Cholinergic innervation of principal neurons in the cochlear nucleus of the Mongolian gerbil

Principal neurons in the ventral cochlear nucleus (VCN) receive powerful ascending excitation and pass on the auditory information with exquisite temporal fidelity. Despite being dominated by ascending inputs, the VCN also receives descending cholinergic connections from olivocochlear neurons and from higher regions in the pontomesencephalic tegmentum. In Mongolian gerbils, acetylcholine acts as an excitatory and modulatory neurotransmitter on VCN neurons, but the anatomical structure of cholinergic innervation of gerbil VCN is not well described. We applied fluorescent immunohistochemical staining to elucidate the development and the cellular localization of presynaptic and postsynaptic components of the cholinergic system in the VCN of the Mongolian gerbil. We found that cholinergic fibers (stained with antibodies against the vesicular acetylcholine transporter) were present before hearing onset at P5, but innervation density increased in animals after P10. Early in development cholinergic fibers invaded the VCN from the medial side, spread along the perimeter and finally innervated all parts of the nucleus only after the onset of hearing. Cholinergic fibers ran in a rostro‐caudal direction within the nucleus and formed en‐passant swellings in the neuropil between principal neurons. Nicotinic and muscarinic receptors were expressed differentially in the VCN, with nicotinic receptors being mostly expressed in dendritic areas while muscarinic receptors were located predominantly in somatic membranes. These anatomical data support physiological indications that cholinergic innervation plays a role in modulating information processing in the cochlear nucleus.     

Cholinergic profiles in the Goettingen miniature pig (Sus scrofa domesticus) brain

Central cholinergic structures within the brain of the even‐toed hoofed Goettingen miniature domestic pig (Sus scrofa domesticus) were evaluated by immunohistochemical visualization of choline acetyltransferase (ChAT) and the low‐affinity neurotrophin receptor, p75^NTR. ChAT‐immunoreactive (‐ir) perikarya were seen in the olfactory tubercle, striatum, medial septal nucleus, vertical and horizontal limbs of the diagonal band of Broca, and the nucleus basalis of Meynert, medial habenular nucleus, zona incerta, neurosecretory arcuate nucleus, cranial motor nuclei III and IV, Edinger‐Westphal nucleus, parabigeminal nucleus, pedunculopontine nucleus, and laterodorsal tegmental nucleus. Cholinergic ChAT‐ir neurons were also found within transitional cortical areas (insular, cingulate, and piriform cortices) and hippocampus proper. ChAT‐ir fibers were seen throughout the dentate gyrus and hippocampus, in the mediodorsal, laterodorsal, anteroventral, and parateanial thalamic nuclei, the fasciculus retroflexus of Meynert, basolateral and basomedial amygdaloid nuclei, anterior pretectal and interpeduncular nuclei, as well as select laminae of the superior colliculus. Double immunofluorescence demonstrated that virtually all ChAT‐ir basal forebrain neurons were also p75^NTR‐positive. The present findings indicate that the central cholinergic system in the miniature pig is similar to other mammalian species. Therefore, the miniature pig may be an appropriate animal model for preclinical studies of neurodegenerative diseases where the cholinergic system is compromised. J. Comp. Neurol. 525:553–573, 2017. © 2016 Wiley Periodicals, Inc.     

Sushi repeat‐containing protein X‐linked 2: A novel phylogenetically conserved hypothalamo‐pituitary protein

Sushi repeat‐containing protein X‐linked 2 (SRPX2) is a novel protein associated with language development, synaptic plasticity, tissue remodeling, and angiogenesis. We investigated the expression and spatial localization of SRPX2 in normal mouse, rat, monkey, and human brain using in situ hybridization and immunohistochemistry. Antibody specificity was determined using in vitro siRNA based silencing of SRPX2. Cell type‐specific expression was verified by double‐labeling with oxytocin or vasopressin. Western blot was used to detect SRPX2 protein in rat and human plasma and cerebrospinal fluid. Unexpectedly, SRPX2 mRNA expression levels were strikingly higher in the hypothalamus as compared to the cortex. All SRPX2 immunoreactive (ir) neurons were localized in the hypothalamic paraventricular, periventricular, and supraoptic nuclei in mouse, rat, monkey, and human brain. SRPX2 colocalized with vasopressin or oxytocin in paraventricular and supraoptic neurons. Hypothalamic SRPX2‐ir positive neurons gave origin to dense projections traveling ventrally and caudally toward the hypophysis. Intense axonal varicosities and terminal arborizations were identified in the rat and human neurohypophysis. SRPX2‐ir cells were also found in the adenohypophysis. Light SRPX2‐ir projections were observed in the dorsal and ventral raphe, locus coeruleus, and the nucleus of the solitary tract in mouse, rat and monkey. SRPX2 protein was also detected in plasma and CSF. Our data revealed intense phylogenetically conserved expression of SRPX2 protein in distinct hypothalamic nuclei and the hypophysis, suggesting its active role in the hypothalamo‐pituitary axis. The presence of SRPX2 protein in the plasma and CSF suggests that some of its functions depend on secretion into body fluids.     

Quantitative mapping reveals age and sex differences in vasopressin,but not oxytocin,immunoreactivity in the rat social behavior neural network

The neuropeptides vasopressin (AVP) and oxytocin (OT) have been implicated in the regulation of numerous social behaviors in adult and juvenile animals. AVP and OT signaling predominantly occur within a circuit of interconnected brain regions known collectively as the “social behavior neural network” (SBNN). Importantly, AVP and OT signaling within the SBNN has been shown to differentially regulate diverse social behaviors, depending on the age and/or sex of the animal. We hypothesized that variation in the display of these behaviors is due in part to age and sex differences in AVP and OT synthesis within the SBNN. However, a thorough characterization of AVP and OT‐immunoreactive (ir) fibers and cell bodies across age and sex within the SBNN has been lacking in rats. We therefore quantified AVP‐ and OT‐ir fibers and cell bodies in 22 subregions of the forebrain SBNN in juvenile and adult, male and female rats. We found numerous age (16 subregions) and sex (10 subregions) differences in AVP‐ir fiber fractional areas, and AVP‐ir cell body numbers, which were mainly observed in the medial amygdala/bed nucleus of the stria terminalis to lateral septum circuit. In contrast to AVP, we observed no age or sex differences in OT‐ir fiber fractional areas or cell bodies in any of the 22 subregions of the forebrain SBNN. Thus, unlike the static pattern observed for OT, AVP innervation of the forebrain SBNN appears to undergo developmental changes, and is highly sexually dimorphic, which likely has significant functional consequences for the regulation of social behavior.     

Immunocytochemical organization and sour taste activation in the rostral nucleus of the solitary tract of mice

Sensory inputs from the oropharynx terminate in both the trigeminal brainstem complex and the rostral part of the nucleus of the solitary tract (nTS). Taste information is conveyed via the facial and glossopharyngeal nerves, while general mucosal innervation is carried by the trigeminal and glossopharyngeal nerves. In contrast, the caudal nTS receives general visceral information largely from the vagus nerve. Although the caudal nTS shows clear morphological and molecularly delimited subdivisions, the rostral part does not. Thus, linking taste‐induced patterns of activity to morphological subdivisions in the nTS is challenging. To test whether molecularly defined features of the rostral nTS correlate with patterns of taste‐induced activity, we combined immunohistochemistry for markers of various visceral afferent and efferent systems with c‐Fos–based activity maps generated by stimulation with a sour tastant, 30 mM citric acid. We further dissociated taste‐related activity from activity arising from acid‐sensitive general mucosal innervation by comparing acid‐evoked c‐Fos in wild‐type and “taste blind” P2X₂/P2X₃ double knockout (P2X‐dbl KO) mice. In wild‐type mice, citric acid stimulation evoked significant c‐Fos activation in the central part of the rostral nTS—activity that was largely absent in the P2X‐dbl KO mice. P2X‐dbl KO mice, like wild‐type mice, did exhibit acid‐induced c‐Fos activity in the dorsomedial trigeminal brainstem nucleus situated laterally adjacent to the rostral nTS. This dorsomedial nucleus also showed substantial innervation by trigeminal nerve fibers immunoreactive for calcitonin gene‐related peptide (CGRP), a marker for polymodal nociceptors, suggesting that trigeminal general mucosal innervation carries information about acids in the oral cavity. J. Comp. Neurol. 525:271–290, 2017. © 2016 Wiley Periodicals, Inc.     

Connectivity and ultrastructure of dopaminergic innervation of the inner ear and auditory efferent system of a vocal fish

Dopamine (DA) is a conserved modulator of vertebrate neural circuitry, yet our knowledge of its role in peripheral auditory processing is limited to mammals. The present study combines immunohistochemistry, neural tract tracing, and electron microscopy to investigate the origin and synaptic characteristics of DA fibers innervating the inner ear and the hindbrain auditory efferent nucleus in the plainfin midshipman, a vocal fish that relies upon the detection of mate calls for reproductive success. We identify a DA cell group in the diencephalon as a common source for innervation of both the hindbrain auditory efferent nucleus and saccule, the main hearing endorgan of the inner ear. We show that DA terminals in the saccule contain vesicles but transmitter release appears paracrine in nature, due to the apparent lack of synaptic contacts. In contrast, in the hindbrain, DA terminals form traditional synaptic contacts with auditory efferent neuronal cell bodies and dendrites, as well as unlabeled axon terminals, which, in turn, form inhibitory‐like synapses on auditory efferent somata. Our results suggest a distinct functional role for brain‐derived DA in the direct and indirect modulation of the peripheral auditory system of a vocal nonmammalian vertebrate.     

Expression of the cold thermoreceptor TRPM8 in rodent brain thermoregulatory circuits

The cold‐ and menthol‐activated ion channel transient receptor potential channel subfamily M member 8 (TRPM8) is the principal detector of environmental cold in mammalian sensory nerve endings. Although it is mainly expressed in a subpopulation of peripheral sensory neurons, it has also been identified in non‐neuronal tissues. Here, we show, by in situ hybridization (ISH) and by the analysis of transgenic reporter expression in two different reporter mouse strains, that TRPM8 is also expressed in the central nervous system. Although it is present at much lower levels than in peripheral sensory neurons, we found cells expressing TRPM8 in restricted areas of the brain, especially in the hypothalamus, septum, thalamic reticular nucleus, certain cortices and other limbic structures, as well as in some specific nuclei in the brainstem. Interestingly, positive fibers were also found traveling through the major limbic tracts, suggesting a role of TRPM8‐expressing central neurons in multiple aspects of thermal regulation, including autonomic and behavioral thermoregulation. Additional ISH experiments in rat brain demonstrated a conserved pattern of expression of this ion channel between rodent species. We confirmed the functional activity of this channel in the mouse brain using electrophysiological patch‐clamp recordings of septal neurons. These results open a new window in TRPM8 physiology, guiding further efforts to understand potential roles of this molecular sensor within the brain.     

Brain mapping of the gonadotropin-inhibitory hormone-related peptide 2 with a novel antibody suggests a connection with emotional reactivity in the Japanese quail (Coturnix japonica,Temminck & Schlegel, 1849)

Gonadotropin-inhibitory hormone (GnIH) is a neuropeptide first discovered in the quail brain that is involved in the control of reproductive physiology and behaviors, and stress response. GnIH gene encodes a second peptide, GnIH-related peptide-2 (RP2), the distribution and function of which remain unknown. We therefore studied GnIH-RP2 distribution by immunohistochemistry using a novel antibody capable of discriminating between GnIH and GnIH-RP2. The overall distribution of GnIH-RP2 is similar to that of GnIH. The vast majority of labeled neurons is located in the paraventricular nucleus (PVN) of the hypothalamus. Labeling of fibers is conspicuous in the diencephalon, but present also in the mesencephalon and telencephalon. Several regions involved in the control of reproduction and stress response (the PVN, septum, bed nucleus of the stria terminalis and nucleus commissura pallii) showed a dense network of immunolabeled fibers. To investigate the potential function of GnIH-RP2 we compared its expression in two quail lines genetically selected for divergence in their emotional reactivity. A quantitative analysis in the above-mentioned brain regions showed that the density of fibers was similar in the two lines. However, the number of GnIH-RP2 labeled neurons was higher in the median portion of the PVN in birds with higher emotional reactivity. These results point to a possible involvement of GnRH-RP2 in modulating stress response and/or emotional reactivity.     

A comparative examination of neural circuit and brain patterning between the lamprey and amphioxus reveals the evolutionary origin of the vertebrate visual center

Vertebrates are equipped with so‐called camera eyes, which provide them with image‐forming vision. Vertebrate image‐forming vision evolved independently from that of other animals and is regarded as a key innovation for enhancing predatory ability and ecological success. Evolutionary changes in the neural circuits, particularly the visual center, were central for the acquisition of image‐forming vision. However, the evolutionary steps, from protochordates to jaw‐less primitive vertebrates and then to jawed vertebrates, remain largely unknown. To bridge this gap, we present the detailed development of retinofugal projections in the lamprey, the neuroarchitecture in amphioxus, and the brain patterning in both animals. Both the lateral eye in larval lamprey and the frontal eye in amphioxus project to a light‐detecting visual center in the caudal prosencephalic region marked by Pax6, which possibly represents the ancestral state of the chordate visual system. Our results indicate that the visual system of the larval lamprey represents an evolutionarily primitive state, forming a link from protochordates to vertebrates and providing a new perspective of brain evolution based on developmental mechanisms and neural functions. J. Comp. Neurol. 523:251–261, 2015. © 2014 Wiley Periodicals, Inc.     

Characterization of the axon initial segment of mice substantia nigra dopaminergic neurons

The axon initial segment (AIS) is the site of initiation of action potentials and influences action potential waveform, firing pattern, and rate. In view of the fundamental aspects of motor function and behavior that depend on the firing of substantia nigra pars compacta (SNc) dopaminergic neurons, we identified and characterized their AIS in the mouse. Immunostaining for tyrosine hydroxylase (TH), sodium channels (Na_v) and ankyrin‐G (Ank‐G) was used to visualize the AIS of dopaminergic neurons. Reconstructions of sampled AIS of dopaminergic neurons revealed variable lengths (12–60 μm) and diameters (0.2–0.8 μm), and an average of 50% reduction in diameter between their widest and thinnest parts. Ultrastructural analysis revealed submembranous localization of Ank‐G at nodes of Ranvier and AIS. Serial ultrathin section analysis and 3D reconstructions revealed that Ank‐G colocalized with TH only at the AIS. Few cases of synaptic innervation of the AIS of dopaminergic neurons were observed. mRNA in situ hybridization of brain‐specific Na_v subunits revealed the expression of Na_v1.2 by most SNc neurons and a small proportion expressing Na_v1.6. The presence of sodium channels, along with the submembranous location of Ank‐G is consistent with the role of AIS in action potential generation. Differences in the size of the AIS likely underlie differences in firing pattern, while the tapering diameter of AIS may define a trigger zone for action potentials. Finally, the conspicuous expression of Na_v1.2 by the majority of dopaminergic neurons may explain their high threshold for firing and their low discharge rate.     

Distribution of LPXRFa,a gonadotropin‐inhibitory hormone ortholog peptide,and LPXRFa receptor in the brain and pituitary of the tilapia

In vertebrates, gonadotropin‐releasing hormone (GnRH) and gonadotropin‐inhibitory hormone (GnIH), respectively, regulate reproduction in positive and negative manners. GnIH belongs to the LPXRFa family of peptides previously identified in mammalian and nonmammalian vertebrates. Studying the detailed distribution of LPXRFa as well as its receptor (LPXRFa‐R) in the brain and pituitary is important for understanding their multiple action sites and potential functions. However, the distribution of LPXRFa and LPXRFa‐R has not been studied in teleost species, partially because of the lack of fish‐specific antibodies. Therefore, in the present study, we generated specific antibodies against LPXRFa and its receptor from Nile tilapia (Oreochromis niloticus), and examined their distributions in the brain and pituitary by immunohistochemistry. Tilapia LPXRFa‐immunoreactive neurons lie in the posterior ventricular nucleus of the caudal preoptic area, whereas LPXRFa‐R‐immunoreactive cells are distributed widely. Double immunofluorescence showed that neither LPXRFa‐immunoreactive fibers nor LPXRFa‐R is closely associated or coexpressed with GnRH1, GnRH3, or kisspeptin (Kiss2) neurons. In the pituitary, LPXRFa fibers are closely associated with gonadotropic endocrine cells [expressing luteinizing hormone (LH) and follicle‐stimulating hormone (FSH)], with adrenocorticomelanotropic cells [corticotropin (ACTH) and α‐melanotropin (α‐MSH)], and with somatolactin endocrine cells. In contrast, LPXRFa‐R are expressed only in LH, ACTH, and α‐MSH cells. These results suggest that LPXRFa and LPXRFa‐R signaling acts directly on the pituitary cells independent from GnRH or kisspeptin and could play multiple roles in reproductive and nonreproductive functions in teleosts. J. Comp. Neurol. 524:2753–2775, 2016. © 2016 Wiley Periodicals, Inc.     

A comparative study of the neural stem cell niche in the adult hypothalamus of human,mouse, rat and gray mouse lemur (Microcebus murinus)

The adult brain contains niches of neural stem cells that continuously add new neurons to selected circuits throughout life. Two niches have been extensively studied in various mammalian species including humans, the subventricular zone of the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus. Recently, studies conducted mainly in rodents have identified a third neurogenic niche in the adult hypothalamus. In order to evaluate whether a neural stem cell niche also exists in the adult hypothalamus in humans, we performed multiple immunofluorescence labeling to assess the expression of a panel of neural stem/progenitor cell (NPC) markers (Sox2, nestin, vimentin, GLAST, GFAP) in the human hypothalamus and compared them with the mouse, rat and a non‐human primate species, the gray mouse lemur (Microcebus murinus). Our results show that the adult human hypothalamus contains four distinct populations of cells that express the five NPC markers: (a) a ribbon of small stellate cells that lines the third ventricular wall behind a hypocellular gap, similar to that found along the lateral ventricles, (b) ependymal cells, (c) tanycytes, which line the floor of the third ventricle in the tuberal region, and (d) a population of small stellate cells in the suprachiasmatic nucleus. In the mouse, rat and mouse lemur hypothalamus, co‐expression of NPC markers is primarily restricted to tanycytes, and these species lack a ventricular ribbon. Our work thus identifies four cell populations with the antigenic profile of NPCs in the adult human hypothalamus, of which three appear specific to humans.     

Altered expression of IGF‐I system in neurons of the inflamed spinal cord during acute experimental autoimmune encephalomyelitis

There is growing evidence that the impaired IGF‐I system contributes to neurodegeneration. In this study, we examined the spinal cords of the EAE, the animal model of multiple sclerosis, to see if the expression of the IGF‐I system is altered. To induce EAE, C57/BL6 mice were immunized with the Hooke lab MOG kit, sacrificed at the peak of the disease and their spinal cords were examined for the immunoreactivities (ir) of the IGF‐I, IGF binding protein‐1 (IGFBP‐1) and glycogen synthase kinase 3β (GSK3β), as one major downstream molecule in the IGF‐I signaling. Although neurons in the non EAE spinal cords did not show the IGF‐I immunoreactivity, they were numerously positive for the IGFBP‐1. In the inflamed EAE spinal cord however, the patterns of expressions were reversed, that is, a significant increased number of IGF‐I expressing neurons versus a reduced number of IGFBP‐1 positive neurons. Moreover, while nearly all IGF‐I‐ir neurons expressed GSK3β, some expressed it more intensely. Considering our previous finding where we showed a significant reduced number of the inactive (phosphorylated) but not that of the total GSK3β expressing neurons in the EAE spinal cord, it is conceivable that the intense total GSK3β expression in the IGF‐I‐ir neurons belongs to the active form of GSK3β known to exert neuroinflammatory effects. We therefore suggest that the altered expression of the IGF‐I system including GSK3β in spinal cord neurons might involve in pathophysiological events during the EAE.     

Phenotyping of nNOS neurons in the postnatal and adult female mouse hypothalamus

Neurons expressing nitric oxide (NO) synthase (nNOS) and thus capable of synthesizing NO play major roles in many aspects of brain function. While the heterogeneity of nNOS‐expressing neurons has been studied in various brain regions, their phenotype in the hypothalamus remains largely unknown. Here we examined the distribution of cells expressing nNOS in the postnatal and adult female mouse hypothalamus using immunohistochemistry. In both adults and neonates, nNOS was largely restricted to regions of the hypothalamus involved in the control of bodily functions, such as energy balance and reproduction. Labeled cells were found in the paraventricular, ventromedial, and dorsomedial nuclei as well as in the lateral area of the hypothalamus. Intriguingly, nNOS was seen only after the second week of life in the arcuate nucleus of the hypothalamus (ARH). The most dense and heavily labeled population of cells was found in the organum vasculosum laminae terminalis (OV) and the median preoptic nucleus (MEPO), where most of the somata of the neuroendocrine neurons releasing GnRH and controlling reproduction are located. A great proportion of nNOS‐immunoreactive neurons in the OV/MEPO and ARH were seen to express estrogen receptor (ER) α. Notably, almost all ERα‐immunoreactive cells of the OV/MEPO also expressed nNOS. Moreover, the use of EYFP^Vglut2, EYFP^Vgat, and GFP^Gad67 transgenic mouse lines revealed that, like GnRH neurons, most hypothalamic nNOS neurons have a glutamatergic phenotype, except for nNOS neurons of the ARH, which are GABAergic. Altogether, these observations are consistent with the proposed role of nNOS neurons in physiological processes.     

Dopaminergic modulation of olfactory-evoked motor output in sea lampreys (Petromyzon marinus L.)

Detection of chemical cues is important to guide locomotion in association with feeding and sexual behavior. Two neural pathways responsible for odor-evoked locomotion have been characterized in the sea lamprey (Petromyzon marinus L.), a basal vertebrate. There is a medial pathway originating in the medial olfactory bulb (OB) and a lateral pathway originating from the rest of the OB. These olfactomotor pathways are present throughout the life cycle of lampreys, but olfactory-driven behaviors differ according to the developmental stage. Among possible mechanisms, dopaminergic (DA) modulation in the OB might explain the behavioral changes. Here, we examined DA modulation of olfactory transmission in lampreys. Immunofluorescence against DA revealed immunoreactivity in the OB that was denser in the medial part (medOB), where processes were observed close to primary olfactory afferents and projection neurons. Dopaminergic neurons labeled by tracer injections in the medOB were located in the OB, the posterior tuberculum, and the dorsal hypothalamic nucleus, suggesting the presence of both intrinsic and extrinsic DA innervation. Electrical stimulation of the olfactory nerve in an in vitro whole-brain preparation elicited synaptic responses in reticulospinal cells that were modulated by DA. Local injection of DA agonists in the medOB decreased the reticulospinal cell responses whereas the D2 receptor antagonist raclopride increased the response amplitude. These observations suggest that DA in the medOB could modulate odor-evoked locomotion. Altogether, these results show the presence of a DA innervation within the medOB that may play a role in modulating olfactory inputs to the motor command system of lampreys.     

Identification of bladder and colon afferents in the nodose ganglia of male rats

The sensory neurons innervating the urinary bladder and distal colon project to similar regions of the central nervous system and often are affected simultaneously by various diseases and disorders, including spinal cord injury. Anatomical and physiological commonalities between the two organs involve the participation of shared spinally derived pathways, allowing mechanisms of communication between the bladder and colon. Prior electrophysiological data from our laboratory suggest that the bladder also may receive sensory innervation from a nonspinal source through the vagus nerve, which innervates the distal colon as well. The present study therefore aimed to determine whether anatomical evidence exists for vagal innervation of the male rat urinary bladder and to assess whether those vagal afferents also innervate the colon. Additionally, the relative contribution to bladder and colon sensory innervation of spinal and vagal sources was determined. By using lipophilic tracers, neurons that innervated the bladder and colon in both the nodose ganglia (NG) and L6/S1 and L1/L2 dorsal root ganglia (DRG) were quantified. Some single vagal and spinal neurons provided dual innervation to both organs. The proportions of NG afferents labeled from the bladder did not differ from spinal afferents labeled from the bladder when considering the collective population of total neurons from either group. Our results demonstrate evidence for vagal innervation of the bladder and colon and suggest that dichotomizing vagal afferents may provide a neural mechanism for cross‐talk between the organs. J. Comp. Neurol. 522:3667–3682, 2014. © 2014 Wiley Periodicals, Inc.     

Development of glycine immunoreactivity in the brain of the sea lamprey: Comparison with γ‐aminobutyric acid immunoreactivity

The development of glycine immunoreactivity in the brain of the sea lamprey was studied by use of immunofluorescence techniques at embryonic to larval stages. Glycine distribution was also compared with that of γ‐aminobutyric acid (GABA) by use of double immunofluorescence. The first glycine‐immunoreactive (ir) cells appeared in the caudal rhombencephalon of late embryos, diencephalon of early prolarvae, and mesencephalon of late prolarvae, in which glycine‐ir cells were observed in several prosencephalic regions (preoptic nucleus, hypothalamus, ventral thalamus, dorsal thalamus, pretectum, and nucleus of the medial longitudinal fascicle), mesencephalon (M5), isthmus, and rhombencephalon. In larvae, glycine‐ir populations were observed in the olfactory bulbs, preoptic nucleus and thalamus (prosencephalon), M5 and oculomotor nucleus (mesencephalon), dorsal isthmic gray, isthmic reticular formation, and various alar and basal plate rhombencephalic populations. No glycine‐ir cells were observed in the larval optic tectum or torus semicircularis, which contain glycine‐ir populations in adults. A wide distribution of glycine‐ir fibers was observed, which suggests involvement of glycine in the function of most lamprey brain regions. Colocalization of GABA and glycine in prolarvae was found mainly in cell groups of the diencephalon, in the ventral isthmic group, and in trigeminal populations. In larvae, colocalization of GABA and glycine was principally observed in the M5 nucleus, the reticular formation, and the dorsal column nucleus. The present results reveal for the first time the complex developmental pattern of the glycinergic system in lamprey, including early glycine‐ir populations, populations transiently expressing glycine, and late‐appearing populations, in relation to maturation changes that occur during metamorphosis. J. Comp. Neurol. 512:747–767, 2009. © 2008 Wiley‐Liss, Inc.     

Neuroanatomical details of the lateral neurons of Drosophila melanogaster support their functional role in the circadian system

Drosophila melanogaster is a long‐standing model organism in the circadian clock research. A major advantage is the relative small number of about 150 neurons, which built the circadian clock in Drosophila. In our recent work, we focused on the neuroanatomical properties of the lateral neurons of the clock network. By applying the multicolor‐labeling technique Flybow we were able to identify the anatomical similarity of the previously described E2 subunit of the evening oscillator of the clock, which is built by the 5th small ventrolateral neuron (5th s‐LN_v) and one ITP positive dorsolateral neuron (LN_d). These two clock neurons share the same spatial and functional properties. We found both neurons innervating the same brain areas with similar pre‐ and postsynaptic sites in the brain. Here the anatomical findings support their shared function as a main evening oscillator in the clock network like also found in previous studies. A second quite surprising finding addresses the large lateral ventral PDF‐neurons (l‐LN_vs). We could show that the four hardly distinguishable l‐LN_vs consist of two subgroups with different innervation patterns. While three of the neurons reflect the well‐known branching pattern reproduced by PDF immunohistochemistry, one neuron per brain hemisphere has a distinguished innervation profile and is restricted only to the proximal part of the medulla‐surface. We named this neuron “extra” l‐LN_v (l‐LN_vx). We suggest the anatomical findings reflect different functional properties of the two l‐LN_v subgroups.