Neuropeptides in the antennal lobe of the yellow fever mosquito,Aedes aegypti

For many insects, including mosquitoes, olfaction is the dominant modality regulating their behavioral repertoire. Many neurochemicals modulate olfactory information in the central nervous system, including the primary olfactory center of insects, the antennal lobe. The most diverse and versatile neurochemicals in the insect nervous system are found in the neuropeptides. In the present study, we analyzed neuropeptides in the antennal lobe of the yellow fever mosquito, Aedes aegypti, a major vector of arboviral diseases. Direct tissue profiling of the antennal lobe by matrix‐assisted laser desorption ionization time‐of‐flight (MALDI‐TOF) mass spectrometry indicated the presence of 28 mature products from 10 different neuropeptide genes. In addition, immunocytochemical techniques were used to describe the cellular location of the products of up to seven of these genes within the antennal lobe. Allatostatin A, allatotropin, SIFamide, FMRFamide‐related peptides, short neuropeptide F, myoinhibitory peptide, and tachykinin‐related peptides were found to be expressed in local interneurons and extrinsic neurons of the antennal lobe. Building on these results, we discuss the possible role of neuropeptide signaling in the antennal lobe of Ae. aegypti. J. Comp. Neurol. 522:592–608, 2014. © 2013 The Authors. The Journal of Comparative Neurology Published by Wiley Periodicals, Inc.     

Multiple olfactory receptor neurons and their axonal projections in the antennal lobe of the honeybee Apis mellifera

The poreplate sensilla of honeybees are equipped with multiple olfactory receptor neurons (ORNs), which innervate glomeruli of the antennal lobe (AL). We investigated the axonal projection pattern in glomeruli of the AL (glomerular pattern), formed by the multiple ORNs of individual poreplate sensilla. We used the different glomerular patterns to draw conclusions about the equipment of poreplate sensilla with different ORN types. ORNs of single poreplate sensilla were stained and analyzed by laser-scanning confocal microscopy and 3D software (AMIRA). In 13 specimens we found between 7 and 23 ORNs. This is in accordance with data found in the literature (5-35 ORNs) suggesting that all ORNs of the single poreplate sensilla were stained. The ORNs innervate the AL via all four sensory tracts (T1-T4), and glomeruli of the anterior part of the AL are more often innervated. Each ORN innervates a single glomerulus (uniglomerular), and all ORNs of one poreplate sensillum project to different glomeruli. Visual inspection and individual identification of glomeruli, based on the honeybee digital AL atlas, were used to evaluate mapping of glomeruli by a rigid transformation of the experimental ALs onto a reference AL. ORNs belonging to individual poreplate sensilla form variable glomerular patterns, and we did not find a common organization of glomerular patterns. We conclude that poreplate sensilla are equipped with different ORN types but that the same ORN types can be found in different poreplate sensilla. The equipment of poreplate sensilla with ORNs is overlapping. The mapping of glomeruli by rigid transformation is revealed to be a powerful tool for comparative neuroanatomy.     

Sensillum-specific, topographic projection patterns of olfactory receptor neurons in the antennal lobe of the cockroach Periplaneta americana

In vertebrates and many invertebrates, olfactory signals detected by peripheral olfactory receptor neurons (ORNs) are conveyed to a primary olfactory center with glomerular organization in which odor-specific activity patterns are generated. In the cockroach, Periplaneta americana, ORNs in antennal olfactory sensilla project to 205 unambiguously identifiable antennal lobe (AL) glomeruli that are classified into 10 glomerular clusters (T1-T10 glomeruli) innervated by distinct sensory tracts. In this study we employed single sensillum staining techniques and investigated the topographic projection patterns of individual ORNs to elucidate the relationship between sensillum types and glomerular organization in the AL. Axons of almost all ORNs projected to individual glomeruli. Axons of ORNs in perforated basiconic sensilla selectively innervated the anterodorsal T1-T4 glomeruli, whereas those in trichoid and grooved basiconic sensilla innervated the posteroventral T5-T9 glomeruli. About 90% of stained ORNs in trichoid sensilla sent axons to the T5 glomeruli and more than 90% of ORNs in grooved basiconic sensilla innervated the T6, T8, and T9 glomeruli. The T5 and T9 glomeruli exclusively receive sensory inputs from the trichoid and grooved basiconic sensilla, respectively. All investigated glomeruli received convergent input from a single type of sensillum except F11 glomerulus in the T6 glomeruli, which was innervated from both trichoid and grooved basiconic sensilla. These results suggest that ORNs in distinct sensillum types project to glomeruli in distinct glomerular clusters. Since ORNs in distinct sensillum types are each tuned to distinct subsets of odorant molecules, the AL is functionally compartmentalized into groups of glomeruli.     

Central projections of the antennal cold receptor neurons and hygroreceptor neurons of the cockroach Periplaneta americana

The central projections of the cold receptor axons were examined by filling two types of cold receptive sensilla with cobalt lysine—a cold and hygroreceptive (C/H) sensillum and a cold receptive and olfactory (C/O) sensillum—on the antennae of the cockroach, Periplaneta americana L. When the dye filled a single C/H sensillum, four axons were stained. Three of these axons terminate in the ipsilateral antennal lobe, while the other branches in the ipsilateral dorsal lobe. One of the branches passed through the tritocerebrum to terminate in the suboesophageal ganglion, while the other branches end in the lobe. When a single C/O sensillum is dye filled, all axons of the four receptor neurons terminate exclusively in the ipsilateral antennal lobe. One axon from the C/H sensillum and one axon from the C/O sensillum terminate in a particular glomerulus in the ventroposterior region of the antennal lobe. Each of these axons also has a tuft in separate giomeruli situated just dorsal to the glomerulus in which both axons terminate. This set of three glomeruli have indistinct boundaries and appear to form a complex of glomeruli similar to the macroglomerular complex of male moths. Assuming modality-specific convergence of antennal afferents, these axons appear to belong to the cold receptor neurons, and the set of glomeruli appear to function in cold reception. Two other neurons stained from C/H sensilla always terminate in theglom-eruli distinct from the set of glomeruli mentioned earlier. These neurons are assigned to the pair of hygroreceptor neurons, and their glomeruli are thought to function in hygroreception. © 1995 Wiley-Liss, Inc.     

Projection patterns of gustatory neurons in the suboesophageal ganglion and tritocerebrum of mosquitoes

Mosquitoes are heavily dependent on gustatory information when feeding. Following the recent elucidation of the molecular basis of gustation in the malaria mosquito, we present a detailed study of primary central projections of gustatory receptor neurons into the brain in the malaria (Anopheles gambiae) and yellow fever (Aedes aegypti) mosquito. In the brain we provide a detailed map of the areas targeted and describe a number of intrinsic neural elements connecting primary taste areas to higher brain levels. The morphological features described are discussed and compared to earlier reports in other insects as, e.g., the fruitfly, Drosophila.     

Expression of a vomeronasal receptor gene (V1r) and G protein alpha subunits in goat, Capra hircus, olfactory receptor neurons

Most mammals have two distinct olfactory epithelia, the olfactory epithelium (OE) and vomeronasal epithelium (VNE), containing, respectively, olfactory receptor neurons (ORNs) and vomeronasal receptor neurons (VRNs). Olfactory receptors (ORs), which couple to G alpha olf, are generally expressed by ORNs, whereas two vomeronasal receptor families (V1rs and V2rs) coupled respectively to G alpha i2 and G alpha o, are expressed by VRNs. Previously, we reported that one goat V1rs (gV1ra1) is expressed by ORNs and VRNs. To investigate the characteristics of vomeronasal-receptor-expressing ORNs in mammals we performed double-label in situ hybridization for gV1ra1, G alpha i2, G alpha olf, olfactory marker protein (OMP), and growth association protein 43 (GAP43). Goat V1r-expressing ORNs are categorized into two types situated in different areas of the epithelium. The first type of V1r-expressing ORN coexpressed G alpha i2, but not OMP or GAP43. The second type of V1r-expressing ORN expresses G alpha olf and OMP, but not G alpha i2 or GAP43. These findings suggest that the two types of V1r-expressing ORN in goat OE function using different G protein alpha subunits for chemoreception.     

Localized ablation of olfactory receptor neurons induces both localized regeneration and widespread replacement of neurons in spiny lobsters

The peripheral olfactory system of the spiny lobster Panulirus argus--located on paired antennules--undergoes continual postembryonic development. This process includes continuous addition of olfactory receptor neurons (ORNs) related to indeterminate growth, continuous replacement, and regeneration when necessitated by damage. We have shown previously that new olfactory tissue is continually added to the proximal margin of these populations, called the proximal proliferation zone (PPZ). Here, we show that focal damage to mature portions of the olfactory system causes localized degeneration of ORNs over 1-10 days after damage. Studies using the cell proliferation marker 5-bromo-2'-deoxyuridine show that localized degeneration was followed by rapid and localized regeneration of olfactory tissue. Rapidly dividing cells were recorded up to 40 days after damage, with regeneration of ORN clusters complete within 80 days. Focal damage appeared to stimulate widespread cell replacement (cell death and proliferation) within mature, undamaged ORN clusters. This response was observed in ORN clusters outside the damaged zone, including mature clusters in the contralateral antennule. The degree of widespread cell replacement was less than local repair after local damage, but it increased with more extensive damage. However, changes in on-going proliferation in the PPZ were not detected, at least not 20 days or longer after damage, suggesting damage-induced widespread proliferation may be specific to mature populations of ORNs. We speculate that localized regeneration involves activity of resident precursor cells not destroyed by the ablation and that unidentified regulatory signals released in response to localized damage induce widespread ORN replacement.     

Hypophysiotropic neurons of the paraventricular nucleus respond in spatially, temporally, and phenotypically differentiated manners to acute vs. repeated restraint stress: rapid publication

The antennules of decapod crustaceans are covered with thousands of chemosensilla that mediate odor discrimination and orientation behaviors. Most studies on chemoreception in decapods have focused on the prominent aesthetasc sensilla. However, previous behavioral studies on lobsters following selective sensillar ablation have revealed that input from nonaesthetasc antennular chemosensilla is sufficient for many odor-mediated behaviors. Our earlier examination of the setal types on the antennules of the Caribbean spiny lobster Panulirus argus revealed three types of nonaesthetasc chemosensilla. The most abundant and widely distributed of these is the hooded sensillum. The present study describes the detailed ultrastructure of antennular hooded sensilla and the physiological response properties of their receptor neurons. Light and scanning and transmission electron microscopy were used to examine structural characteristics, and electrophysiology was used to examine single-unit responses elicited by focal chemical and mechanical stimulation of antennular hooded sensilla. Hooded sensilla have a porous cuticle and are innervated by 9-10 chemosensory and 3 mechanosensory neurons whose dendrites project to the distal end of the sensillum. Hooded sensillar chemosensory neurons responded to waterborne chemicals, were responsive to only one of the six tested single compounds, and had different specificities. Hooded sensillar mechanosensory neurons were not spontaneously active. They had low sensitivity in that they responded to tactile but not waterborne vibrations, and they responded to sensillar deflection with phasic bursts of activity. These results support the idea that hooded sensilla are bimodal chemo-mechanosensilla and are receptors in an antennular chemosensory pathway that parallels the well-described aesthetasc chemosensory pathway.     

Two closely located areas in the suboesophageal ganglion and the tritocerebrum receive projections of gustatory receptor neurons located on the antennae and the proboscis in the moth Heliothis virescens

Sucrose stimulation of gustatory receptor neurons on the antennae, the tarsi, and the mouthparts elicits the proboscis extension reflex in many insect species, including lepidopterans. The sensory pathways involved in this reflex have only partly been investigated, and in hymenopterans only. The present paper concerns the pathways of the gustatory receptor neurons on the antennae and on the proboscis involved in the proboscis extension reflex in the moth Heliothis virescens (Lepidoptera; Noctuidae). Fluorescent dyes were applied to the contact chemosensilla, sensilla chaetica on the antennae, and sensilla styloconica on the proboscis, permitting tracing of the axons of the gustatory receptor neurons in the central nervous system. The stained axons showed projections from the two appendages in two closely located but distinct areas in the suboesophageal ganglion (SOG)/tritocerebrum. The projections of the antennal gustatory receptor neurons were located posterior-laterally to those from the proboscis. Electrophysiological recordings from the receptor neurons in s. chaetica during mechanical and chemical stimulation were performed, showing responses of one mechanosensory and of several gustatory receptor neurons. Separate neurons showed excitatory responses to sucrose and sinigrin. The effect of these two tastants on the proboscis extension reflex was tested by repeated stimulations with solutions of the two compounds. Whereas sucrose elicited extension in 100% of the individuals in all repetitions, sinigrin elicited extension in fewer individuals, a number that decreased with repeated stimulation.     

Localization of putative nitrergic neurons in peripheral chemosensory areas and the central nervous system of Aplysia californica

The distribution of putative nitric oxide synthase (NOS)-containing cells in the opisthobranch mollusc Aplysia californica was studied by using NADPH-diaphorase (NADPH-d) histochemistry in the CNS and peripheral organs. Chemosensory areas (the mouth area, rhinophores, and tentacles) express the most intense staining, primarily in the form of peripheral highly packed neuropil regions with a glomerular appearance as well as in epithelial sensory-like cells. These epithelial NADPH-d-reactive cells were small and had multiple apical ciliated processes exposed to the environment. NADPH-d processes were also found in the salivary glands, but there was no or very little staining in the buccal mass and foot musculature. In the CNS, most NADPH-d reactivity was associated with the neuropil of the cerebral ganglia, with the highest density of glomeruli-like NADPH-d-reactive neurites in the areas of the termini and around F and C clusters. A few NADPH-d-reactive neurons were also found in other central ganglia, including paired neurons in the buccal, pedal, and pleural ganglia and a few asymmetrical neurons in the abdominal ganglion. The distribution patterns of NADPH-d-reactive neurons did not overlap with other known neurotransmitter systems. The highly selective NADPH-d labeling revealed here suggests the presence of NOS in sensory areas both in the CNS and the peripheral organs of Aplysia and implies a role for NO as a modulator of chemosensory processing.     

Synaptic connection between olfactory receptor cells and uniglomerular projection neurons in the antennal lobe of the American cockroach,Periplaneta americana

Both antennal receptor cell axons and uniglomerular projection neurons of the antennal lobe were specifically labeled, and their synaptic relationship was studied at the fine structural level. The labelings were applied in different combinations: i) Experimentally induced anterograde degeneration of sensory-afferent axons was combined with injection of horseradish peroxidase into uniglomerular projection neurons. ii) Lucifer Yellow was injected into uniglomerular projection neurons, and receptor cell axons were anterogradely labeled with the lipophilic dye DiI. The fluorescent dyes were transformed by immuno- or photochemical treatment into electron-dense markers. In both types of preparations, a considerable number of monosynaptic output synapses from antennal receptor neurons onto processes of uniglomerular projection neurons were identified within the glomeruli of the lobe. In most cases, the receptor axon was connected in a dyadic fashion, firstly to a process of a projection neuron and secondly to a nonlabeled process. The results clearly demonstrate a direct connection between receptor cells and output neurons of the cockroach antennal lobe which exists in parallel to the already proposed and demonstrated polysynaptic connection via inhibitory local interneurons. © 1996 Wiley-Liss, Inc.     

Central processing of sex pheromone,host odour,and oviposition deterrent information by interneurons in the antennal lobe of female Spodoptera littoralis (Lepidoptera: Noctuidae)

Physiological and anatomical characteristics of antennal lobe interneurons in female Spodoptera littoralis (Boisd.) were investigated using intracellular recording and staining techniques. Responses of local interneurons and projection neurons to female sex pheromone components, host plant odours, and behaviourally active oviposition deterrents were recorded. We found local interneurons and projection neurons that responded specifically to only one or two of the tested odours, but we also found less specific cells, and neurons that responded to most of the tested odourants. These findings show that there are not only specific olfactory pathways in female moths up to the protocerebral level, but also that integration can begin in the antennal lobe. No correlation was found between the degree of specificity of either local interneurons or projection neurons and their respective morphological characteristics. Specialized and unspecialized local interneurons arborized throughout the antennal lobe. Specialized and unspecialized projection neurons had uniglomerular arborizations in the antennal lobe and sent their axons to the calyces of the mushroom body, and to the lateral horn of the protocerebrum. One specific projection neuron had multiglomerular arborizations and projected only to the lateral horn of the protocerebrum. Projection neurons arborizing in the glomeruli closest to the entrance of the antennal nerve always responded to pheromone components. No other correlations were found between the arborization pattern of projection neurons in the antennal lobe or in the protocerebrum and their response characteristics. The sensitivity of local interneurons and projection neurons was in the same range as that of receptor neurons in olfactory sensilla on the antennae, suggesting a much lower convergence in the central nervous system in females than in the pheromone-processing pathway in males. © 1994 Wiley-Liss, Inc.     

17 beta-estradiol inhibits cytokine, chemokine, and chemokine receptor mRNA expression in the central nervous system of female mice with experimental autoimmune encephalomyelitis

Cytokines and chemokines govern leukocyte trafficking, thus regulating inflammatory responses. In this study, the anti-inflammatory effects of low dose 17 beta-estradiol were evaluated on chemokine, chemokine receptor, and cytokine expression in the spinal cords (SC) of BV8S2 transgenic female mice during acute and recovery phases of experimental autoimmune encephalomyelitis (EAE). In EAE protected mice, 17 beta-estradiol strongly inhibited mRNA expression of the chemokines RANTES, MIP-1 alpha, MIP-2, IP-10, and MCP-1, and of the chemokine receptors CCR1, CCR2 and CCR5 at both time points. Conversely, ovariectomy, which abrogated basal 17 beta-estradiol levels and increased the severity of EAE, enhanced the expression of MIP-1 alpha and MIP-2 that were over-expressed by inflammatory mononuclear cells in SC. 17 beta-estradiol inhibited expression of LT-beta, TNF-alpha, and IFN-gamma in SC, but had no effect on IL-4 or IL-10, indicating reduced inflammation but no deviation toward a Th2 response. Interestingly, elevated expression of CCR1 and CCR5 by lymph node cells was also inhibited in 17 beta-estradiol treated mice with EAE. Low doses of 17 beta-estradiol added in vitro to lymphocyte cultures had no direct effect on the activation of MBP-Ac1-11 specific T cells, and only at high doses diminished production of IFN-gamma, but not IL-12 or IL-10. These results suggest that the beneficial effects of 17 beta-estradiol are mediated in part by strong inhibition of recruited inflammatory cells, resulting in reduced production of inflammatory chemokines and cytokines in CNS, with modest effects on encephalitogenic T cells that seem to be relatively 17 beta-estradiol insensitive.     

Expression and vesicular localization of mouse Trpml3 in stria vascularis, hair cells, and vomeronasal and olfactory receptor neurons

TRPML3 is a member of the mucolipin branch of the transient receptor potential cation channel family. A dominant missense mutation in Trpml3 (also known as Mcoln3) causes deafness and vestibular impairment characterized by stereocilia disorganization, hair cell loss, and endocochlear potential reduction. Both marginal cells of the stria vascularis and hair cells express Trpml3 mRNA. Here we used in situ hybridization, quantitative RT-qPCR, and immunohistochemistry with several antisera raised against TRPML3 to determine the expression and subcellular distribution of TRPML3 in the inner ear as well as in other sensory organs. We also use Trpml3 knockout tissues to distinguish TRPML3-specific from nonspecific immunoreactivities. We find that TRPML3 localizes to vesicles of hair cells and strial marginal cells but not to stereociliary ankle links or pillar cells, which nonspecifically react with two antisera raised against TRPML3. Upon cochlear maturation, TRPML3 protein is redistributed to perinuclear vesicles of strial marginal cells and is augmented in inner hair cells vs. outer hair cells. Mouse somatosensory neurons, retinal neurons, and taste receptor cells do not appear to express physiologically relevant levels of TRPML3. Finally, we found that vomeronasal and olfactory sensory receptor cells do express TRPML3 mRNA and protein, which localizes to vesicles in their somas and dendrites as well as at apical dendritic knobs.     

Cellular prion protein/laminin receptor: distribution in adult central nervous system and characterization of an isoform associated with a subtype of cortical neurons

The 67-kDa LR protein was originally discovered as a non-integrin laminin receptor. Several more recent in vitro studies demonstrated the function of 67-kDa LR and its related 'precursor' form 37-kDa LRP as receptors of cellular prion protein and their implication in abnormal prion protein propagation in vitro. In addition, expression of both proteins was shown to increase considerably in the brain of scrapie-infected mice and hamsters. While LRP/LR are thus likely to play important roles in neuronal cell adhesion, survival and homeostasis and during pathological disorders, little is known so far about their fine cellular distribution in adult central nervous system. Using immunocytochemistry and western blotting, we show here that the 67-kDa LR is the major receptor form in adult rat brain and spinal cord, expressed within the cytoplasm and at the plasma membrane of most neurons and in a subset of glial cells. The overall distribution of LR correlates well with that reported for laminin-1 but also with brain regions classically associated with prion-related neurodegeneration. In contrast to LR, the 37-kDa LRP form is much less abundant in adult than in postnatal central nervous system. Characterization of a novel antibody allowed us to study the distribution across tissues of cell membrane-associated LRP. Interestingly, this form is almost exclusively found on a subclass of parvalbumin-immunoreactive cortical interneurons known to degenerate during the early stages of Creutzfeldt-Jakob disease. Our demonstration of local differences in the expression of particular LRP/LR isoforms may be a first step towards unraveling their specific molecular interactions.     

Histaminergic neurons in the central and peripheral nervous system of gastropods (Helix, Lymnaea): an immunocytochemical, biochemical, and electrophysiological approach

Distribution, chemical-neuroanatomy, concentration, and uptake-release properties of histamine (HA)-containing neurons and the possible physiological effects of HA in the central and peripheral nervous system of the pulmonate snails, Helix pomatia and Lymnaea stagnalis, are described. In the CNS of both species, the distribution pattern of HA-immunoreactive (HA-IR) neurons was similar. In both species the majority were located in the buccal, cerebral, and pedal ganglia. In Helix, approximately 400 HA-IR neurons were seen, whereas in Lymnaea approximately 130 labeled cells were visualized. The neuropils, connectives, commissures, several peripheral nerves, and a part of the peripheral tissues (lip and foot of both species and the upper tentacles of Helix) were innervated by HA-IR elements. Numerous sensory cells were found in the tentacles, lip, and statocysts. The HA concentration values assayed by HPLC ranged from 4.8 to 47.4 pmol/mg in the different central ganglia of Helix, and from 4.3 to 18.6 pmol/mg in Lymnaea CNS, whereas the peripheral tissues contained 0.33-1 pmol/mg HA in Helix and 0.26-0.46 pmol/mg in Lymnaea. In the Lymnaea CNS, a high-affinity (37.6 microM), single component 3H-HA uptake system was demonstrated. 3H-HA release evoked by either electrical stimulation or 100 mM K+ could be prevented in Ca2+-free physiological solution. Voltage-clamp experiments indicated specific changes caused by HA in the membrane conductance of identified central neurons of Helix and Lymnaea. Exogenously applied 10(-5) M HA resulted in the acceleration of locomotion (gliding by foot cilia) of Lymnaea. The findings suggest an important signaling role of HA, described here for the first time, in the nervous system of higher-order, pulmonate, gastropods, involving efferent, integrative, and sensory functions. The data can also be applied as a background for further specification of HA in the regulation of different behaviors in these species.     

Peptidergic neurons in the snail Helix pomatia: Distribution of neurons in the central and peripheral nervous systems that react with an Antibody raised to the insect neuropeptide,leucokinin I

In this study, antiserum raised against an insect myotropic peptide, leucokinin I (DPAFNSWGamide), was: used for mapping leucokinin-like immunoreactive (LK-LI) neurons in the gastropod mollusc, Helix pomatia. Immunocytochemistry performed on both whole-mounts and cryostat sections demonstrated LK-LI neurons in all ganglia of the central nervous system (CNS), except the visceral ganglion. Altogether about 700 immunolabelled neurons have been found, with nearly one-half (46%) in the cerebral ganglia. A large proportion of the LK-LI neurons have small cell bodies and are likely to be interneurons. The most prominent LK-LI cell group is represented by the entire neuron population of the mesocerebri, which is the major source of a thick fiber bundle system, encircling and innervating the whole CNS. One single LK-LI giant neuron was found, which is located in the left pedal ganglion and is termed GLPdLKC (giant left pedal leucokinin immunoreactive cell). This cell has not been identified previously. The ganglion neuropils are heavily innervated by varicose LK-LI fiber arborizations. Some integrative centers, such as the medullary neuropil of the procerebri, reveal an extreme density of LK-LI innervation. All major peripheral nerves contain a large number of LK-LI axons, and LK-LI innervation is found in the musculature of different peripheral organs (buccal mass, lip, tentacles, oviduct, intestine). Among the peripheral organs investigated, the intestine contains a rich varicose LK-LI network, composed of both intrinsic and extrinsic elements. Radioimmunoassay (RIA) demonstrates a very high content of LK-LI material in Helix ganglion extracts (about 50 pmol/CNS). This is the first report on the occurrence of a substance resembling the myotropic neuropeptide leucokinin I in a phylum outside arthropods. Based on our immunocytochemical observations, a role for leucokinin-like peptides in both central and peripheral regulatory processes in Helix is suggested. According to double-labelling experiments, only a small number of the LK-LI neurons are labelled with an antibody to the vertebrate tachykinin substance P.     

The distribution of the mRNA and protein products of the melanin-concentrating hormone (MCH) receptor gene, slc-1, in the central nervous system of the rat

Melanin-concentrating hormone (MCH), a 19 amino acid cyclic peptide, is largely expressed in the hypothalamus. It is implicated in the control of general arousal and goal-orientated behaviours in mammals, and appears to be a key messenger in the regulation of food intake. An understanding of the biological actions of MCH has been so far hampered by the lack of information about its receptor(s) and their location in the brain. We recently identified the orphan G-protein-coupled receptor SLC-1 as a receptor for the neuropeptide MCH. We used in situ hybridization histochemistry and immunohistochemistry to determine the distribution of SLC-1 mRNA and its protein product in the rat brain and spinal cord. SLC-1 mRNA and protein were found to be widely and strongly expressed throughout the brain. Immunoreactivity was observed in areas that largely overlapped with regions mapping positive for mRNA. SLC-1 signals were observed in the cerebral cortex, caudate-putamen, hippocampal formation, amygdala, hypothalamus and thalamus, as well as in various nuclei of the mesencephalon and rhombencephalon. The distribution of the receptor mRNA and immunolabelling was in good general agreement with the previously reported distribution of MCH itself. Our data are consistent with the known biological effects of MCH in the brain, e.g. modulation of the stress response, sexual behaviour, anxiety, learning, seizure production, grooming and sensory gating, and with a role for SLC-1 in mediating these physiological actions.     

Relationship of μ- and δ-opioid receptors to GABAergic neurons in the central nervous system,including antinociceptive brainstem circuits

Inhibition of neurons containing γ-aminobutyric acid (GABA) may underlie some of the excitatory effects of opioids in the central nervous system (CNS). In the present study, we examined the relationship of the cloned μ- and δ-opioid receptors (MOR1 and DOR1, respectively) to GABAergic neurons in brain and spinal cord. This was done by combining immunofluorescent staining for MOR1 or DOR1 with that for GABA or glutamic acid decarboxylase (GAD); fluorescent retrograde tract-tracing was used in some cases to identify neurons with particular projections. In rats, cells double labeled for GABA and MOR1 were observed in layers II–VI of the parietal cortex and in layers II–IV of the piriform cortex. In the hippocampus, double labeling was observed in the dentate gyrus and in regions CA1 and CA3. Double labeling was very prominent in the striatum and in the reticular nucleus of the thalamus; it was also observed in other portions of the diencephalon. However, double labeling for GABA and MOR1 was never observed in the cerebellar cortex. Cells double labeled for GABA and MOR1 were common in the periaqueductal gray (PAG) and the medial rostral ventral medulla (RVM) of both rats and monkeys, suggesting that involvement of GABAergic neurons with supraspinal opioid antinociception may extend to primates. In the RVM of rats, many of those double-labeled neurons were retrogradely labeled from the dorsal spinal cord. In contrast, double-labeled neurons in the PAG were almost never retrogradely labeled from the RVM. No unequivocal examples of double labeling for DOR1 and GAD were found in any region of the CNS that we examined in either rats or monkeys. However, GABAergic neurons were often apposed by DOR1 immunoreactive varicosities. Our findings suggest that activation of μ-opioid receptors directly modulates the activity of GABAergic neurons throughout the CNS, including neurons involved in the supraspinal component of opioid analgesia. In contrast, δ-opioid receptors appear to be positioned to modulate the activity of GABAergic neurons indirectly. J. Comp. Neurol. 392:528–547, 1998. © 1998 Wiley-Liss, Inc.