One of the main mechanisms used by neurons and glial cells to promote repair following brain injury is to upregulate activity-dependent molecules such as insulin-like growth factor 1 (IGF-1) and interleukin-1β (IL-1β). In the auditory system, IGF-1 is crucial for restoring synaptic transmission following hearing loss; however, whether IL-1β is also involved in this process is unknown. In this study, we evaluated the expression of IGF-1 and IL-1β within neurons and glial cells of the ventral cochlear nucleus in adult rats at 1, 7, 15 and 30 days following bilateral cochlear ablation. After the lesion, significant increases in both the overall mean gray levels of IGF-1 immunostaining and the mean gray levels within cells of the cochlear nucleus were observed at 1, 7 and 15 days compared with control animals. The expression and distribution of IL-1β in the ventral cochlear nucleus of ablated animals was temporally and spatially correlated with IGF-1. We also observed a lack of colocalization between IGF-1 and IL-1β with either astrocytes or microglia at any of the time points following ablation. These results suggest that the upregulation of IGF-1 and IL-1β levels within neurons – but not within glial cells – may reflect a plastic mechanism involved in repairing synaptic homeostasis of the overall cellular environment of the cochlear nucleus following bilateral cochlear ablation. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.

In this study, we investigated the time-window during which dysfunctional neuron-glial signaling – caused by bilateral cochlear ablation – may lead to increased synthesis and release of IGF-1 and IL-1β within the cochlear nucleus. Our findings indicate that there is an upregulation of IGF-1 (arrows in A-C) and IL-1β (arrows in G-I) levels within neurons (D, J-L) but not within glial cells (E, F, M, N) in the cochlear nucleus which may reflect a synaptic reparative mechanism following bilateral cochlear ablation.

Microglia are the brain resident macrophages tasked with the defense and maintenance of the central nervous system (CNS). The hematopoietic origin of microglia has warranted a therapeutic potential for the hematopoietic system in treating diseases of the CNS. However, migration of bone marrow derived cells into the CNS is a marginal event under normal healthy conditions. A busulfan-based chemotherapy regimen was used for bone marrow transplantation in WT mice before subjecting them to a hypoxic-ischemic brain injury or in APP/PS1 mice prior to the formation of amyloid plaques. The cells were tracked and analyzed throughout the development of the pathology. The efficacy of a preventive M-CSF treatment was also studied to highlight the effects of circulating monocytes in hypoxic-ischemic brain injury. Such an injury induces a strong migration of BMDC into the CNS, without the need for irradiation. These migrating cells do not replace the entire microglia pool but are rather confined to the sites of injury for several weeks, suggesting that they could perform specific functions. M-CSF showed neuroprotective effects as a preventive treatment. In APP/PS1 mice, the formation of amyloid plaques was sufficient to induce the entry of cells into the parenchyma, though in low numbers. This study confirms that bone marrow-derived cells infiltrate the CNS in animal models for stroke and Alzheimer's disease and that peripheral cells can be targeted to treat affected regions of the CNS. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.

Using a myeloablative chemotherapy regimen, the authors have investigated the capacity of bone marrow derived cells to enter into the central nervous system. These cells migrated specifically to sites of injuries in models of stroke and Alzheimer's disease but did not repopulate the entire parenchyma.

Horizontal head rotation evokes significant responses from trochlear motoneurons of turtle that suggests they have a functional role in abduction of the eyes like that in frontal-eyed mammals. The finding is unexpected given that the turtle is generally considered lateral-eyed and assumed to have eye movements instead like that of lateral-eyed mammals, where innervation of the superior oblique muscle by the trochlear nerve (nIV) produces intorsion, elevation, and adduction (not abduction). Using an isolated turtle head preparation with the brain removed, glass suction electrodes were used to stimulate nIV with trains of current pulses. Eyes were monitored via an infrared camera with the head placed in a gimble to quantify eye rotations and their directions. Stimulations of nIV evoked intorsion, elevation, and abduction. Dissection of the superior oblique muscle identified lines of action and a location of insertion on the eye, which supported kinematics evoked by nIV stimulation. Eye positions in alert behaving turtles with their head-extended were compared to that when their heads were retracted in the carapace. When the head was retracted, there was a reduction in interpupillary distance and an increase in binocular overlap. Occlusion of peripheral fields by the carapace forces the turtle to a more frontal-eyed state, perhaps the reason for the action of abduction by the superior oblique muscle. These findings support why trochlear motoneurons in turtle respond in the same way as abducens motoneurons to horizontal rotations, an unusual characteristic of vestibulo-ocular physiology in comparison to other mammalian lateral-eyed species. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.

Unlike the rabbit, trochlear nerve (nIV) stimulation in turtle evokes ocular abduction, like that observed in frontal-eyed mammals. Interpupillary distance also decreases due to lateral occlusion when turtles retract their heads into the carapace. This may be the evolutionary reason that the turtle's trochlear activation resembles frontal-eyed mammals.

In the present study, we used microelectrode recordings of multiunit responses to evaluate patterns of the reactivation of somatosensory cortex after sensory loss produced by spinal cord lesions in the common marmoset (Callithrix jacchus). These New World monkeys have become a popular model in studies of cortical organization and function. Primary somatosensory cortex and adjoining somatosensory areas can become extensively deactivated by lesions of somatosensory afferents as they ascend in the dorsal columns of the cervical spinal cord. Six to seven weeks after complete lesions of the cuneate fasciculus subserving the forelimb at cervical levels 5-6, the hand region in contralateral areas 3b and 1 were reactivated by inputs from the forelimb, but excluded representations of some or all digits. In a similar manner, recording sites from the forelimb region of areas 2-5 responded to parts of the forelimb but not to digits after an extensive lesion of the contralateral cuneate fasciculus at C5-C6. Lesions that damaged only the gracile fasciculus or a small percentage of the cuneate fasciculus did not produce changes in the gross hand representation in contralateral areas 3b, 3a, 1, and 2. Finally, a complete but lower lesion of the cuneate fasciculus at C8 produced some abnormalities in the reactivation, but the digits were represented. The results indicate that areas 3a, 3b, 1, and 2-5 of the somatosensory cortex are extensively reactivated after large, apparently complete lesions of the contralateral cuneate fasciculus, but afferents from the digits may not contribute to their reactivation. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.

Microelectrode recording results from cortex caudal to area 3b of a marmoset that received a cervical dorsal column lesion indicate that the 2-5 region is responsive to tactile stimulation, and the somatotopy of the region may be altered by long-standing sensory loss.

The presubiculum (PrS) plays critical roles in spatial information processing and memory consolidation and has also been implicated in temporal lobe epileptogenesis. Despite its involvement in these processes, a basic structure-function analysis of PrS cells remains far from complete. To this end, we performed whole-cell recording and biocytin-labeling of PrS neurons in layer (L) II and LIII to examine their electrophysiological and morphological properties. We characterized the cell types based on electrophysiological criteria, correlated their gross morphology, and classified them into distinct categories using unsupervised hierarchical cluster analysis. We identified seven distinct cell types: regular-spiking (RS), irregular-spiking (IR), initially-bursting (IB), stuttering (Stu), single-spiking (SS), fast-adapting (FA), and late-spiking (LS) cells, of which RS and IB cells were common to LII and LIII, LS cells were specific to LIII, and the remaining cell types were identified exclusively in LII. Recorded neurons were either pyramidal or non-pyramidal shaped, and barring Stu cells, displayed spine-rich dendrites. The RS, IB and IR cells appeared to be projection neurons based on extension of their axons into LIII of the medial entorhinal area (MEA) and/or angular bundle. We conclude that layers II and III of PrS are distinct in their neuronal populations and together constitute a more diverse population of neurons than previously suggested. PrS neurons serve as major drivers of circuits in superficial (LII-III) entorhinal cortex (ERC) and couple neighboring structures through robust afferentation thereby substantiating presubiculum's critical role in the parahippocampal region. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.

This study characterizes/classifies neuron types in superficial layers of the rat presubiculum based on physiological properties assayed via whole-cell recordings while correlating their gross morphology through biocytin-labeling. The identification of seven physiologically distinct neuron types suggests the presubiculum as being a more neuronally-diverse structure than previously envisaged.

Genetic factors that influence seizure susceptibility can act transiently during the development of neural circuits, or may be necessary for the proper functioning of existing circuits. We provide evidence that the Drosophila seizure-sensitive mutant easily shocked, (eas), represents a neurological disorder in which abnormal functioning of existing neural circuits leads to seizure-sensitivity. The eas⁺ gene encodes for the protein Ethanolamine Kinase, involved in phospholipid biosynthesis. We show that induction of eas⁺ in adult mutant flies rescues seizure-sensitivity despite previously known developmental defects in brain morphology. Additionally, through cell-type specific rescue, our results suggest a specific role for eas⁺ in excitatory rather than inhibitory neural transmission. Overall, our findings emphasize an important role for proper phospholipid metabolism in normal brain function, and suggest that certain classes of epilepsy syndromes could have the potential to be treated with gene therapy techniques. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.

We show that seizure-sensitivity in the Drosophila mutant easily shocked (eas), a gene encoding Ethanolamine Kinase, can be rescued using conditional expression of the wild-type gene in adult flies. Additionally, expression in cholinergic rather than GABAergic neurons is sufficient for rescue, indicating distinct neuronal circuits are affected in the mutant.

Cholinergic neurons are the major excitatory neurons of the enteric nervous system (ENS), and include intrinsic sensory neurons, interneurons and excitatory motor neurons. Cholinergic neurons have been detected in the embryonic ENS, however, the development of these neurons has been difficult to study as they are difficult to detect prior to birth using conventional immunohistochemistry. In this study, we used ChAT-Cre;R26R-YFP mice to examine the development of cholinergic neurons in the gut of embryonic and postnatal mice. Cholinergic (YFP+) neurons were first detected at embryonic day (E)11.5, and the proportion of cholinergic neurons gradually increased during pre- and postnatal development. At birth, myenteric cholinergic neurons comprised less than half of their adult proportions in the small intestine (25% of myenteric neurons were YFP+ at P0 compared to 62% in adults). The earliest cholinergic neurons appear to mainly project anally. Projections into the presumptive circular muscle were first observed at E14.5. A subpopulation of cholinergic neurons co-express calbindin through embryonic and postnatal development, but only a small proportion co-expressed neuronal nitric oxide synthase. Our study shows that cholinergic neurons in the ENS develop over a protracted period of time. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.

Using ChAT-Cre;R26R-YFP mice, the authors were able to examine the embryonic and postnatal development of myenteric cholinergic neurons. The authors show that although some cholinergic neurons differentiate very early during the development of the enteric nervous system, they do not reach adult proportions until around post-natal day 10.

Tanycytes are highly specialized ependymal cells that form a blood-cerebrospinal fluid (CSF) barrier at the level of the median eminence (ME), a circumventricular organ (CVO) located in the tuberal region of the hypothalamus. This ependymal layer harbors well-organized tight junctions, a hallmark of central nervous system barriers that is lacking in the fenestrated portal vessels of the ME. The displacement of barrier properties from the vascular to the ventricular side allows the diffusion of blood-borne molecules into the parenchyma of the ME while tanycyte tight junctions control their diffusion into the CSF, thus maintaining brain homeostasis. In the present work, we combined immunohistochemical and permeability studies to investigate the presence of tanycyte barriers along the ventricular walls of other brain CVOs. Our data indicate that, unlike cuboidal ependymal cells, ependymal cells bordering the CVOs possess long processes that project into the parenchyma of the CVOs to reach the fenestrated capillary network. Remarkably, these tanycyte-like cells display well-organized tight junctions around their cell bodies. Consistent with these observations, permeability studies show that this ependymal layer acts as a diffusion barrier. Together, our results suggest that tanycytes are a characteristic feature of all CVOs and yield potential new insights into their involvement in regulating the exchange between the blood, the brain and the CSF within these “brain windows”. J. Comp. Neurol. , 2013. © 2013 Wiley Periodicals, Inc.

Combining immunohistochemical with permeability studies, the authors provide evidence that tanycyte-like cells make up a blood-cerebrospinal fluid barrier at the ventricular wall of the circumventricular organs. Tight junction-bearing tanycyte-like cells facing the fenestrated vessels of the circumventricular organs ensure brain homeostasis by confining blood-borne molecules to these “brain windows”.

While physiological studies suggested convergence of chorda tympani and glossopharyngeal afferent axons onto single neurons of the rostral nucleus of the solitary tract (rNTS), anatomical evidence has been elusive. The current study uses high-magnification confocal microscopy to identify putative synaptic contacts from afferent fibers of the two nerves onto individual projection neurons. Imaged tissue is re-visualized with electron microscopy, confirming that overlapping fluorescent signals in confocal z-stacks accurately identify appositions between labeled terminal and dendrite pairs. Monte Carlo modeling reveals that the probability of overlapping fluorophores is stochastically unrelated to the density of afferent label suggesting that convergent innervation in the rNTS is selective rather than opportunistic. Putative synaptic contacts from each nerve are often compartmentalized onto dendrite segments of convergently innervated neurons. These results have important implications for orosensory processing in the rNTS, and the techniques presented here have applications in investigations of neural microcircuitry with an emphasis on innervation patterning. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.

Using high resolution confocal and electron microscopy, the authors demonstrate monosynaptic convergence of the chorda tympani and glossopharyngeal nerves onto individual ascending projection neurons in the rostral nucleus of the solitary tract. This circuit arrangement is likely crucial in the initial processing of orosensory information.

The projection pattern of the olivocerebellar (OC) axons, which terminate mainly as climbing fibers (CFs) in the cerebellar cortex, tightly reflects the compartmental and developmental organization of the cerebellum as revealed by mapping and reconstruction studies in the rat. The avian cerebellum is well lobulated and longitudinally-compartmentalized like the mammalian cerebellum. However, the projection pattern of the OC axons has not been studied in detail for most areas of the avian cerebellum. In the present study, we reconstructed labeled chick OC axons resulting from a small focal injection of biotinylated dextran amine into the inferior olive to investigate their morphological characteristics, and to determine their relationship to the general morphology of the chick cerebellum.

Labeled CFs were distributed basically in a single longitudinally-elongated narrow band-shaped area in lobules I-VIII, but in multiple, transversely widened, band-shaped areas in lobules IX-X. Three of the four reconstructed OC axons terminated in a single longitudinally band-shaped area in lobules IXa-c, while the other one that terminated in multiple mediolaterally separated areas in lobule IXc, which is part of the flocculus. Single OC axons branched into 14 CFs on the average. Two CFs occasionally merged together to form a single terminal arbor. Axons also had thin, non-CF collaterals that projected either to a cerebellar nucleus or to the cortex. The results indicate that the morphological characteristics of OC axons, including branching and termination, are basically conserved between the chick and the rat. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.

We reconstructed labeled chick olivocerebellar axons resulting from a small focal injection of biotinylated dextran amine into the inferior olive. Morphological characteristics of olivocerebellar axons, including branching and termination, are basically conserved between the chick and the rat. Climbing fibers were distributed usually in a single longitudinally-elongated narrow band-shaped area.

The neural crest is a population of mesenchymal cells that after migrating from the neural tube give rise to a structures and cell-types: jaw, part of the peripheral ganglia and melanocytes. Although much is known about neural crest development in jawed vertebrates, a clear picture of trunk neural crest development for elasmobranchs is yet to be developed. Here we present a detailed study of trunk neural crest development in the bamboo shark, Chiloscyllium punctatum. Vital labeling with DiI and in situ hybridization using cloned Sox8 and Sox9 probes demonstrated that trunk neural crest cells follow a pattern similar to the migratory paths already described in zebrafish and amphibians. We found shark trunk neural crest along the rostral side of the somites, the ventromedial pathway, branchial arches, gut, sensory ganglia and nerves. Interestingly, Chiloscyllium punctatum Sox8 and Sox9 sequences aligned with vertebrate SoxE genes, but appeared to be more ancient than the corresponding vertebrate paralogs. The expression of these two SoxE genes in trunk neural crest cells, especially Sox9, matched the Sox10 migratory patterns observed in teleosts. Interestingly, we observed DiI cells and Sox9 labeling along the lateral line, suggesting that in C. punctatum, glial cells in the lateral line are likely of neural crest origin. Though this has been observed in other vertebrates, we are the first to show that the pattern is present in cartilaginous fishes. These findings demonstrate that trunk neural crest cell development in Chiloscyllium punctatum follows the same highly conserved migratory pattern observed in jawed vertebrates J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.

Using live DiI labeling and Sox8 and Sox9 in situ the authors show that migration of trunk neural crest cells in sharks is highly conserved across evolution. They found cells over the rostral somites and in ventral portion of the embryo (purple for Sox9 labeling and red for DiI labeling).

Nuclei in the central nervous system are three-dimensional (3D) aggregates of neurons that have common physiological properties, functionalities and connectivities. To form specific nuclei, neurons migrate from their birthplace towards the presumptive nuclear region where they change their dynamics to aggregate and re-arrange into a distinct 3D structure, a process that we term nucleogenesis. Nuclei, together with the laminar structure, form the basic cytoarchetectonic unit for information processing. However, in contrast to much studied laminar structures, the neuronal dynamics that contribute to the aggregation process to form nuclei are poorly understood. Here, we analyze nucleogenesis by observing the mouse precerebellar pontine nucleus (PN), and provide the first four-dimensional view of nucleogenesis by tracking neuronal behaviors along the three spatial axes over time. Early- and late-born PN neurons were labeled by in utero electroporation and their behaviors on cultured brain slices were recorded by time-lapse imaging. We find that when PN neurons migrate medially into the nuclear region, many of them switch to migrate radially and laterally, to populate the dorsal and lateral PN regions, respectively. The tendency to switch to radial migration is much less in later-born neurons, whereas that to switch to lateral migration is comparable between the two groups. In contrast to the radial and mediolateral axes, very few PN neurons switch to migrate rostrocaudally. These results could thus provide a framework for understanding the mechanisms that regulate this complex yet important process. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.

We performed systematic analysis of neuronal dynamics during pontine nucleus (PN) formation along the three spatial axes over time. PN neurons display several modes of migration after entering nuclear region. There are distinct differences of neuronal behaviors between neurons of early and late birthdates, as well as along the three spatial dimensions.

The central and medial extended amygdala comprises the central (CEA) and medial nuclei of the amygdala (MEA), respectively, together with anatomically connected regions of the bed nucleus of the stria terminalis (BST). To reveal direct and multisynaptic connections within the central and medial extended amygdala, monosynaptic and transneuronal viral tracing experiments were performed in adult male rats. In the first set of experiments, a cocktail of anterograde and retrograde tracers was iontophoretically delivered into the medial CEA (CEAm), anterodorsal MEA (MEAad), or posterodorsal MEA (MEApd), revealing direct, topographically-organized projections between distinct amygdalar and BST subnuclei. In the second set of experiments, the retrograde transneuronal tracer pseudorabies virus (PRV) was microinjected into the CEAm or MEAad. After 48 hr survival, there were no significant differences between monosynaptic and PRV cases in the subnuclear distribution or proportions of retrogradely-labeled BST neurons. However, after 60 hr survival, CEAm-injected cases displayed an increased proportion of labeled neurons within the anteromedial group of BST subnuclei (amgBST) and within the posterior BST, which do not directly innervate the CEA. MEApd-injected 60 hr cases displayed a significantly increased proportion of retrograde labeling in the amgBST compared to monosynaptic and 48 hr cases, whereas MEAad-injected cases displayed no proportional changes over time. Thus, multisynaptic circuits within the medial extended amygdala overlap the direct connections comprising this anatomical unit, whereas the multisynaptic boundaries of the central extended amygdala extend into BST subnuclei previously identified as part of the medial extended amygdala. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.

Using iontophoretic delivery of monosynaptic tracers and pressure injections of transneuronal viral tracers, the authors map resulting labeling patterns to demonstrate that direct and multisynaptic pathways link disctinct subregions of the bed nucleus of stria terminalis with distinct subregions of the central and medial amygdala in the adult male rat brain.

Despite its anatomical prominence, the function of primate pulvinar is poorly understood. A few electrophysiological studies in simian primates have investigated the functional organization of pulvinar by examining visuotopic maps. Multiple visuotopic maps have been found in all studied simians, with differences in organization reported between New and Old World simians. Given that prosimians are considered closer to the common ancestors of New and Old World primates, we investigated the visuotopic organization of pulvinar in the prosimian bush baby (Otolemur garnettii). Single electrode extracellular recording was used to find the retinotopic maps in the lateral (PL) and inferior (PI) pulvinar. Based on recordings across cases a 3D model of the map was constructed. From sections stained for Nissl bodies, myelin, acetylcholinesterase, calbindin or cytochrome oxidase, we identified three PI chemoarchitectonic subdivisions, lateral central (PIcl), medial central (PIcm) and medial (PIm) inferior pulvinar. Two major retinotopic maps were identified that cover PL and PIcl, the dorsal one in dorsal PL and the ventral one in PIcl and ventral PL. Both maps represent the central vision at the posterior end of the border between the maps, the upper visual field in the lateral half and the lower visual field in the medial half. They share many features with the maps reported in the pulvinar of simians, including location in pulvinar and the representation of the upper-lower and central-peripheral visual field axes. The second order representation in the lateral map and a laminar organization are likely features specific to Old World simians. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.

Two retinotopic maps were identified electrophysiologically in bush baby lateral (PL) and inferior (PI) pulvinar. In PI three additional chemoarchitectonic subdivisions were identified (Not shown). The location and orientation of these maps in pulvinar are similar but not identical between prosimians and simians. The maps are depicted on the drawing of a coronal section in which + and – represent upper and lower visual fields and numerals represent eccentricity. LGN, lateral geniculate nucleus.

Proopiomelanocortin (POMC) and agouti-related protein (AGRP) neurons in the hypothalamus regulate various aspects of energy homeostasis and metabolism. POMC and AGRP neurons, respectively, agonize and antagonize melanocortin receptors on their common downstream neurons. However, it is unknown whether they also reciprocally stimulate and inhibit the same neurons by amino acid transmitters. While AGRP neurons are mostly GABAergic, surprisingly, only a small population of POMC neurons has been found to be glutamatergic, and a significantly larger subpopulation to be GABAergic. To further examine amino acid phenotypes of POMC neurons, we studied mRNA expression for the glutamatergic marker, type 2 vesicular glutamate transporter (VGLUT2), and the GABA synthetic enzyme, GAD67, in POMC neurons of both rats and mice using in situ hybridization techniques. In rats, approximately 58% of POMC neurons were labeled for VGLUT2 and 37% for GAD67 mRNA. In mice, approximately 43% of POMC neurons contained VGLUT2, and 54% contained GAD67 mRNA. In both species, a prominent medio-lateral distribution pattern was observed at rostral and mid levels of the POMC cell group with VGLUT2-POMC neurons dominating in lateral portions and GAD67-POMC neurons in medial portions. These data demonstrate that both glutamatergic and GABAergic cells are present in comparably significant numbers among POMC neurons. Their glutamatergic or GABAergic phenotype may represent a major functional division within the POMC cell group. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.

The mRNA expression of the glutamatergic marker, VGLUT2, and the GABAergic marker, GAD67, was studied in hypothalamic POMC neurons. In rats, VGLUT2 and GAD67 were expressed in 58% and 37% of POMC neurons, respectively. In mice, 43% of POMC neurons were labeled for VGLUT2 and 54% for GAD67 mRNA. These data demonstrate that significant glutamatergic and GABAergic subpopulations of POMC neurons both exist.

Vesicular glutamate transporters (VGLUT1-3) carry glutamate into synaptic vesicles. VGLUT3 has been reported to be localized in non-glutamatergic neuronal populations in the brain. However, detailed subcellular localization of VGLUT3 has not been shown. In particular, the identity of synaptic vesicles expressing VGLUT3 remains to be revealed. Here we present novel electron microscopic postembedding immunogold data from mouse and rat brains showing that small, clear and round synaptic vesicles in GABAergic nerve terminals contain labeling for both VGLUT3 and the vesicular GABA transporter (VGAT). Immunoisolation of synaptic vesicles confirmed the immunogold data and show vesicular co-localization of VGLUT3 and VGAT. Moreover, we show that gold particles signaling VGLUT3 are present in synaptic vesicles in acetylcholinergic nerve terminals in the striatum. Quantitative immunogold analyses reveal that the density of VGLUT3 gold particles is similar in GABAergic terminals in the hippocampus and the neocortex as in cholinergic terminals in the striatum. In contrast to in the hippocampus and the neocortex, VGLUT3 was absent from VGAT positive terminals in the striatum. The labeling pattern produced by the VGLUT3 antibodies was found to be specific; there was no labeling in VGLUT3 knockout tissue, and the observed labeling throughout the rat brain corresponds to the known light-microscopic distribution of VGLUT3. From the present results we infer that glutamate is released with GABA from inhibitory terminals and acetylcholine from cholinergic terminals. J. Comp. Neurol. , 2013. © 2013 Wiley Periodicals, Inc.

Prior to release glutamate is packed into synaptic vesicles by a family of vesicular glutamate transporters (VGLUT1-3). VGLUT3 is known to be located in non-glutamatergic neurons. Here we show that VGLUT3 is localized on synaptic vesicles together with the vesicular GABA transporter (VGAT) in GABAergic nerve terminals in the neocortex and the hippocampus. In the striatum VGLUT3 is present on synaptic vesicles in cholinergic, but not in GABAergic terminals. This suggests that glutamate and GABA are co-released in certain brain regions, leading to a more sophisticated modulation of brain signaling than previously thought.

The midbrain periaqueductal gray (PAG) is involved in many basic survival behaviors that affect respiration. We hypothesized that the PAG promotes these behaviors by changing the firing of pre-inspiratory (pre-I) neurons in the pre-Bötzinger complex, a cell group, which is thought to be important in generating respiratory rhythm. We tested this hypothesis by recording single unit activity of pre-Bötzinger pre-I neurons during stimulation in different parts of the PAG. Stimulation in the dorsal PAG increased the firing of pre-I neurons resulting in tachypnea. Stimulation in the medial part of the lateral PAG converted the pre-I neurons into inspiratory phase-spanning cells resulting in inspiratory apneusis. Stimulation in the lateral part of the lateral PAG generated an early onset of the pre-I neuronal discharge, which continued throughout the inspiratory phase, while at the same time attenuating diaphragm contraction. Stimulation in the ventral part of the lateral PAG induced tachypnea, but inhibited pre-I cell firing, while stimulation in the ventrolateral PAG not only inhibited pre-I cells but also the diaphragm leading to apnea. These findings show that PAG stimulation changes the activity of the pre-Bötzinger pre-I neurons. These changes are in line with the different behaviors generated by the PAG, such as the dorsal PAG generating avoidance behavior, the lateral PAG generating fight and flight and the ventrolateral PAG generating freezing and immobility. J. Comp. Neurol. , 2013. © 2013 Wiley Periodicals, Inc.

The midbrain periaqueductal gray (PAG) is known to generate those behaviors necessary to survive the circumstances in which the animals find themselves. The authors show that within this context the PAG excites the pre-I neurons of the pre-Bötzinger complex both phasically and tonically, or even silences them, depending on which behavior is necessary.

The glycinergic cell populations in the brain of the lesser spotted dogfish were studied by a glycine immunofluorescence method. Numerous glycine-immunoreactive (Gly-ir) neurons were observed in different brain nuclei. In the telencephalon, Gly-ir cells were observed in the olfactory bulb, telencephalic hemispheres and preoptic region. In the hypothalamus, cerebrospinal fluid-contacting Gly-ir neurons were observed in the lateral and posterior recess nuclei. Coronet cells of the saccus vasculosus were Gly-ir. In the diencephalon, Gly-ir neurons were observed in the prethalamus and pretectum. In the midbrain, both the optic tectum and lateral mesencephalic nucleus contained numerous Gly-ir neurons. In the cerebellum, many Golgi cells were Gly-ir. In the rhombencephalon, Gly-ir cells were observed in the medial and ventral octavolateral nuclei, vagal lobe, visceromotor nuclei and reticular formation, including the inferior raphe nucleus. In the spinal cord, some neurons of the marginal nucleus and some cells of the dorsal and ventral horns were Gly-ir. Comparison of dogfish Gly-ir cell populations with those reported in the sea lamprey, Siberian sturgeon and zebrafish revealed some shared features but also notable differences. For example, Gly-ir cells were observed in the dogfish cerebellum unlike in the Siberian sturgeon and zebrafish, whereas the absence of Gly-ir neurons in the isthmus is shared by all these species, except lampreys. Gly-ir populations in the dogfish hypothalamus and telencephalon are notable in comparison with those of the other jawed vertebrates investigated to date. Together, these results reveal a complex and divergent evolution of glycinergic systems in the major groups of fishes. J. Comp. Neurol. , 2013. © 2013 Wiley Periodicals, Inc.

Using glycine immunofluorescence, we describe for the first time the putative glycinergic populations of the brain of a shark. The dogfish shared some glycinergic populations with other fishes but showed notable differences in the telencephalic lobes and cerebellum, which reveals divergent evolution of glycinergic systems of early vertebrate groups.

Abbreviated title: CB2R expression in Müller cells

The presence of the cannabinoid receptor type 1 (CB1R) has been largely documented in the rodent and primate retinas in recent years. There is however some controversy concerning the presence of the CB2 receptor (CB2R) within the central nervous system. Only recently, CB2R has been found in the rodent retina, but its presence in the primate retina has not yet been demonstrated. The aim of this study was twofold: 1) to characterize the distribution patterns of CB2R in the monkey retina and compare this distribution to that previously reported for CB1R and 2) to resolve the controversy on the presence of CB2R in the neural component of the retina. We therefore thoroughly examined the cellular localization of CB2R in the vervet monkey (Chlorocebus sabeus) retina, using confocal microscopy. Our results demonstrate that CB2R, like CB1R, is present throughout the retinal layers with however striking dissimilarities. Double labeling of CB2R and glutamine synthetase shows that CB2R is restricted to Müller cell processes, extending from the internal limiting membrane with very low staining, to the external limiting membrane with heavy labeling. We conclude that CB2R is indeed present in the retina but exclusively in the retinal glia whereas CB1R is only expressed in the neuro-retina. These results extend our knowledge on the expression and distribution of cannabinoid receptors in the monkey retina, although further experiments are still needed in order to clarify their role in retinal functions. J. Comp. Neurol. , 2013. © 2013 Wiley Periodicals, Inc.

Using confocal microscopy, we show that cannabinoid receptor CB2 (CB2R) is expressed in retinal Müller cells of the vervet monkey. CB2R expression (magenta) is found throughout glutamine synthetase (GS, green) positive Müller cells with a higher polarization towards the outer retina. OLM, outer limiting membrane; HFL, Henle fiber layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar = 75 μm.

Muscarinic modulation of mesolimbic dopaminergic neurons in the ventral tegmental area (VTA) plays an important role in reward, potentially mediated through the M5 muscarinic acetylcholine receptor (M5R). However, the key sites for M5R mediated control of dopamine neurons within this region are still unknown. To address this question we examined the electron microscopic immunocytochemical localization of antipeptide antisera against M5R and the plasmalemmal dopamine transporter (DAT) in single sections through the rat VTA. M5R was located mainly to VTA somatodendritic profiles (71%; n=627), at least one-third (33.2%; n=208) of which also contained DAT. The M5R immunoreactivity was distributed along cytoplasmic tubulovesicular endomembrane systems in somata and large dendrites, but more often located at plasmalemmal sites in small dendrites, the majority of which did not express DAT. The M5R-immunoreactive dendrites received a balanced input from unlabeled terminals forming either asymmetric or symmetric synapses. As compared with dendrites, M5R was less often seen in axon terminals, comprising only 10.8% (n=102) of the total M5R-labeled profiles. These terminals were usually presynaptic to unlabeled dendrites, suggesting M5R activation can indirectly modulate non-DAT containing dendrites through presynaptic mechanisms.Our results provide the first ultrastructural evidence that in the VTA, M5R has a subcellular location conducive to major involvement in postsynaptic signaling in many dendrites, only some of which express DAT.These findingssuggest that cognitive and rewarding effects ascribed to muscarinic activation in the VTA are primarilycredited to M5R activation at postsynaptic plasma membranes distinct fromdopamine transport. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.

We have shown that muscarinic M5 receptor subtype is located primarily in somatodendritic compartments of midbrain ventral tegmental area neurons, but also in some of their afferent axon terminals. These potential activation sites confer the M5R subtype a critical position for fine adjustment of DA-related mesocorticolimbic-guided behaviors.

Ciliary neurotrophic factor (CNTF) administration maintains, protects and promotes the regeneration of both motor neurons (MNs) and skeletal muscle in a wide variety of models. Expression of CNTF receptor α (CNTFRα), an essential CNTF receptor component, is greatly increased in skeletal muscle following neuromuscular insult. Together the data suggest that muscle CNTFRα may contribute to neuromuscular maintenance, protection and/or regeneration in vivo.

To directly address the role of muscle CNTFRα, we selectively depleted it in vivo by using a “floxed” CNTFRα mouse line and a gene construct (mlc1f-Cre) that drives the expression of Cre specifically in skeletal muscle. The resulting mice were challenged with sciatic nerve crush. Counting of nerve axons and retrograde tracing of MNs indicated that muscle CNTFRα contributes to MN axonal regeneration across the lesion site. Walking track analysis indicated that muscle CNTFRα is also required for normal recovery of motor function. However, the same muscle CNTFRα depletion unexpectedly had no detected effect on the maintenance or regeneration of the muscle itself, even though exogenous CNTF has been shown to affect these functions. Similarly MN survival and lesion-induced terminal sprouting were unaffected.

Therefore, muscle CNTFRα is an interesting new example of a muscle growth factor receptor which, in vivo under physiological conditions, contributes much more to neuronal regeneration than to the maintenance or regeneration of the muscle itself. This novel form of muscle-neuron interaction also has implications in the therapeutic targeting of the neuromuscular system in MN disorders and following nerve injury. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.

We selectively depleted muscle CNTFRα, an essential CNTF receptor component, in vivo and found it contributes to motor neuron axonal regeneration across the site of peripheral nerve lesion and normal recovery of motor function. Surprisingly, we found no role for muscle CNTFRα in maintenance or regeneration of the muscle itself.

Many brain structures project to both the anteroventral thalamic nucleus and the anteromedial thalamic nucleus. In the present study, pairs of different tracers were placed into these two thalamic sites in the same rats to determine the extent to which these nuclei receive segregated inputs. Only inputs from the laterodorsal tegmental nucleus, the principal extrinsic cholinergic source for these thalamic nuclei, showed a marked degree of collateralisation, with approximately 13% of all cells labelled in this tegmental area projecting to both nuclei. Elsewhere, double labelled cells were very scarce, comprising ∼1% of all labelled cells. Three general patterns of anterior thalamic innervation were detected in these other areas. In some sites, e.g., prelimbic cortex, anterior cingulate cortex, and secondary motor area, cells projecting to the anteromedial and anteroventral thalamic nuclei were closely intermingled, with often only subtle distribution differences. These same projections were also often intermingled with inputs to the mediodorsal thalamic nucleus, but again there was little or no collaterisation. In other sites, e.g., the subiculum and retrosplenial cortex, there was often less overlap of cells projecting to the two anterior thalamic nuclei. A third pattern related to the dense inputs from the medial mammillary nucleus, where well defined topographies ensured little intermingling of the neurons that innervate the two thalamic nuclei. The finding, that a very small minority of cortical and limbic inputs bifurcate to innervate both anterior thalamic nuclei, highlights the potential for parallel information streams to control their functions, despite arising from common regions. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.

Double-fluorescent retrograde neuronal tracing techniques reveal the organisation of projections into the anteroventral and anteromedial thalamic nuclei. Results show that despite projections often originating from shared regions, there is a very high level of segregation in cortical and limbic inputs into these nuclei with very few bifurcating neurones innervating both nuclei.

In the avian cervical spinal cord, there are motoneurons (MNs) that send their axons through the dorsal roots. They have been called dorsal motoneurons (dMNs) and assumed to correspond to MNs of the accessory nerve that innervate the cucullaris muscle (SAN-MNs). However, their target muscles have not been elucidated to date. In the present study, we tried to determine the targets and the specific combination of transcription factors expressed by dMNs and SAN-MNs, and to describe the detailed development of dMNs. Experiments with tracing techniques confirmed that axons of dMNs innervated the cucullaris muscle. Retrogradely labeled dMNs were distributed in the ventral horn of C3 and more caudal segments. In most cases, some dMNs were also observed in the C2 segment. It was also demonstrated that SAN-MNs existed in the ventral horn of the C1-C2 segments and the adjacent caudal hindbrain. Both SAN-MNs and dMNs expressed Isl1 but did not express Isl2, MNR2 or Lhx3. Rather these MNs expressed Phox2b, a marker for branchial motoneurons (brMNs), although the intensity of expression was weaker. Dorsal MNs and SAN-MNs were derived from the Nkx2.2-positive precursor domain and migrated dorsally. Dorsal MNs remain in the ventral domain of the neural tube unlike brMNs in the brain stem. These results indicate that dMNs and SAN-MNs belong to a common MN population innervating the cucullaris muscle, and also suggest that they are similar to brMNs of the brain stem, although there are differences in Phox2b expression and in the final location of each population. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.

Dorsal motoneurons, that send peripheral axons through the dorsal root, have been repeatedly described in the cervical spinal cord of avian embryos for more than one hundred years. However, their target muscles have not been revealed until today. Here we show that their target muscle is the cucullaris muscle, and that these motoneurons express molecular markers that are similar to those of the branchial motoneurons in the brain stem.

In naked mole-rat (NMR) colonies, breeding is monopolized by the queen and her consorts. Subordinates experience gonadal development if separated from the queen. To elucidate the neuroendocrine factors underlying reproductive suppression/development in NMRs, we have quantified plasma gonadal steroids and GnRH-1- and kisspeptin-immunoreactive (ir) neurons in subordinate adults and in those allowed to develop into breeders, with or without subsequent gonadectomy. In males and females, respectively, plasma testosterone and progesterone are higher in breeders than in subordinates. No such distinction occurs for plasma estradiol; its presence after gonadectomy and its positive correlation with adrenal estradiol suggest an adrenal source. Numbers of GnRH-1-ir cell bodies do not differ between gonad-intact breeders and subordinates within or between the sexes. As in phylogenetically-related guinea pigs, kisspeptin-ir processes pervade the internal and external zones of the median eminence. Their distribution is consistent with actions on GnRH-1 neurons at perikaryal and/or terminal levels. In previously investigated species, numbers of kisspeptin-ir cell bodies vary from substantial to negligible according to sex and/or reproductive state. NMRs are exceptional: irrespective of sex, reproductive state or presence of gonads, substantial numbers of kisspeptin-ir cell bodies are detected in the rostral periventricular region of the third ventricle (RP3V) and in the anterior periventricular (PVa), arcuate and dorsomedial hypothalamic nuclei. Nevertheless, the greater number in the RP3V/PVa of female breeders compared with female subordinates or male breeders suggests that emergence from a hypogonadotrophic state in females may involve kisspeptin-related mechanisms similar to those underlying puberty or seasonal breeding in other species. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.

In naked mole-rat (NMR) colonies, the queen and her consorts monopolize breeding. This study has quantified GnRH-1- and kisspeptin-immunoreactive (ir) neurons and plasma gonadal steroids in breeders and subordinates. Extensive species comparisons show NMRs to be novel: substantial numbers of kisspeptin-ir cell bodies are detected in the rostral periventricular region (including the AVPV) and arcuate and dorsomedial hypothalamic nuclei, irrespective of reproductive status, plasma gonadal steroids or sex. Nevertheless, rostral periventricular kisspeptin-ir cell bodies are more numerous in female breeders than female subordinates.

RFamide-related peptide-3 (RFRP-3) neurons have been shown to inhibit gonadotropin releasing hormone (GnRH) neuronal activity and hence reproduction in birds and eutherian mammals. They have also been proposed to have a direct hypophysiotropic effect on pituitary gonadotropin release. We used a new RFRP-3 antibody to characterize the cell body distribution and fiber projections of RFRP-3 neurons in the adult female brushtail possum brain. RFRP-3-immunoreactive cell bodies were found scattered within the dorsomedial hypothalamus and the dorsomedial half of the ventromedial hypothalamus, while GnRH neurons were observed scattered rostro-caudally along the lateral septum, rostral to the medial septum. There was a significant twofold increase in the RFRP-3 cell body number during the non-breeding season (summer) compared to the breeding season (winter). Immunoreactive RFRP-3 fibers were distributed throughout the thalamus, preoptic area and hypothalamus. Very few fibers were observed in the median eminence, especially in the external zone. Intraperitoneal injection of the retrograde tracer Fluoro-Gold resulted in the labeling of 40% of hypophysiotropic tuberoinfundibular dopaminergic (tyrosine hydroxylase positive) neurons; however <10% of zona incerta dopaminergic neurons (which are not hypophysiotropic) or RFRP-3 neurons were labeled with this tracer. These observations suggest that RFRP-3 exhibits a seasonal fluctuation in cell numbers as seen in sheep and birds, which is consistent with an increased inhibitory tone during the non-breeding season. The lack of RFRP-3 fibers in the median eminence and of Fluoro-Gold uptake from the periphery imply that the actions of this peptide occur primarily centrally rather than at the anterior pituitary gland. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.

RFRP-3 exhibits a pronounced seasonal fluctuation in cell numbers in the brushtail possum hypothalamus, suggesting an increased inhibitory tone during the non-breeding season. The lack of RFRP-3 fibers in the median eminence and of Fluoro-Gold uptake from the periphery imply its actions occur centrally rather than at the pituitary gland.

Estrogens play a salient role in the development and maintenance of both male and female nervous systems and behaviors. The plainfin midshipman (Porichthys notatus), a teleost fish, has two male reproductive morphs that follow alternative mating tactics and diverge in multiple somatic, hormonal and neural traits, including the central control of morph-specific vocal behaviors. After we identified duplicate estrogen receptors (ERβ1 and ERβ2) in midshipman, we developed antibodies to localize protein expression in the central vocal-acoustic networks and saccule, the auditory division of the inner ear. As in other teleost species, ERβ1 and ERβ2 were robustly expressed in the telencephalon and hypothalamus in vocal-acoustic and other brain regions shown previously to exhibit strong expression of ERα and aromatase (estrogen synthetase, CYP19) in midshipman. Like aromatase, ERβ1 label co-localized with glial fibrillary acidic protein (GFAP) in telencephalic radial glial cells. Quantitative PCR revealed similar patterns of transcript abundance across reproductive morphs for ERβ1, ERβ2, ERα and aromatase in the forebrain and saccule. In contrast, transcript abundance for ERs and aromatase varied significantly between morphs in and around the sexually polymorphic vocal motor nucleus (VMN). Together, the results suggest that VMN is the major estrogen target within the estrogen-sensitive hindbrain vocal network that directly determines the duration, frequency and amplitude of morph-specific vocalizations. Comparable regional differences in steroid receptor abundances likely regulate morph-specific behaviors in males and females of other species exhibiting alternative reproductive tactics. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.

Estrogens are critical in nervous system and behavioral regulation. Plainfin midshipman fish have alternative male reproductive morphs with divergent, hormonally-regulated vocal behaviors. We identified duplicate estrogen receptors and localized them to central vocal and auditory networks. Quantification of estrogen receptors and aromatase transcripts revealed unique expression profiles in the vocal motor nucleus (VMN) of male and female morphs, suggesting VMN is a major estrogen target. Differential receptor expression is likely crucial in maintaining behavioral differences in alternative reproductive tactics.

To understand manual tactile functions in primates it is essential to explore the interactions between the finger pad representations in somatosensory cortex. To this end, we used optical imaging and electrophysiological mapping to guide neuroanatomical tracer injections into distal digit tip representations of Brodmann area 3b in the squirrel monkey. Retrogradely labeled cell densities and anterogradely labeled fibers and terminal patches in somatosensory areas were plotted and quantified with respect to tangential distribution. Within area 3b, reciprocal patchy distribution of anterograde and retrograde labeling spanned the representation of the distal pad of multiple digits indicating strong cross-digit connectivity. Inter-areal connections revealed bundles of long-range fibers projecting anteroposteriorly, connecting area 3b with clusters of labeled neurons and terminal axon arborizations in area 1. Inter-areal linkage appeared to be largely confined to the representation of the injected finger. These findings provide the neuroanatomical basis for the interaction between distal finger pad representations observed by recent electrophysiological studies. We propose that intra-areal connectivity may be heavily involved in interdigit integration such as shape discrimination, while long-range inter-areal connections may subserve active touch in a digit-specific manner. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.

Using bidirectional tract tracing combined with intrinsic signal optical imaging, the authors show strong connectivity between neighboring distal finger pad representations in area 3b of the somatosensory cortex of the squirrel monkey. In contrast, inter-areal connections are apparently digit specific. We propose that intra-areal connectivity is heavily involved in interdigit integration, while long-range inter-areal connections may act in a digit-specific manner. Scale bar = 1 mm.

Cochlear implants restore hearing cues in the severe-profoundly deaf by electrically stimulatingspiral ganglion neurons (SGNs). However, SGNs degeneratefollowing loss of cochlear hair cells, due at least in partto a reduction in the endogenous neurotrophin (NT) supply, normally provided by hair cells and supporting cellsof the organ of Corti. Deliveringexogenous NTs to the cochlea can rescue SGNs from degenerationand canalsopromote the ectopic growthof SGN neurites.This resprouting maydisruptthe cochleotopic organizationupon which cochlear implants rely to impart pitch cues. Using retrograde labeling and confocal imaging of SGNs we determined the extent of neurite growthfollowing 28 days of exogenous NT treatment in deafened guinea pigs with and without chronic electrical stimulation (ES). On completion of this treatment we measured the spread of neural activation to intracochlear ES by recording neural responses across thecochleotopically organized inferior colliculus using multichannel recording techniques. Although NT treatment significantly increased both the length and the lateral extent of growth of neurites along the cochlea compared to deafened controls, these anatomical changes did not affect the spread of neural activationwhen examined immediately after 28 days of NT treatment.NT treatment did, however, result in lower excitation thresholdscompared with deafened controls. These data support the application of NTs for improved clinical outcomes for cochlear implant patients. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.

Using multichannel recording techniques combined with neural labelling the authors show that spiral ganglion neurites in deafened cochleae chronically treated with exogenous neurotrophins and electrical stimulation exhibit ectopic growth compared with the highly organized neurite projections evident in hearing cochleae. Importantly, these anatomical changes did not affect the spread of neural activation to electrical stimulation via a cochlear implant.

The formation and refinement of synaptic connections are dependent upon the activity that emerges from nascent synaptic connections. Such activity has the effect of regulating the production and release of specific neurotransmitters. To determine the role of activity in regulating the production of peptide-positive synapses, we used antibodies against Phe-Met-Arg-Phe-NH₂ and acetylated α-tubulin as well as intracellular injections of Neurobiotin to examine varicosities belonging to heart excitor (HE) neurons on the heart tubes of medicinal leeches, Hirudo spp. We found that the production of peptide-positive varicosities increased considerably during the last week of embryogenesis, which coincided with the emergence of rhythmic activity of the heart tube. When we compromised central input to HE neurons with bicuculline or by surgical ablation of the central pattern generator during early embryogenesis, we found that activity in the heart tubes and its rhythmicity were greatly diminished. Furthermore, the activity of HE neurons has also lost its rhythmicity and appeared tonic while production of peptide-positive varicosities was substantially reduced as well. Partial surgical ablations that preserved rhythmic activity in the heart tube while disrupting heart tube innervation by some HE neurons still resulted in peptide-positive varicosity production. Taken together, we suggest that postsynaptic rhythmic activity of the heart tube is necessary and sufficient for the development and maturation of peptide-positive synapses. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.

The formation of peptide-containing presynaptic varicosities in a motor neuron depends upon rhythmic activity of the target and motor neuron. Disruption of rhythmic central pattern generator (CPG) drive during early development results in a pronounced deficit of motor synapses.

Compared to proprioceptive afferent collateral projections, less is known about the anatomical, neurochemical and functional basis of nociceptive collateral projections modulating lumbar central pattern generators (CPG). Quick response times are critical to ensure rapid escape from aversive stimuli. Furthermore, sensitization of nociceptive afferent pathways can contribute to a pathological activation of motor circuits. We investigated the extent and role of collaterals of capsaicin-sensitive nociceptive sacrocaudal afferent (nSCA) nerves that directly ascend several spinal segments in Lissauer's tract and the dorsal column and regulate motor activity. Anterograde tracing demonstrated direct multi-segmental projections of the sacral dorsal root 4 (S4) afferent collaterals in Lissauer's tract and in the dorsal column. Subsets of the traced S4 afferent collaterals expressed transient receptor potential vanilloid 1 (TRPV1), which transduces a nociceptive response to capsaicin. Electrophysiological data revealed that S4 dorsal root stimulation could evoke regular rhythmic bursting activity, and our data suggest that capsaicin sensitive collaterals contribute to CPG activation across multiple segments. Capsaicin's effect on S4 evoked locomotor activity was potent until the lumbar 5 (L5) segments; and diminished in rostral segments. Using calcium imaging we found elevated calcium transients within the Lissauer's tract and dorsal column at L5 segments when compared to the calcium transients only within the dorsal column at the lumbar 2 (L2) segments which were desensitized by capsaicin. We conclude that lumbar locomotor networks in the neonatal mouse spinal cord are targets for modulation by direct multisegmental nSCA, subsets of which express TRPV1 in the Lissauer's tract and the dorsal column. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.

Text for graphical abstract It has been recognized for many years than long-range afferents travel multiple segments within the spinal cord but their function has been elusive. Here we present anatomical and electrophysiological evidence that molecularly defined TRPV1 nociceptive afferents that project multiple segments within the lumbosacral spinal cord can functionally affect rhythmic motor behaviors.

We investigated the frequency-related topography of connections of the primary auditory cortical field (AI) in the Mongolian gerbil with subcortical structures of the auditory system by means of the axonal transport of two bidirectional tracers, which were simultaneoulsy injected into regions of AI with different best frequencies (BFs).

We found topographic, most likely frequency-matched (tonotopic) connections as well as non-topographic (non-tonotopic) connections. AI projects in a tonotopic way to the ipsilateral ventral (MGv) and dorsal divisions (MGd) of the medial geniculate body (MGB), reticular thalamic nucleus and dorsal nucleus of the lateral lemniscus as well as to the ipsi- and contralateral dorsal cortex of the inferior colliculus (IC) and central nucleus of the IC. AI receives tonotopic inputs from MGv and MGd. Projections from different BF regions of AI terminate in a non-tonotopic way in the ipsilateral medial division of the MGB (MGm), suprageniculate thalamic nucleus (SG) and brachium of the IC (bic) as well as in the ipsi- and contralateral external cortex and pericollicular areas of the IC. The anterograde labeling in the intermediate and ventral nucleus of the lateral lemniscus, parts of the superior olivary complex and divisions of the cochlear nucleus was generally sparse, thus a clear topographic arrangement of the labeled axons could not be ruled out. AI receives non-tonotopic inputs from the ipsilateral MGm, SG and bic.

In conclusion, the tonotopic and non-tonotopic corticofugal connections of AI can potentially serve for both, conservation and integration of frequency-specific information in the respective target structures. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.

The Mongolian gerbil (Meriones unguiculatus) has become a frequently used animal model in auditory neuroscience. Here, we investigated the frequency-related topography of connections of the primary auditory cortical field (AI) with subcortical structures of the auditory system in this species. We found topographic, most likely frequency-matched (tonotopic) connections as well as non-topographic (non-tonotopic) connections. The former may serve for a conservation of frequency-specific information in the respective target structures of AI whereas the latter could be involved in frequency-integration processes.

Spinal lamina I is a key area for relaying and integrating information from nociceptive primary afferents with various other sources of inputs. While lamina I projection neurons have been intensively studied, much less attention has been given to local-circuit neurons (LCNs), which form the majority of the lamina I neuronal population. In this work the infrared light-emitting diode (IR-LED) oblique illumination technique was used to visualize and label LCNs, allowing reconstruction and analysis of their dendritic and extensive axonal trees.

We show that the majority of lamina I neurons with locally branching axons fall into the multipolar (with ventrally protruding dendrites) and flattened (dendrites limited to lamina I) somatodendritic categories. Analysis of their axons revealed that the initial myelinated part gives rise to several unmyelinated small-diameter branches that have a high number of densely packed large varicosities and an extensive rostrocaudal (2-3 segments), mediolateral and dorsoventral (reaching laminae III-IV) distribution. The extent of the axon and the occasional presence of long solitary branches suggest that LCNs may also form short and long propriospinal connections. We also found that the distribution of axon varicosities and terminal field locations show substantial heterogeneity and that a substantial portion of LCNs is inhibitory.

Our observations indicate that LCNs of lamina I form intersegmental as well as interlaminar connections and may govern large numbers of neurons, providing anatomical substrate for rostrocaudal “processing units” in the dorsal horn. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.

Using an intact spinal cord preparation the authors describe axon architecture of lamina I local circuit neurons (red and green). Extensive local- and long rostrocaudal axon branches suggest information feed towards deeper laminae as well as neighboring segments, providing anatomical substrate for interlaminar and intersegmental processing in the dorsal horn.

We analyzed the mouth of three species, representative of the three orders of the class Thaliacea (Tunicata) - Pyrosoma atlanticum (Pyrosomatida), Doliolum nationalis (Doliolida) and Thalia democratica (Salpida) – to verify the presence of mechanoreceptors, particularly hair cells. In vertebrates, hair cells are well known mechanoreceptors of the inner ear and lateral line, typically exhibiting an apical hair bundle composed of a cilium and stereovilli but lacking an axon. For a long time, hair cells were thought to be exclusive to vertebrates. However, evidence of a mechanosensory organ (the coronal organ) employing hair cells in the mouth of tunicates, considered the sister group of vertebrates, suggests that tunicate and vertebrate hair cells may share a common origin. This study on thaliaceans, a tunicate group not yet investigated, shows that both P. atlanticum and D. nationalis possess a coronal organ, in addition to sensory structures containing peripheral neurons (i.e., cupular organs and triads of sensory cells). In contrast, in T. democratica, we did not recognize any oral multicellular sensory organ. We hypothesize that in T. democratica, hair cells were secondarily lost, concomitantly with the loss of branchial fissures, the acquisition of a feeding mechanism based on muscle activity and a mechanosensory apparatus based on excitable epithelia. Our data are consistent with the hypothesis that hair cells were present in the common ancestor of tunicates and vertebrates, from which hair cells progressively evolved. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.

Tunicates, the closest relatives of vertebrates, possess a mechanoreceptor, the coronal organ, based on hair cells. We analyzed three thaliacean species that have evolved different solutions for mechanoreception. Some solutions are unique within tunicates, others are shared; some are based on primary sensory cells, others on secondary sensory cells.

Sexually dimorphic sensory systems are common in Hymenoptera and are considered to result from sex-specific selection pressures. An extreme example of sensory dimorphism is found in the solitary bee tribe Eucerini. Males of long-horned bees bear antennae that exceed body length. In this study we investigated the pronounced sexual dimorphism of the peripheral olfactory system and its representation in higher brain centers of the species Eucera berlandi. Eucera males have elongated antennae with 10 times more poreplates and three times more olfactory receptor neurons than females. The male antennal lobe (AL) comprises fewer glomeruli than the female AL (∼100 vs. ∼130), of which four are male-specific macroglomeruli. No sex-differences were found in the relative volume of the mushroom bodies, a higher order neuropil essential for learning and memory in Hymenoptera. Compared with the Western honeybee, the degree of sexual dimorphism in Eucera is more pronounced at the periphery. In contrast, sex-differences in glomerular numbers are higher in the eusocial honeybee and a sexual dimorphism of the relative investment in mushroom body tissue is only observed in Apis. The observed differences between the eusocial and solitary bee species may reflect differences in (male)-specific behavioral traits and associated selection pressures, which are discussed in brief. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.

The solitary long-horned bee Eucera berlandi exhibits a spectacular sexual dimorphism in their antennae. In this study we describe the dimorphism in the peripheral olfactory system and its representation in higher order neuropils of the olfactory path. Further, we compare it to the eusocial honeybee and discuss similarities and differences in a physiological and evolutionary context.

Kainate receptors mediate fast, excitatory synaptic transmission for a range of inner neurons in the mammalian retina. However, allocation of functional kainate receptors to known cell types and their sensitivity remains unresolved. Using the cation channel probe 1-amino-4-guanidobutane agmatine (AGB), we investigated kainate sensitivity of neurochemically identified cell populations within the structurally intact rat retina. Most inner retinal neuron populations responded to kainate in a concentration dependent manner. OFF cone bipolar cells demonstrated the highest sensitivity of all inner neurons to kainate. Immunocytochemical localization of AGB and macromolecular markers confirmed Type 2 BCs as part of this kainate sensitive population. The majority of amacrine (ACs) and ganglion cells (GCs) showed kainate responses with different sensitivities between major neurochemical classes (GABA/Glycine ACs > Glycine ACs > GABA ACs; Glu/weakly GABA GCs > Glu GCs). Conventional and displaced cholinergic ACs were highly responsive to kainate whilst dopaminergic ACs do not appear to express functional kainate receptors. These findings further contribute to our understanding of neuronal networks in complex multi-cellular tissues. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.

Using the probe agmatine, we found kainate sensitivity is highest in OFF bipolar cells (particularly type 2) followed by ganglion cells. GABA/glycine amacrine cells (ACs) are the most sensitive ACs, followed by glycine and GABA ACs. Cholinergic ACs are highly responsive but dopaminergic ACs do not express functional kainate receptors.

We studied the anatomical remodeling and gliosis of retinal Müller cells in the rd/rd mouse model of photoreceptor degeneration. A computational calculation of glutamine synthetase immunoreactivity was developed so we could specifically quantify changes in Müller cell anatomy between control mice (C57Bl/6) and the dystrophic strain. We found no change in number of Müller cell somata between mice strains indicating no cell proliferation as a function of development and degeneration. The retinal area occupied by the total Müller cell body (soma and processes) was significantly less in the rd/rd mouse retina compared with control mice. When only the outer retina was considered, we found rd/rd Müller cell processes were dramatically reduced during the cone phase of photoreceptor degeneration. However, at older ages, an increase Müller cell processes was seen. Conversely, glial fibrillary acidic protein (GFAP) expression showed a significant increase during cone degeneration followed by a reduction in older ages. Müller cell electrophysiology, particularly K⁺ currents and membrane potential was similar between rd/rd and control Müller cells during cone degeneration. Together, these results show that glial remodeling in the rd/rd retina follows separate phases - an initial conservative glial response involving the loss of Müller cells processes, hyper-expression of GFAP and preservation of normal electrophysiology followed by an active growth of Müller cell processes, glial seal formation and attenuation of GFAP expression after complete photoreceptor loss. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.

In the rd/rd mouse, Muller cell processes are reduced in the outer retina during early degeneration but increase after total photoreceptor loss. Conversely, GFAP expression increases at early stages then decreases at older ages. Muller cell electrophysiology remains normal during early degeneration. Thus, rd/rd gliosis has an initial conservative response followed by active remodeling.

Prior studies have identified two anatomically and neurochemically distinct cellular compartments within the mammalian striatum, termed striosomes and matrix, which express μ-opioid receptors (μOR) and EphA4, respectively. Here we identify and characterize an additional compartment in the rat striatum composed of neurons that express EphA7. In situ hybridization and immunohistochemical data indicate that neurons expressing EphA7 mRNA and protein are arranged in a banded “matrisome-like” pattern confined to the matrix in the dorsal striatum. Within the ventral striatum, EphA7-positive (+) neurons have a less organized mosaic pattern that partially overlaps areas expressing μOR. Immunolabeling data demonstrate that EphA7⁺striatofugal axons form distinct fascicles leaving the striatum. Within the globus pallidus, EphA7⁺ axons terminate primarily within ventromedial areas of the nucleus and along its striatal border. EphA7⁺ axons avoid regions containing dopamine neurons within the substantia nigra and preferentially innervate areas near the rostral and caudal margins of the nucleus. Within both nuclei, EphA7⁺ axons have similar but more restricted terminal fields than the entire population of EphA4⁺ matrix axons, indicating that EphA7⁺axons comprise a subpopulation of matrix axons. Ligand binding data demonstrate that ephrin-A5 selectively binds areas of the striatum, globus pallidus and substantia nigra containing EphA7⁺ neurons and axons, but not areas expressing only EphA4. Our findings demonstrate that EphA7 expression identifies a novel “matrisome” compartment within the matrix that binds ephrin-A5 and possesses unique axonal projections. Our findings also suggest that EphA7 and ephrin-A5 may participate in the formation of this matrisome subcompartment and its striatofugal projections. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.

The EphA7 receptor is a member of the Eph/ephrin family of molecules, which are known to participate in axonal guidance and cell boundary formation during development. In the postnatal striatum, EphA7 expression delineates a novel striatal compartment. These “matrisome” compartments are located entirely within the striatal matrix and are organized into bands paralleling the curvature of the external capsule. The postnatal expression pattern of EphA7 suggests it plays a role in striatal compartment formation and/or the formation of striatal connections.

There is ambiguity concerning the distribution of neurons that express the ghrelin receptor (GHSR) in the medulla oblongata. In the current study we have used a sensitive non-radioactive method to investigate GHSR mRNA distribution by in situ hybridization. Strong expression of the GHSR gene was confirmed in neurons of the facial nucleus (FacN, 7), the dorsal vagal complex (DVC) and the semi-compact (but not compact) nucleus ambiguus (AmbSC and AmbC). In addition, expression of GHSR was found in other regions, where it had not been described before. GHSR-positive neurons were observed in the gustatory rostral nucleus tractus solitarius and in areas involved in vestibulo-ocular processing (such as the medial vestibular nucleus and the nucleus abducens). GHSR expression was also noted in ventral areas associated with cardio-respiratory control, including the gigantocellular reticular nucleus, the lateral paragigantocellular nucleus, the rostral and caudal ventrolateral medulla, the (pre)-Bötzinger complex and the rostral and caudal ventrolateral respiratory group. However, GHSR-positive neurons in ventrolateral areas did not express markers for cardiovascular presympathetic vasomotor neurons, respiratory propriobulbar rhythmogenic neurons or sensory interneurons. GHSR-positive cells were intermingled with catecholamine neurons in the dorsal vagal complex but these populations did not overlap.

Thus, the ghrelin receptor occurs in the medulla oblongata in i) second order sensory neurons processing gustatory, vestibulo-ocular and visceral sensation; ii) cholinergic somatomotor neurons of the FacN and autonomic preganglionic neurons of the DMNX and AmbSC; iii) cardiovascular neurons in the DVC, Gi and LPGi; iv) neurons of as yet unknown function in the ventrolateral medulla. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.

A detailed survey of ghrelin receptor (GHSR) mRNA expression in the rat medulla reveals expression in areas involved in gustatory, vestibulo-ocular, visceral sensory processing, and cardio-respiratory control. GHSR mRNA is found in the caudal and ventral aspects of the nucleus ambiguus and retroambiguus, but not in the rostral compact formation, ruling out control of esophagal musculature by ghrelin. GHSR mRNA expressing neurons (white) are distinct from catecholaminergic TH-expressing neurons (green) in the dorsal vagal complex (pictured) and the RVLM.

Glucoprivation or hypoglycemia induces a range of counter regulatory responses including glucose mobilization, reduced glucose utilization and de novo glucose synthesis. These responses are mediated in part by the sympathetic nervous system. The aim of this study was to determine the chemical codes of sympathetic preganglionic neurons (SPN) activated by glucoprivation, induced by 2-Deoxy-D-glucose (2DG). SPN controlling the adrenal glands and celiac ganglia, which ultimately can innervate the liver and pancreas, were targeted together with the superior cervical ganglia (control). 23.9±1.3% of SPN in the T4-T11 region contained c-Fos immunoreactivity following 2DG. 70.3±1.8% of SPN innervating the adrenal glands and 37.4±3% of SPN innervating celiac ganglia were activated. 14.8±3.5% of SPN (C8-T3) innervating superior cervical ganglia were activated. In the C8-T3 region 55±10% of SPN activated contained PPCART with only 12±3% expressing PPE mRNA whereas in the T4-T11 region 78±4% contained PPE with only 6.0±0.6% expressing PPCART mRNA. Thus CART is not involved in glucose mobilization. Two chemically distinct populations of SPN (PPE+ (57.4±5%), PPE- (˜40%)) were identified to regulate adrenaline release in response to glucoprivation. Multiple chemically distinct SPN populations innervating a specific target could suggest their graded recruitment. The two distinct populations of SPN (PPE+ (67.6±9%), PPE- (˜30%)) projecting to celiac ganglia activated by glucoprivation could direct pancreatic and hepatic or other counter regulatory responses. Nearly all SPN that expressed PPE mRNA and projected to the adrenal glands or celiac ganglia were activated suggesting a role for the inhibitory peptide enkephalin in responses evoked by glucoprivation. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.

A quarter of T4-T11 sympathetic preganglionic neurons (SPN) contribute to counterregulatory responses following glucoprivation. Of the SPN controlling adrenaline release, 60% contain prepro-enkephalin (PPE) and 40% do not. SPN also innervate the celiac ganglia (CG), whose function includes controlling glucagon release and glycogenolysis, and 70% of this population also contains PPE. In contrast, 3% of activated SPN innervating the superior cervical ganglia contain PPE. A single function such as adrenaline release is controlled by a neurochemically heterogeneous population of SPN.

Somesthesis-guided exploration of the external world requires cortical processing of both cutaneous and proprioceptive information and their integration into motor commands toguide further haptic movement. In the past, attention has been givenmostlyto thecortical circuits processing cutaneous information for somatic motor integration. By comparison, little has been examined about how cortical circuits are organized for higher-order proprioceptive processing. Using the rat cortex as a model, we characterized the intrinsic and corticocortical circuits arising in the major proprioceptive region of the primary somatosensory cortex (SI) that is conventionally referred to as the dysgranular zone (DSZ). We made small injections of biotinylated dextran amine (BDA) as ananterograde tracer in various parts of the DSZ, revealing three distinct principles of its cortical circuit organization. First, its intrinsic circuits extend mainly along the major axis of DSZ to organize multiple patches of interconnections. Second, the central and peripheral regions of DSZ produce differential patterns of intra-areal and corticocortical circuits. Third, theprojection fields of DSZencompass only selective regions of the second somatic (SII), posterior parietal (PPC), and primary motor (MI) cortices. These projection fields are at least partially separated from those of SI cutaneous areas.

Based on these observations, we hypothesize that the cortical circuits of DSZ facilitate a modular integration ofproprioceptive information along its major axisanddisseminatethis informationto only selective parts of higher-order somatic and MIcortices inparallel to cutaneousinformation. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.

As shown in the tangential section of rat frontal cortex, the proprioceptive area of primary somatosensory cortex (SI) that is also known as the dysgranular zone (DSZ) projects to selective regions of the second somatic (SII), posterior parietal (PPC), and primary motor cortex (MI). These regions in each cortex are separated from the areas that receive SI input from the whisker area. These observations indicate that parallel circuits arise from the SI proprioceptive and cutaneous areas to the surrounding cortices.

Throughout development both the body and the brain change at remarkable rates. Specifically, the number of cells in the brain undergoes dramatic non-linear changes; first exponentially increasing in cell number and then decreasing in cell number. Importantly, different cell types, such as neurons and glia, undergo these changes at different stages of development. In the current investigation we used the isotropic fractionator method to examine the changes in cellular composition at multiple developmental milestones in the short-tailed opossum, Monodelphis domestica. Here we report several novel findings concerning marsupial brain development and organization. First, during the later stages of neurogenesis (P18) neurons comprise most of the cells in the neocortex, although the total number of neurons remains the same throughout the lifespan. In contrast, in the subcortical regions the number of neurons decreases dramatically after P18, and a converse relationship is observed for non-neuronal cells. In the cerebellum, the total number of cells gradually increases until P180 and then remains constant, and then the number of neurons is consistent across the developmental ages examined. For the three major structures examined, neuronal density and the percent of neurons within a structure is highest during neurogenesis and then decreases after this time point. Finally, the total number of neurons in the opossum brain is relatively low compared to other small-brained mammals such as mice. The relatively low number of neurons and correspondingly high number of non-neurons suggests that in the marsupial brain non-neurons may play a significant role in signal processing. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.

Throughout development the number of neurons and glia in the brain undergo dramatic non-linear changes. We examined the cellular composition of the short-tailed opossum brain at multiple developmental milestones. Neuronal density and percentage are highest during neurogenesis and then decrease, and the total number of neurons in the opossum brain is relatively low compared to other mammals. The low number of neurons and high number of glia suggests that in marsupials glia may play a significant role in signal processing.

Elevating levels of nerve growth factor (NGF) can have pronounced effects on the survival and maintenance of distinct populations of neurons. We have generated a line of transgenic mice in which NGF is expressed under the control of the smooth muscle α-actin promoter. These transgenic mice have augmented levels of NGF protein in the descending colon and urinary bladder, and as a consequence these tissues display increased densities of NGF-sensitive sympathetic efferents and sensory afferents. Here, we provide a thorough examination of sympathetic and sensory axonal densities in the descending colon and urinary bladder of NGF transgenic mice with and without the expression of the p75 neurotrophin receptor (p75NTR). In response to elevated NGF levels, sympathetic axons (immunostained for tyrosine hydroxylase) undergo robust collateral sprouting in the descending colon and urinary bladder of adult transgenic mice (i.e., those tissues having smooth muscle cells); this sprouting is not augmented in the absence of p75NTR expression. As for sensory axons (immunostained for calcitonin gene-related peptide) in the urinary bladders of transgenic mice, fibers undergo sprouting that is further increased in the absence of p75NTR expression. Sympathetic axons are also seen invading the sensory ganglia of transgenic mice; these fibers form perineuronal plexuses around a subpopulation of sensory somata. Our results reveal that elevated levels of NGF in target tissues stimulate sympathetic and sensory axonal sprouting, and that an absence of p75NTR by sensory afferents (but not by sympathetic efferents) leads to a further increase of terminal arborization in certain NGF-rich peripheral tissues. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.

Overexpression of nerve growth factor (NGF) leads to robust sprouting by sympathetic axons in the descending colon and urinary bladder of transgenic mice. Axonal growth by sympathetic neurons is enhanced in the urinary bladder of those transgenic mice carrying two mutated alleles for the p75 neurotrophin receptor (p75).

Polypteriform fishes are believed to be basal to other living ray-finned bony fishes, and they may be useful for providing information of the neural organization that existed in the brain of the earliest ray-finned fishes. The calcium-binding proteins calretinin (CR) and calbindin-D28k (CB) have been widely used to characterize neuronal populations in vertebrate brains. Here, the distribution of the immunoreactivity against CR and CB was investigated in the olfactory organ and brain of Polypterus senegalus and compared to the distribution of these molecules in other ray-finned fishes. In general, CB-immunoreactive (ir) neurons were less abundant than CR-ir cells. CR immunohistochemistry revealed segregation of CR-ir olfactory receptor neurons in the olfactory mucosa and their bulbar projections. Our results confirmed important differences between pallial regions in terms of CR immunoreactivity of cell populations and afferent fibers. In the habenula, these calcium-binding proteins revealed right-left asymmetry of habenular subpopulations and segregation of their interpeduncular projections. CR immunohistochemistry distinguished between some thalamic, pretectal and posterior tubercle-derived populations. Abundant CR-ir populations were observed in the midbrain, including the tectum. CR immunoreactivity was also useful for characterizing a putative secondary gustatory/visceral nucleus in the isthmus, and for distinguishing territories in the primary viscerosensory column and octavolateral region. Comparison of the data obtained within a segmental neuromeric context indicates that some CB-ir and CR-ir populations in polypteriform fishes are shared with other ray-finned fishes, but other positive structures appear to have evolved following the separation between polypterids and other ray-finned fishes. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.

The distribution of calretinin and calbindin is investigated in the brain of Polypterus, a basal ray-finned fish. Among others, it allowed studying the segregation of olfactory neurons in the olfactory system, distinguishing between pallial regions, characterizing asymmetry and segregation of habenular populations and projections, characterizing a secondary gustatory/visceral nucleus and distinguishing among territories in the viscerosensory column and octavolateral region. Characterization of positive populations in Polypterus provided further criteria for analyzing evolution of the brain in ray-finned fishes.

Highvoltage-activated Ca channels participate in multiple cellular functions, including transmitter release, excitation, and gene transcription. Ca channels are heteromeric proteins consisting of a pore-forming α₁ subunit andauxiliary α₂δ and β subunits. Although there are reports of α₂δ₄subunit mRNA in the mouse retina and the localization of the α₂δ₄ subunit immunoreactivity to salamander photoreceptor terminals, there is a limited overall understanding of itsexpression and localization in the retina. α₂δ₄ subunitexpression and distribution in the mouse and ratretina were evaluated using RT-PCR, Western blot and immunohistochemistry with specific primers and awell-characterizedantibody to theα₂δ₄ subunit.α₂δ₄subunit mRNA and protein are present in mouse and rat retina, brain, and liver homogenates. Immunostaining for the α₂δ₄ subunit is mainly localized toMüller cell processes and endfeet, photoreceptor terminals and photoreceptor outer segments. This subunit is also expressed in a fewdisplaced ganglion cells and bipolar cell dendrites. These findings suggest that the α₂δ₄ subunit participates in the modulation of L-typeCa²⁺current regulating neurotransmitter release from photoreceptor terminals and Ca²⁺-dependent signaling pathwaysin bipolar and Müller cells. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.

The expression and distribution of the α₂δ₄ subunit in the retina was evaluated using RT-PCR, Western blot and immunohistochemistry. α₂δ₄ subunit mRNA and protein are present in brain, retina and liver homogenates. Immunostaining for the α₂δ₄ subunit is mainly localized to Müller cell processes and endfeet, photoreceptor terminals and photoreceptor outer segments. In addition, this subunit is expressed in a few displaced ganglion cells and bipolar cell dendrites. These findings suggest that the α₂δ₄ subunit participates in the modulation of L-typeCa²⁺ current regulating neurotransmitter release from photoreceptors and bipolar cells, and regulates Ca²⁺ dependent signaling pathways in Müller cells.

Striatal cholinergic interneurons (ChIs) are involved in reward-dependent learning and the regulation of attention. The activity of these neurons is modulated by intrinsic and extrinsic GABAergic and glutamatergic afferents, but the source and relative prevalence of these diverse regulatory inputs remain to be characterized. To address this issue, we performed a quantitative ultrastructural analysis of the GABAergic and glutamatergic innervation of ChIs in the post-commissural putamen of rhesus monkeys to address this issue. Post-embedding immunogold localization of GABA combined with peroxidase immunostaining for choline acetyltransferase showed that 60% of all synaptic inputs to ChIs originate from GABAergic terminals, while 21% are from putatively glutamatergic terminals that establish asymmetric synapses, and 19% from other (non-GABAergic) sources of symmetric synapses. Double pre-embedding immunoelectron microscopy using substance P and Met-/Leu-enkephalin antibodies to label GABAergic terminals from collaterals of “direct” and “indirect” striatal projection neurons, respectively, revealed that 47% of the indirect pathway terminals and 36% of the direct pathway terminals target ChIs. Together, substance P- and enkephalin-positive terminals represent 24% of all synapses onto ChIs in the monkey putamen. These findings show that ChIs receive prominent GABAergic inputs from multiple origins, including a significant contingent from axon collaterals of direct and indirect pathway projection neurons. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.

We performed a quantitative ultrastructural analysis of the GABAergic innervation of cholinergic interneurons (ChIs) in the monkey post-commissural putamen. Our findings show that ChIs receive prominent GABAergic inputs from multiple origins, including a significant contingent (24%) from axon collaterals of direct (substance P-expressing) and indirect (enkephalin-expressing) pathway projection neurons, along their entire somatodendritic domain. Our data suggest that the activity of striatal ChIs is tightly regulated by local connections of GABAergic axon collaterals of projection neurons in primates.

Parkinson's disease is a neurodegenerative disorder characterized by Lewy bodies and neurites composed mainly of the presynaptic protein, α-synuclein. Frequently, Lewy bodies and neurites are identified in the gut of Parkinson's disease patients and may underlie associated gastrointestinal dysfunctions. We recently reported selective expression of α-synuclein in the axons of cholinergic neurons in the guinea-pig and human distal gut; however, it is not clear if α-synuclein expression varies along the gut, nor how closely expression is associated with other synaptic proteins. We used multiple-labeling immunohistochemistry to quantify which neurons in the guinea-pig ileum expressed α-synuclein, cysteine string protein-α (CSPα), synaptophysin, synaptotagmin-1, or synaptobrevin-2 in their axons. Of the 10 neurochemically-defined axonal populations, a significantly greater proportion of vesicular acetylcholine transporter-immunoreactive (VAChT-IR) varicosities (80 ± 1.7%, n=4, P < 0.001) contained α-synuclein-immunoreactivity, and a significantly greater proportion of α-synuclein-IR axons also contained VAChT-immunoreactivity (78 ± 1.3%, n=4), compared to any of the other 9 populations (P < 0.001 in all cases). Of synaptophysin-, synaptotagmin-1-, synaptobrevin-2-, and CSPα-IR varicosities, 98 ± 0.7%, 96 ± 0.7%, 88 ± 1.6% and 85 ± 2.9% (n=4) contained α-synuclein-immunoreactivity, respectively. Of α-synuclein-IR varicosities, 96 ± 0.9%, 99 ± 0.6%, 83 ± 1.9% and 87 ± 2.3% (n=4) contained synaptophysin-, synaptotagmin-1-, synaptobrevin-2-, and CSPα-immunoreactivity, respectively. We report a close association between the expression of α-synuclein and other synaptic proteins in cholinergic axons in the guinea-pig ileum. Selective expression of α-synuclein may relate to the neurotransmitter system utilized and predispose cholinergic enteric neurons to neurodegeneration in Parkinson's disease. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.

Mishandling of α-synuclein contributes to formation of Lewy bodies and Lewy neurites in Parkinson's disease. Here we show that the synaptic chaperones, α-synuclein and cysteine string protein-α, and synaptic vesicle proteins, synaptophysin, synaptotagmin-1 and synaptobrevin-2, are selectively co-expressed in cholinergic axons, marked by vesicular acetylcholine transporter, in the enteric nervous system of the guinea-pig ileum. We speculate that the neurotransmitter-release mechanism utilized by cholinergic neurons in the gut may predispose them to degeneration in Parkinson's disease.

The perirhinal cortex plays a critical role in recognition and associative memory. However, the network properties that support perirhinal contributions to memory are unclear. To shed light on this question, we compared the synaptic articulation of short- and long-range inputs from the perirhinal cortex or temporal neocortex with perirhinal neurons in rats. Iontophoretic injections of the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHAL) were performed at different rostrocaudal levels of the ventral temporal neocortex or perirhinal cortex, and electron microscopic observations of anterogradely labeled (PHAL+) axon terminals found at perirhinal sites adjacent to or rostrocaudally distant from the injection sites were performed. Following neocortical injections, the density of PHAL+ axons in the perirhinal cortex decreased steeply with rostrocaudal distance from the injection sites, much more so than following perirhinal injections. Otherwise, similar results were obtained with neocortical and perirhinal injections. In both cases, most (76-86%) PHAL+ axon terminals formed asymmetric synapses, typically with spines (Type-A,83-89%), and less frequently with dendritic profiles (Type-B,11-17%). The remaining terminals formed symmetric synapses with dendritic profiles (Type-C,14-23%). Importantly, Type-B and C synapses were 2.4-2.6 times more frequent in short- than long-range connections. The postsynaptic elements in Type A-C synapses were identified with immunocytochemistry for CAMKIIα, a marker of glutamatergic cortical neurons. Type-A and C terminals contacted CAMKIIα-positive principal cells, whereas Type-B synapses contacted presumed inhibitory neurons. Overall, these results suggest that principal perirhinal neurons are subjected to significantly more inhibition from short- than long-range cortical inputs, an organization that likely impacts on perirhinal contributions to memory. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.

Extrinsic and intrinsic cortical inputs to the perirhinal cortex involve more inhibition at rostrocaudal levels adjacent to the neurons at the origin of the projection (short-range) than at longitudinally distant sites (long-range). This differential connectivity likely shapes perirhinal contributions to memory.

Naturally-occurring cell death is essential to the development of the mammalian nervous system. Although the importance of developmental cell death has been appreciated for decades, there is no comprehensive account of cell death across brain areas in the mouse. Moreover, several regional sex differences in cell death have been described inthe ventral forebrain and hypothalamus, but it is not known how widespread the phenomenon is. We used immunohistochemical detection of activated caspase-3 to identify dying cells in the brains of male and female mice from postnatal day (P)1 to P11. Cell death density, total number of dying cells, and regional volume were determined in 16 regions of the hypothalamus and ventral forebrain(the anterior hypothalamus, arcuate nucleus, anteroventral periventricular nucleus, medial preoptic nucleus, paraventricular nucleus, suprachiasmatic nucleus and ventromedial nucleus of the hypothalamus;the basolateral, central and medial amygdala; the lateral and principal nuclei of the bed nuclei of the striaterminalis; the caudate-putamen, globuspallidus, lateral septum, and Islands of Calleja). All regions showed a significant effect of age on cell death. The timing of peak cell death varied between P1 to P7 and the average rate of cell death varied10-fold among regions. Several significant sex differences in cell death and/or regional volume were detected. These data address large gaps in the developmental literature and suggest interesting region-specific differences in the prevalence and timing of cell death in the hypothalamus and ventral forebrain. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.

We used immunocytochemistry for activated caspase-3 (AC3) to quantify developmental cell death in the hypothalamus and ventral forebrain of male and female mice between postnatal day (P) 1 and P11. The peak of cell death varied between P1 and P7 and rate of cell death varied over 5-fold among the 16 regions examined. Several sex differences in cell death or regional volume were observed and for almost all regions a significant reduction in cell death occurred between P5 and P7.

The waggle dancers of honeybees encode roughly the distance and direction to the food source as the duration of the waggle phase and the body angle during the waggle phase. It is believed that hive-mates detect airborne vibrations produced during the waggle phase to acquire distance information, and simultaneously detect the body axis during the waggle phase to acquire direction information. It has been further proposed that the orientation of the body axis on the vertical comb is detected by neck hairs (NHs) on the prosternal organ. The afferents of the NHs project into the prothoracic and mesothoracic ganglia, and the dorsal subesophageal ganglion (dSEG). This study demonstrates somatotopic organization within the dSEG of the central projections of the mechanosensory neurons of the NHs. The terminals of the NH afferents in dSEG are in close apposition to those of Johnston's organ (JO) afferents. The sensory axons of both terminate in a region posterior to the crossing of the ventral intermediate tract (VIT) and the maxillary dorsal commissures I and III (MxDCI, III) in the subesophageal ganglion. These features of the terminal areas of the NH and JO afferents are common to the worker, drone, and queen castes of honeybees. Analysis of the spatial relationship between the NH neurons and the morphologically and physiologically characterized vibration-sensitive interneurons DL-Int-1 and DL-Int-2 demonstrated that several branches of DL-Int-1 are in close proximity to the central projection of the mechanosensory neurons of the NHs in the dSEG. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.

Graphical Abstract: We demonstrate that the topological organization of sensory afferents within the dorsal subesophageal ganglion (dSEG) reflects the subregions of the peripheral neck hairs (NHs, detector of direction). The terminals of the NH afferents within the dSEG are in close apposition to those of Johnston's organ(detector of distance) afferents.

The loss of cognitive function in Alzheimer's disease (AD) patients is strongly correlated with the loss of neurons in various regions of the brain. We have created a new fluorescent bigenic mouse model of AD by crossing “H-line” yellow fluorescent protein (YFP) mice with the 5xFAD mouse model, which we call the 5XY mouse model. The 5xFAD mouse has been shown to have significant loss of L5 pyramidal neurons by 12 months of age. These neurons are transgenically labeled with YFP in the 5XY mouse, which enable longitudinal imaging of structural changes. In the 5XY mice, we observed an appearance of axonal dystrophies, with two distinct morphologies in the early stages of the disease progression. Simple swelling dystrophies are transient in nature and are not directly associated with amyloid plaques. Rosette dystrophies are more complex structures that remained stable throughout all imaging sessions, and always surrounded an amyloid plaque. Plaque growth was followed over 4 weeks, and significant growth was seen between weekly imaging sessions. In addition to axonal dystrophy appearance and plaque growth, we were able to follow spine stability in 4-month old 5XY mice, which revealed no significant loss of spines. 5XY mice also showed a striking shrinkage of the neocortex at older ages (12–14 months). The 5XY mouse model may be a valuable tool for studying specific events in the degeneration of the neocortex, and may suggest new avenues for therapeutic intervention. J. Comp. Neurol. 521:2181–2194, 2013. © 2012 Wiley Periodicals, Inc.

The loss of cognitive function in Alzheimer's disease (AD) patients is strongly correlated with the loss of neurons in various regions of the brain. We have created a new fluorescent bigenic mouse model of AD by crossing “H-line” yellow fluorescent protein (YFP) mice with the 5xFAD mouse model, which we call the 5XY mouse model. The 5xFAD mouse has been shown to have significant loss of L5 pyramidal neurons by 12 months of age. These neurons are transgenically labeled with YFP in the 5XY mouse, which enable longitudinal imaging of structural changes. In the 5XY mice, we observed an appearance of axonal dystrophies, with two distinct morphologies in the early stages of the disease progression. Simple swelling dystrophies are transient in nature and are not directly associated with amyloid plaques. Rosette dystrophies are more complex structures that remained stable throughout all imaging sessions, and always surrounded an amyloid plaque. Plaque growth was followed over 4 weeks, and significant growth was seen between weekly imaging sessions. In addition to axonal dystrophy appearance and plaque growth, we were able to follow spine stability in 4-month old 5XY mice, which revealed no significant loss of spines. 5XY mice also showed a striking shrinkage of the neocortex at older ages (12–14 months). The 5XY mouse model may be a valuable tool for studying specific events in the degeneration of the neocortex, and may suggest new avenues for therapeutic intervention. J. Comp. Neurol. 521:2181–2194, 2013. © 2012 Wiley Periodicals, Inc.

We have used longitudinal in vivo two-photon microscopy to study a new bigenic mouse model of Alzheimer's disease (5xFAD-YFP) that exhibits massive loss of fluorescently labeled layer 5 pyramidal neurons. Imaging of this transgenic mouse enabled the study of spine stability 500-600 μm below the pia mater, progressive atrophy of axons, appearance and growth of amyloid plaques, and eventual loss of neocortical pyramidal neurons.

The brainstem nucleus locus coeruleus (LC) is the sole source of norepinephrine (NE)-containing fibers in the mammalian cortex. Previous studies suggest that the density of noradrenergic fibers in rat is relatively uniform across cortical regions and that cells in the nucleus discharge en masse. This implies that activation of the LC results in equivalent release of NE throughout the cortex. However, it is possible that there could be differences in the density of axonal varicosities across regions, and that these differences, rather than a difference in fiber density, may contribute to the regulation of NE efflux. Quantification of dopamine β-hydroxylase (DβH)-immunostained varicosities was performed on several cortical regions and in the ventral posterior medial (VPM) thalamus by using unbiased sampling methods. The density of DβH varicosities is greater in the prefrontal cortex than in motor, somatosensory, or piriform cortices, greater in superficial than in deep layers of cortex, and greater in the VPM than in the somatosensory cortex. Our results provide anatomical evidence for non-uniform release of NE across functionally discrete cortical regions. This morphology may account for a differential, region-specific, impact of LC output on different cortical areas. J. Comp. Neurol. 521:2195–2207, 2013. © 2012 Wiley Periodicals, Inc.

The conventional view has been that norepinephrine (NE)-containing projections from the locus coeruleus (LC) are uniform across the cortical mantle. Although NE fiber density may be equivalent among regions, the density of NE varicosities is not uniform. We found that NE varicosities are greater in the prefrontal cortex than in the motor, somatosensory, or piriform cortices. This suggests that activation of the LC may produce a differential release of NE across the cortex, with the greatest amounts of NE released within the prefrontal cortex.

Melanin-concentrating hormone (MCH) is a hypothalamic neuropeptide that acts via MCH receptor 1 (MCHR1) in the mouse. It promotes positive energy balance; thus, mice lacking MCH or MCHR1 are lean, hyperactive, and resistant to diet-induced obesity. Identifying the cellular targets of MCH is an important step to understanding the mechanisms underlying MCH actions. We generated the Mchr1-cre mouse that expresses cre recombinase driven by the MCHR1 promoter and crossed it with a tdTomato reporter mouse. The resulting Mchr1-cre/tdTomato progeny expressed easily detectable tdTomato fluorescence in MCHR1 neurons, which were found throughout the olfactory system, striatum, and hypothalamus. To chemically identify MCH-targeted cell populations that play a role in energy balance, MCHR1 hypothalamic neurons were characterized by colabeling select hypothalamic neuropeptides with tdTomato fluorescence. TdTomato fluorescence colocalized with dynorphin, oxytocin, vasopressin, enkephalin, thyrothropin-releasing hormone, and corticotropin-releasing factor immunoreactive cells in the paraventricular nucleus. In the lateral hypothalamus, neurotensin, but neither orexin nor MCH neurons, expressed tdTomato. In the arcuate nucleus, both Neuropeptide Y and proopiomelanocortin cells expressed tdTomato. We further demonstrated that some of these arcuate neurons were also targets of leptin action. Interestingly, MCHR1 was expressed in the vast majority of leptin-sensitive proopiomelanocortin neurons, highlighting their importance for the orexigenic actions of MCH. Taken together, this study supports the use of the Mchr1-cre mouse for outlining the neuroanatomical distribution and neurochemical phenotype of MCHR1 neurons. J. Comp. Neurol. 521:2208–2234, 2013. © 2012 Wiley Periodicals, Inc.

Melanin-concentrating hormone (MCH) is an orexigenic neuropeptide that acts on MCH receptors 1 (MCHR1) in rodents. This study uses the Mchr1-cre/tdTomato mouse to readily identify MCHR1-expressing neurons by red fluorescence. We found that MCHR1 is expressed in different cell types throughout the hypothalamus. Thus we provide a neuroanatomical basis for MCH to regulate neuroendocrine and autonomic functions, including energy balance and appetite.

Glucagon-like-peptide 1 (GLP-1) is expressed not only in gut endocrine cells, but also in cells in the caudal brainstem and taste buds. To better understand the functions of central GLP-1, GLP-1 expression was immunohistochemically profiled in normal rat brain and its distribution correlated with FOS induction following systemic administration of a GLP-1 receptor agonist, exendin-4. In the present study, only a small number of GLP-1-immunoreactive cell bodies were observed in the nucleus of the solitary tract (NTS). However, these neurons send abundant projections to other regions of the brain, in particular the forebrain, including the paraventricular and dorsomedial nuclei of the hypothalamus, the central nucleus of the amygdala, the oval nucleus of the bed nuclei of the stria terminalis, and the paraventricular nucleus of the thalamus. Intraperitoneal administration of exendin-4 resulted in extensive FOS expression in areas of the forebrain and the hindbrain. In the forebrain, FOS expression was largely confined to regions where a high density of GLP-1-immunoreactive terminals was also localized. The majority of GLP-1-immunoreactive cells in the NTS were not FOS-positive. FOS-positive cells appeared to represent a different population from those expressing GLP-1. Thus, GLP-1-containing neurons in the brainstem may not be involved in receiving and relaying to other regions of the brain the physiological signals of prandial GLP-1 secreted by intestinal L-cells. Projections of GLP-1-containing neurons to the distinctive structures in the forebrain imply that central GLP-1 may play an important role in the behavioral and metabolic integration of autonomic control and arousal in the rat. J. Comp. Neurol. 521:2235–2261, 2013. © 2012 Wiley Periodicals, Inc.

Glucagon-like-peptide 1 (GLP-1) is not only secreted by gut endocrine cells but also expressed in a small number of neurons in the caudal brainstem. These neurons send massive outputs to other areas of the brain, particularly forebrain, that are known to be involved in metabolic homeostasis. Peripheral administration of GLP-1 receptor agonist does not appear to activate GLP-1-containing neurons directly, but activates areas in the forebrain where abundant GLP-1-containing terminals are found, suggesting central GLP-1-mediated activity is independent of peripheral GLP-1.

Nonmammalian vertebrates have a remarkable capacity to regenerate brain tissue in response to central nervous system (CNS) injury. Nevertheless, it is not clear whether animals recover lost function after injury or whether injury-induced cell proliferation mediates recovery. We address these questions using the visual system and visually-guided behavior in Xenopus laevis tadpoles. We established a reproducible means to produce a unilateral focal injury to optic tectal neurons without damaging retinotectal axons. We then assayed a tectally-mediated visual avoidance behavior to evaluate behavioral impairment and recovery. Focal ablation of part of the optic tectum prevents the visual avoidance response to moving stimuli. Animals recover the behavior over the week following injury. Injury induces a burst of proliferation of tectal progenitor cells based on phospho-histone H3 immunolabeling and experiments showing that Musashi-immunoreactive tectal progenitors incorporate the thymidine analog chlorodeoxyuridine after injury. Pulse chase experiments indicate that the newly-generated cells differentiate into N-β-tubulin-immunoreactive neurons. Furthermore, in vivo time-lapse imaging shows that Sox2-expressing neural progenitors divide in response to injury and generate neurons with elaborate dendritic arbors. These experiments indicate that new neurons are generated in response to injury. To test if neurogenesis is necessary for recovery from injury, we blocked cell proliferation in vivo and found that recovery of the visual avoidance behavior is inhibited by drugs that block cell proliferation. Moreover, behavioral recovery is facilitated by changes in visual experience that increase tectal progenitor cell proliferation. Our data indicate that neurogenesis in the optic tectum is critical for recovery of visually-guided behavior after injury. J. Comp. Neurol. 521:2262–2278, 2013. © 2012 Wiley Periodicals, Inc.

Although amphibians are known for their regenerative response to brain injury, it is unclear whether recovery of behavioral deficits requires neurogenesis. We report that focal injury of the optic tectum Xenopus tadpoles prevents behavioral avoidance to moving stimuli, yet animals recover after one week. Injury induces proliferation of tectal progenitors that generate neurons. Blocking cell proliferation inhibits recovery, and changes in visual experience that increase neurogenesis facilitate recovery, indicating that neurogenesis is critical for recovery of visually-guided behavior after injury.

N-Methyl-D-aspartate receptors (NMDARs) are involved in learning and memory processes in vertebrates and invertebrates. In Neohelice granulata, NMDARs are involved in the storage of associative memories (see references in text). The aim of this work was to characterize this type of glutamate receptor in Neohelice and to describe its distribution in the central nervous system (CNS). As a first step, a detailed study of the CNS of N. granulata was performed at the neuropil level, with special focus on one of the main structures involved in this type of memory, the supraesophageal ganglion, called central brain. The characterization of the NMDAR was achieved by identifying the essential subunit of these receptors, the NR1-like subunit. The NR1-like signals were found via western blot and immunohistochemistry techniques in each of the major ganglia: the eyestalk ganglia, the central brain, and the thoracic ganglion. Western blots yielded two bands for the crab NR1-like subunit, at ∼88 and ∼84 kDa. This subunit is present in all the major ganglia, and shows a strong localization in synaptosomal membranes. NMDARs are distributed throughout the majority of each ganglion but show prominent signal intensity in some distinguishable neuropils and neurons. This is the first general description of the N. granulata nervous system as a whole and the first study of NMDARs in the CNS of decapods. The preferential localization of the receptor in some neuropils and neurons indicates the presence of possible new targets for memory processing and storage. J. Comp. Neurol. 521:2279–2297, 2013. © 2012 Wiley Periodicals, Inc.

This study describes the distribution of the NMDA receptor subunit NR-1 and its characterization in the main ganglia of the central nervous system of the crab Neohelice granulata, including the eyestalk ganglia, central brain and thoracic ganglion. This receptor is a key component involved in memory storage in all species studied. Much of the work performed regarding memory storage in this crab indicates that the central brain is an important structure in this process and the focus of our description.

Astrocytes in the posterodorsal portion of the medial amygdala (MePD) are sexually dimorphic in adult rats: males have more astrocytes in the right MePD and more elaborate processes in the left MePD than do females. Functional androgen receptors (ARs) are required for masculinization of MePD astrocytes, as these measures are demasculinized in adult males carrying the testicular feminization mutation (Tfm) of the AR gene, which renders AR dysfunctional. We now report that the number of astrocytes is already sexually dimorphic in the right MePD of juvenile 25-day-old (P25) rats. Because Tfm males have as many astrocytes as wild-type males at this age, this prepubertal sexual dimorphism is independent of ARs. After P25, astrocyte number increases in the MePD of all groups, but activation of ARs augments this increase in the right MePD, where more astrocytes are added in males than in Tfm males. Consequently, by adulthood, females and Tfm males have equivalent numbers of astrocytes in the right MePD. Sexual dimorphism in astrocyte arbor complexity in the left MePD arises after P25, and is entirely AR-dependent. Thus, masculinization of MePD astrocytes is a result of both AR-independent processes before the juvenile period and AR-dependent processes afterward. J. Comp. Neurol. 521:2298–2309, 2013. © 2012 Wiley Periodicals, Inc.

Astrocytes in a portion of the rodent amygdala are sexually dimorphic in number and arbor complexity. These sex differences emerge at different times during the lifespan through both androgen-dependent and -independent processes. Given their role in synapse formation, this may reflect a “rewiring” of the amygdala during critical periods.

Topographic organization of neurons is a hallmark of brain structure. The establishment of the connections between topographically organized brain regions has attracted much experimental attention, and it is widely accepted that molecular cues guide outgrowing axons to their targets in order to construct topographic maps. In a number of systems afferent axons are organized topographically along their trajectory as well, and it has been suggested that this pre-target sorting contributes to map formation. Neurons in auditory regions of the brain are arranged according to their best frequency (BF), the sound frequency they respond to optimally. This BF changes predictably with position along the so-called tonotopic axis. In the avian auditory brainstem, the tonotopic organization of the second- and third-order auditory neurons in nucleus magnocellularis (NM) and nucleus laminaris (NL) has been well described. In this study we examine whether the decussating NM axons forming the crossed dorsal cochlear tract (XDCT) and innervating the contralateral NL are arranged in a systematic manner. We electroporated dye into cells in different frequency regions of NM to anterogradely label their axons in XDCT. The placement of dye in NM was compared to the location of labeled axons in XDCT. Our results show that NM axons in XDCT are organized in a precise tonotopic manner along the rostrocaudal axis, spanning the entire rostrocaudal extent of both the origin and target nuclei. We propose that in the avian auditory brainstem, this pretarget axon sorting contributes to tonotopic map formation in NL. J. Comp. Neurol. 521:2310–2320, 2013. © 2012 Wiley Periodicals, Inc.

Combining multicell and single-cell labeling with a whole-brainstem imaging technique, the authors show topographic axon arrangement in the avian auditory brainstem. This pre-target axon sorting might contribute to tonotopic map formation in the auditory system.

Defining how arginine vasopressin (AVP) acts centrally to regulate homeostasis and behavior is problematic, as AVP is made in multiple nuclei in the hypothalamus (i.e., paraventricular [PVN], supraoptic [SON], and suprachiasmatic [SCN]) and extended amygdala (i.e., bed nucleus of the stria terminalis [BNST] and medial amygdala [MeA]), and these groups of neurons have extensive projections throughout the brain. To understand the function of AVP, it is essential to know the site of origin of various projections. In mice, we used gonadectomy to eliminate gonadal steroid hormone–dependent expression of AVP in the BNST and MeA and electrolytic lesions to eliminate the SCN, effectively eliminating those AVP-immunoreactive projections; we also quantified AVP-immunoreactive fiber density in gonadectomized and sham-operated male and female mice to examine sex differences in AVP innervation. Our results suggest that the BNST/MeA AVP system innervates regions containing major modulatory neurotransmitters (e.g., serotonin and dopamine) and thus may be involved in regulating behavioral state. Furthermore, this system may be biased toward the regulation of male behavior, given the numerous regions in which males have a denser AVP-immunoreactive innervation than females. AVP from the SCN is found in regions important for the regulation of hormone output and behavior. Innervation from the PVN and SON is found in brain regions that likely work in concert with the well-known peripheral AVP actions of controlling homeostasis and stress response; female-biased sex differences in this system may be related to the heightened stress response observed in females. J. Comp. Neurol. 521:2321–2358, 2013. © 2012 Wiley Periodicals, Inc.

The neuropeptide vasopressin is produced in multiple distinct neuronal populations implicated in a wide variety of functions. Our study identifies, in the mouse, the unique pathways of steroid-sensitive vasopressin projections implicated in social behavior; the suprachiasmatic nucleus (SCN)-derived projections involved in circadian rhythms; and the non-SCN hypothalamic projections that help regulate physiological homeostasis. This vasopressin roadmap will provide direction to future researchers in the quest to understand the unique neural mechanisms involved in complex behavioral and physiological processes.

The corticospinal tract in the macaque and human forms the major descending pathway involved in volitional hand movements. Following a unilateral cervical dorsal root lesion, by which sensory input to the first three digits (D1–D3) is removed, monkeys are initially unable to perform a grasp retrieval task requiring sensory feedback. Over several months, however, they recover much of this capability. Past studies in our laboratory have identified a number of changes in the afferent circuitry that occur as function returns, but do changes to the efferent pathways also contribute to compensatory recovery? In this study we examined the role of the corticospinal tract in pathway reorganization following a unilateral cervical dorsal rhizotomy. Several months after animals received a lesion, the corticospinal pathways originating in the primary somatosensory and motor cortex were labeled, and terminal distribution patterns on the two sides of the cervical cord were compared. Tracers were injected only into the region of D1–D3 representation (identified electrophysiologically). We observed a strikingly different terminal labeling pattern post lesion for projections originating in the somatosensory versus motor cortex. The terminal territory from the somatosensory cortex was significantly smaller compared with the contralateral side (area mean = 0.30 vs. 0.55 mm²), indicating retraction or atrophy of terminals. In contrast, the terminal territory from the motor cortex did not shrink, and in three of four animals, aberrant terminal label was observed in the dorsal horn ipsilateral to the lesion, indicating sprouting. These differences suggest that cortical regions play a different role in post-injury recovery. J. Comp. Neurol. 521:2359–2372, 2013. © 2012 Wiley Periodicals, Inc.

The corticospinal tract in the macaque and human originates from at least nine separate cortical regions, and is the major descending pathway mediating volitional hand movements. Here we compared primary motor and somatosensory corticospinal subcomponents in the recovery process several months following a unilateral cervical dorsal rhizotomy in macaques. Our findings show a very different response from each cortical region, indicating that each plays a very different role post injury. Our findings also indicate that the corticospinal tract response to spinal cord injury is considerably more complex than is generally recognized.

The noradrenergic locus coeruleus (LC) regulates arousal, memory, sympathetic nervous system activity, and pain. Forebrain projections to LC have been characterized in rat, cat, and primates, but not systematically in mouse. We surveyed mouse forebrain LC-projecting neurons by examining retrogradely labeled cells following LC iontophoresis of Fluoro-Gold and anterograde LC labeling after forebrain injection of biotinylated dextran amine or viral tracer. Similar to other species, the central amygdalar nucleus (CAmy), anterior hypothalamus, paraventricular nucleus, and posterior lateral hypothalamic area (PLH) provide major LC inputs. By using mice expressing green fluorescent protein in γ-aminobutyric acid (GABA)ergic neurons, we found that more than one-third of LC-projecting CAmy and PLH neurons are GABAergic. LC colocalization of biotinylated dextran amine, following CAmy or PLH injection, with either green fluorescent protein or glutamic acid decarboxylase (GAD)65/67 immunoreactivity confirmed these GABAergic projections. CAmy injection of adeno-associated virus encoding channelrhodopsin-2-Venus showed similar fiber labeling and association with GAD65/67-immunoreactive (ir) and tyrosine hydroxylase (TH)-ir neurons. CAmy and PLH projections were densest in a pericoerulear zone, but many fibers entered the LC proper. Close apposition between CAmy GABAergic projections and TH-ir processes suggests that CAmy GABAergic neurons may directly inhibit noradrenergic principal neurons. Direct LC neuron targeting was confirmed by anterograde transneuronal labeling of LC TH-ir neurons following CAmy or PLH injection of a herpes virus that expresses red fluorescent protein following activation by Cre recombinase in mice that express Cre recombinase in GABAergic neurons. This description of GABAergic projections from the CAmy and PLH to the LC clarifies important forebrain sources of inhibitory control of central nervous system noradrenergic activity. J. Comp. Neurol. 521:2373–2397, 2013. © 2012 Wiley Periodicals, Inc.

We mapped mouse forebrain projections to the brainstem locus ceruleus, focusing on GABAergic projection neurons. Major GABAergic projections arise in the central amygdala and posterior lateral hypothalamus. These projections largely terminate outside the cellular core but form direct connections with the noradrenergic neurons based on transfer of viral tracer.