurons and the fact that NAcc projection neurons have extensive local collaterals suggest that CART may have effects in both terminal and cell body regions of the accumbens and may therefore affect information processing in the NAcc by modulating accumbal neurons.

Nicotine modulation of stress‐related peptide neurons

Nicotine has been shown to activate stress-related brain nuclei, including the paraventricular nucleus of the hypothalamus (PVN) and the central nucleus of the amygdala (CEA), through complex mechanisms involving direct and indirect pathways. To determine the neurochemical identities of rat brain neurons which are activated by a low dose (0.175 mg/kg) of nicotine given 30 minutes before sacrifice, we have used single- and double-label in situ hybridization. Neuronal activation was quantified by localization of (35)S-labeled probe for the immediate early gene, c-fos. Corticotrophin releasing factor (CRF), enkephalin (ENK), and dynorphin (DYN) mRNAs were colocalized using a colorimetric, digoxigenin-labeled probe. Film autoradiographic studies showed that nicotine significantly increased c-fos mRNA expression in both PVN and CEA. Pretreatment with the centrally acting nicotinic antagonist, mecamylamine (1 mg/kg), blocked nicotine's effects, whereas pretreatment with the peripherally acting antagonist, hexamethonium (5 mg/kg), did not, indicating that c-fos induction was mediated by a central nicotinic receptor. Double labeling studies showed that nicotine induced c-fos expression within CRF cells in the PVN, as well as in a small population of ENK cells, but not in PVN DYN cells. In contrast, there was no significant nicotine-induced increase in c-fos expression in CEA CRF or DYN cells, whereas nicotine treatment did increase c-fos expression within CEA ENK cells.

Distribution of preprodynorphin mRNA and dynorphin-a immunoreactivity in the sheep preoptic area and hypothalamus

Endogenous opioid peptides (EOP) are important modulators in a variety of neuroendocrine systems, including those mediating reproduction, energy balance, lactation, and stress. Recent work in the ewe has implicated the EOP, dynorphin (DYN), in the inhibitory effects of progesterone on pulsatile gonadotropin releasing hormone secretion. Although DYN is involved in a number of hypothalamic functions in the sheep, little is known regarding the localization of preprodynorphin (PPD) expression and its major product DYN A (1–17). In this study, we determined the distribution of PPD mRNA and DYN A-containing cell bodies in the brains of ovary-intact, luteal ewes. To detect PPD mRNA, an ovine PPD mRNA was subcloned by reverse transcription–polymerase chain reaction from sheep hypothalamus and used to create a 35S-labeled riboprobe for in situ hybridization. Neurons that expressed PPD mRNA and DYN A immunoreactivity were widely distributed in the ovine preoptic area and hypothalamus. PPD mRNA-expressing cells were seen in the supraoptic nucleus, paraventricular nucleus, preoptic area, anterior hypothalamus area, bed nucleus of the stria terminalis, ventromedial nucleus (VMN), dorsomedial nucleus of the hypothalamus, and the arcuate nucleus. All of these regions also contained DYN A-positive cell bodies except for the VMN, raising the possibility that PPD is preferentially processed into other peptide products in the VMN. In summary, based on the expression of both mRNA and peptide, DYN cells are located in a number of key hypothalamic regions involved in the neuroendocrine control of homeostasis in sheep.

Colocalization of progesterone receptors in parvicellular dynorphin neurons of the ovine preoptic area and hypothalamus.

Recent evidence suggests that the dynorphin-kappa receptor opioid system acts to mediate the inhibitory effect of progesterone (P) on GnRH pulse frequency during the luteal phase of the ovine estrous cycle. It is known that progesterone receptors (PRs) are required for the actions of P on GnRH secretion. Therefore, if P acts directly on dynorphin (DYN) neurons, then these neurons should contain PRs. To test this hypothesis, we used a dual-label immunoperoxidase procedure to visualize PRs and DYN in the preoptic area (POA) and hypothalamus of ovary-intact ewes killed during the luteal phase of the estrous cycle. The PR was colocalized in more than 90% of parvicellular DYN neurons in the POA, anterior hypothalamus (AHA), and arcuate nucleus (ARC). By contrast, none of magnocellular DYN cells of the paraventricular and supraoptic nuclei coexpressed immunoreactive PRs. The high percentage of colocalization of PRs in parvicellular DYN cells of the POA, AHA, and ARC suggests that these cells are prime targets of P. In addition, DYN cells in the ARC, but not the POA or AHA, were found to receive synaptic inputs from DYN-positive axon terminals. This observation raises the possibility that an ultrashort feedback loop controls the release of DYN from ARC neurons.

Ultrastructural analysis of CGRP and gaba terminals on dynorphin cells in the primate dorsal horn

Ultrastructural analysis of CGRP and gaba terminals on dynorphin cells in the primate dorsal horn

Ultrastructural analysis of dynorphin B–immunoreactive cells and terminals in the superficial dorsal horn of the deafferented spinal cord of the rat

Light microscopic studies have demonstrated important differences in the distribution of enkephalin and dynorphin cells and terminals in the dorsal horn. Most importantly, dynorphin neurons are located in regions almost exclusively associated with the transmission and/or control of nociceptive messages (laminae I, IIo, and V); enkephalin neurons, although located in the same regions, are also found in areas involved in the transmission of nonnociceptive messages, e.g., laminae IIi and III. To determine whether there are also differences in the synaptic organization of the two opioid peptides, we have examined the distribution of dynorphin B immunoreactivity at the ultrastructural level. The studies were performed in colchicine-treated rats that underwent dorsal rhizotomy so that the relationship of dynorphin terminals and cells to primary afferent terminals could be established. Dynorphin B-immunoreactive cell bodies and dendrites in laminae I and IIo receive convergent primary and nonprimary afferent input, which suggests that dynorphin neurons receive a small-diameter, nociceptive input. Dynorphin terminals predominantly contain round, agranular vesicles; some terminals also contain a few dense core vesicles. Most dynorphin terminals are presynaptic to unlabelled dendrites; both asymmetric and symmetrical axonal contacts were noted. Dynorphin-immunoreactive boutons are also presynaptic to unlabelled cell bodies and spines. Twenty-nine percent of dynorphin terminals were associated with axonal profiles, including degenerating primary afferent terminals; only rarely could a synaptic density be detected. Although some degenerating primary afferent terminals were clearly presynaptic to dynorphin-immunoreactive terminals, in most cases, the polarity of the relationship between primary afferents and dynorphin terminals could not be established. These data indicate that synaptic interactions made by and with dynorphin-immunoreactive cells and terminals in the superficial dorsal horn are not very different from those that were previously reported for enkephalin cells and terminals. Thus, it is unlikely that dynorphin terminals provide a significant presynaptic input to primary afferent fibers. On the other hand, the presence of a primary afferent input to dynorphin cell bodies and dendrites in the superficial dorsal horn suggests that dynorphin cells receive a direct input from small-diameter, nociceptive primary afferents. That connection might contribute to the increased levels of dynorphin message and peptide that have been reported in rats experiencing a chronic inflammatory condition.(ABSTRACT TRUNCATED AT 400 WORDS)

Lateral hypothalamic dynorphinergic efferents to the amygdala and brainstem in the rat

Dynorphin is present within perikarya of the lateral hypothalamus (LH) and perifornical nucleus (PeF), and within nerve terminals of the central nucleus of the amygdala, central grey, parabrachial nucleus, and the dorsal vagal complex (nucleus of the solitary tract and dorsal motor nucleus of the vagus). Each of these nuclei receive efferent projections from the LH and PeF. In this study, the possibility that dynorphin cells with LH and PeF innervate each of these nuclei was investigated using a combined retrograde tracing-immunofluorescence technique. As enkephalinergic perikarya have also been localized to LH and PeF, peptide E (an enkephalin precursor fragment) was also studied for comparison. Following injections of fast blue into the central nucleus, parabrachial nucleus, central grey, and dorsal vagal complex, numerous retrogradely-labeled dynorphin-immunoreactive neurons were present within the LH and PeF. In comparison, retrogradely-labeled peptide E-immunoreactive cells were infrequently observed. These results suggest the LH and PeF to be a major source of dynorphin to the forebrain and brainstem.

The distribution of substance P-, enkephalin-and dynorphin-immunoreactive neurons in the medulla of the rat and their contribution to bulbospinal pathways

This study examined the medullary distribution of peptide-containing neurons at the origin of bulbospinal pathways in the rat. Antisera directed against substance P, methionine-enkephalin-arg-glγ-leu and dynorphin B were used on sections in which spinally projecting brainstem neurons had been identified by the retrograde transport of a protein-gold complex that was injected into the spinal cord. Both the relative numbers and distribution of the different peptide-immunoreactive spinally projecting neurons differed. Methionine-enkephalin-immunoreactive neurons were twice as numerous as the substance P-immunoreactive cells and seven times more numerous than the dynorphin B-positive neurons. The methionine-enkephalin cells were found in all medullary raphénuclei, and in the ventromedial and ventrolateral medullary reticular formation. Caudally, the methionine-enkephalin cells were concentrated laterally; more rostrally, they were located more medially. Three major loci of methionine-enkephalin-immunoreactive cells were found: (1) the nucleus reticularis paragigantocellularis lateralis, at levels caudal to the facial nucleus, (2) the B3 cell group (nucleus raphémagnus and the nucleus reticularis magnocellularis, pars alpha) and the most rostral part of the B1 and B2 cell groups (nuclei raphépallidus and obscurus), (3) a dense cluster of cells that flanks the dorsal surface of the dorsal accessory olive (referred to as the nucleus interfascicularis hypoglossi, pars dorsalis). Substance P-like cells were seen in all raphénuclei except for the most anterior portion of the B3 cell group. Substance P-immunoreactive cells were also seen in both the ventromedial (nuclei reticularis ventralis and magnocellularis) and ventrolateral medulla (nucleus reticularis paragigantocellularis lateralis). Finally there was a dense concentration of substance P neurons in the nucleus interfascicularis hypoglossi, pars ventralis. The distribution of dynorphin-immunoreactive neurons differed significantly from that of methionine-enkephalin and substance P. Dynorphin cells were almost exclusively found in the ventrolateral medulla (nucleus reticularis paragigantocellularis lateralis), at all Ievels between the lateral reticular nucleus and the caudal pole of the facial nucleus. The proportion of each of these peptidergic-immunoreactive cells at the origin of bulbospinal pathways differed considerably. Substance P spinally projecting neurons were more numerous than methionine-enkephalin spinally projecting neurons. The majority of the substance P projection neurons were found in the Bl and B2 cell groups, in the adjacent ventromedial medulla and in the nucleus interfascicularis hypoglossi, pars ventralis. A few substance P projection neurons were also found in the caudal reaches of the B3 cell group. Methionine-enkephalin projection neurons were located in the rostral B1/B2 cell groups, B3 cell group and in the nucleus interfascicularis hypoglossi, pars dorsalis. None were found in caudal B1/B2 cell groups. Methionine-enkephalin projecting neurons were thus located rostral to the majority of the double-labelled substance P cells. Only scattered projection neurons were found among the methionine-enkephalin- and substance P-immunoreactive cells of the nucleus reticularis paragigantocellularis lateralis. In contrast to the large numbers of double-labelled methionine-enkephalin and substance P cells, no spinally projecting dynorphin neurons were found. We suggest that there are differential methionine-enkephalin 5-hydroxytryptamine inhibitory and substance P/5 hydroxytryptamine excitatory influences that issue from different medullary areas and are, respectively, exerted upon dorsal horn nociceptive neurons, and visceral/somatic motoneurons and intermediolateral preganglionic neurons.

Immunoreactive pro‐enkephalin and pro‐dynorphin products are differentially distributed within the nucleus of the solitary tract of the rat

In this study we examined the distribution of two different endogenous opioid peptides in the nucleus of the solitary tract of the rat medulla. As a marker for immunoreactive enkephalin, we used an antiserum directed against one of the proenkephalin products, methionine enkephalin-arg-gly-leu (m-Enk). To identify immunoreactive dynorphin we used an antiserum directed against the prodynorphin product, dynorphin B (Dyn B). The PAP method was used on both colchicine and normal animals. Caudal to the obex, within the commissural nucleus, there is extensive overlap of both immunoreactive m-Enk and Dyn B terminals and cells. While the cells are morphologically similar, the immunoreactive dynorphin cells are somewhat larger. Rostral to the obex, there is a marked difference in the distribution of the two compounds. Immunoreactive m-Enk terminals are concentrated medial to the solitary tract; there is minimal staining laterally. In contrast, immunoreactive Dyn B terminals are concentrated lateral to the solitary tract. The rostral cellular distribution of the two opioid peptides follows a similar pattern. The morphology of the medially located m-Enk and laterally located Dyn B cells is also readily distinguished. The former are small, round cells with minimal dendritic labelling; the latter are larger, pyramidal neurons with prominent apical and basal dendrites. Since the medial and lateral nuclei of the solitary tract have been associated with cardiovascular and respiratory control, respectively, these data suggest that different endorphin families have different functional actions within the nucleus of the solitary tract.

Dynorphin is located throughout the CNS and is often co-localized with alpha-neo-endorphin

The opioid peptide dynorphin has been described as widely distributed in CNS when measured by RIA. Our previous immunohistochemical studies have only demonstrated dynorphin cells as those containing AVP. We now report the specific localization of dynorphin throughout the neuraxis. Further, dynorphin and alpha-neo-endorphin have been co-localized to the same magnocellular neurosecretory cells in hypothalamus. We report agreement with the findings of others and extend them to include a cell group in dorsomedial hypothalmus, further strengthening the association between dynorphin and alpha-neo-endorphin.