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We have previously used the differential display method to identify a gene that is expressed preferentially in the mushroom bodies of worker honeybees and to show that it encodes a putative inositol 1,4,5-trisphosphate receptor (IP3R) homologue (Kamikouchi et al. [1998] Biochem. Biophys. Res. Commun. 242:181–186). In the present study, we examined whether the expression of some of the genes for proteins involved in the intracellular Ca²⁺ signal transduction is also concentrated in the mushroom bodies of the honeybee by isolating cDNA fragments that encode the Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and protein kinase C (PKC) homologues of the honeybee. In situ hybridization analysis revealed that the expression of these genes was also concentrated in the mushroom bodies of the honeybee brain: The CaMKII gene was expressed preferentially in the large-type Kenyon cells of the mushroom bodies, whereas that for PKC was expressed in both the large and small types of Kenyon cells. The expression of the genes for IP3R and CaMKII was concentrated in the mushroom bodies of the queen and drone as well as in those of the worker bee. Furthermore, the enzymatic activities of CaMKII and PKC were found to be higher in the mushroom bodies/central bodies than in the optic and antennal lobes of the worker bee brain. These results suggest that the function of the intracellular Ca²⁺ signal transduction is enhanced in Kenyon cells in comparison to other neuronal cell types in the honeybee brain. J. Comp. Neurol. 417:501–510, 2000. © 2000 Wiley-Liss, Inc.

This is the third paper in a series on the morphology, immunohistochemistry, and synaptology of the mormyrid electrosensory lateral line lobe (ELL). The ELL is a highly laminated, cerebellum‐like structure in the rhombencephalon that subserves an active electric sense: Objects in the nearby environment are detected on the basis of changes in the reafferent electrosensory signals that are generated by the animal's own electric organ discharge. This paper concentrates on the intermediate (cell and fiber) layer of the medial zone of the ELL and pays particular attention to the large multipolar neurons of this layer (LMI cells). LMI cells are γ‐aminobutyric acid (GABA)ergic and have one axon and three to seven proximal dendrites that all become myelinated after their last proximal branching point. The axon projects to the contralateral homotopic region and has ipsilateral collaterals. Both ipsilaterally and contralaterally, it terminates in the deep and superficial granular layers. The myelinated dendrites end in the deep granular layer, where they most likely do not make postsynaptic specializations, but do make presynaptic specializations, similar to those of the LMI axons. Because it is not possible to distinguish between axonal and dendritic LMI terminals in the granular layer, the authors refer to both as LMI terminals. These are densely filled with small, flattened vesicles and form large appositions with ELL granular cell somata and dendrites with symmetric synaptic membrane specializations. LMI cells do not receive direct electrosensory input on their somata, but electrophysiological recordings suggest that they nevertheless respond strongly to electrosensory signals (Bell [1990] J. Neurophysiol. 63:303–318). Consequently, the authors speculate that the myelinated dendrites of LMI cells are excited ephaptically (i.e., by electric field effects) by granular cells, which, in turn, are excited via mixed synapses by mormyromast primary afferents. The authors suggest that this ephaptic activation of the GABAergic presynaptic terminals of the myelinated dendrites may trigger immediate synaptic release of GABA and, thus, may provide a very fast local feedback inhibition of the excited granular cells in the center of the electrosensory receptive field. Subsequent propagation of the dendritic excitation down the myelinated dendrites to the somata and axon hillocks of LMI cells probably generates somatic action potentials, resulting in the spread of inhibition through axonal terminals to a wide region around the receptive field center and in the contralateral ELL. Similar presynaptic myelinated dendrites that subserve feedback inhibition, until now, have not been described elsewhere in the brain of vertebrates. J. Comp. Neurol. 431:255–275, 2001. © 2001 Wiley‐Liss, Inc.

The tyrosine kinase TrkB is a receptor for the neurotrophic factors brain‐derived neurotrophic factor (BDNF) and neurotrophin‐4 (NT‐4). Retinal ganglion cells are responsive to BDNF, and TrkB has been localized in ganglion cells as well as in a subpopulation of amacrine cells in the retina of the chicken and the rat. In the present paper, we analyzed the distribution of TrkB immunoreactivity in the retina of marmoset monkeys, ferrets, rabbits, rats, mice, chickens, pigeons, barn owls, Pseudemysturtles, Xenopusfrogs, goldfishes, and carps. TrkB antibodies gave a positive reaction in all of these vertebrates. TrkB immunoreactivity was detected in the majority of retinal ganglion cells. Some amacrine cells also contained TrkB immunoreactivity; they were located mainly at the vitreal border of the inner nuclear layer, and their relative abundance varied in the different species. Until now, no information has been available concerning the neurochemical identity of the amacrine neurons containing TrkB. In some species (marmoset monkeys, rats, pigeons), we observed that the morphology and location of TrkB‐immunoreactive amacrine cells was reminiscent of that of the well‐described dopaminergic cells. To determine whether dopaminergic amacrine cells contained TrkB immunoreactivity, we therefore performed double‐labelling immunohistochemistry by using tyrosine hydroxylase (TH) antibodies in combination with TrkB antibodies in marmoset monkeys, rats, pigeons, Pseudemysturtles, and goldfishes. The most novel finding of the present paper is that, in all of these species, the majority of dopaminergic neurons were found to contain TrkB immunoreactivity. Dopaminergic neurons, on the other hand, represented only a fraction of the TrkB+amacrine cells.

Fiber connections of the carp torus longitudinalis were re‐examined by means of tract‐tracing methods. The torus longitudinalis projected mainly to the stratum marginale of the optic tectum, area pretectalis, and corpus cerebelli. The stratum marginale was anterogradely labeled only by biocytin, but not by horseradish peroxidase. Because the stratum is composed of extremely fine axons of the small toral neurons, this may be ascribed to different molecular weights of the tracers. The main afferent sources to the torus longitudinalis were the nucleus subvalvularis, which was located beneath the nucleus lateralis valvulae, the nucleus subeminentialis pars magnocellularis, and neurons along the posterior mesencephalo‐cerebellar tract. Some labeled cells also appeared in the area pretectalis, nucleus paracommissuralis, optic tectum, and torus semicircularis. In a previous paper, it was incorrectly reported that the valvula cerebelli was the main source of afferents to the torus longitudinalis. Here we report the reason for the previous mistake in relation to the techniques employed. J. Comp. Neurol. 457:202–211, 2003. © 2003 Wiley‐Liss, Inc.

The extracellular matrix of the brain contains large aggregates of chondroitin sulfate proteoglycans (CSPG), which form lattice‐like cell coatings around distinct neuron populations and are termed perineuronal nets. The function of perineuronal nets is not fully understood, but they are often found around neurons containing the calcium‐binding protein parvalbumin, suggesting a function in primarily highly active neurons. In the present paper the distribution of perineuronal nets was studied in two functional cell groups of the primate oculomotor system with well‐known firing properties: 1) the saccadic omnipause neurons in the nucleus raphe interpositus (RIP) exhibit a high tonic firing rate, which is only interrupted during saccades; they are inhibitory and use glycine as a transmitter; and 2) premotor burst neurons for vertical saccades in the rostral interstitial nucleus of the medial longitudinal fascicle (RiMLF) fire with high‐frequency bursts during saccades; they are excitatory and use glutamate and/or aspartate as a transmitter. In the macaque monkey, both cell populations were identified by their parvalbumin immunoreactivity and were studied for the presence of perineuronal nets using CSPG antibodies or lectin binding with Wisteria floribundaagglutinin. In addition, the expression of another calcium‐binding protein, calretinin, was studied in both cell groups. Double‐ and triple‐immunofluorescence methods revealed that both omnipause and burst neurons are selectively ensheathed with strongly labeled perineuronal nets. Calretinin was coexpressed in at least 70% of the saccadic burst neurons, but not in the omnipause neurons. Parallel staining of human tissue revealed strongly labeled perineuronal nets around the saccadic omnipause and burst neurons, in corresponding brainstem regions, which specifically highlighted these neurons within the poorly structured reticular formation. These findings support the hypothesis that perineuronal nets may provide a specialized microenvironment for highly active neurons to maintain their fast‐spiking activity and are not related to the transmitter or the postsynaptic action of the ensheathed neurons. J. Comp. Neurol. 455:341–352, 2003. © 2002 Wiley‐Liss, Inc.

This paper describes the morphology and physiology of five types of local interneurons and three types of ascending intersegmental interneurons in the locust metathoracic ganglion that are points of convergence of sensory information from the wings. Four types of spiking local interneurons are members of a population with somata at the ventral midline. They are depolarised by stimulation of a metathoracic wing nerve, suggesting that they encode a sensory representation of this appendage. Some are also depolarised with short latencies following stimulation of a mesothoracic wing nerve, indicating that they collate intersegmental as well as local information. All the local interneurons have branches in the anterior ventral association centre or around the roots of the nerve that carries wing sensory neurons. This distinguishes them from other interneurons in the population. A fifth type of local interneuron that has unusual bilateral branching and is not a member of this population is described for the first time. The ascending interneurons are members of three populations. Neurons of each population have a characteristic pattern of responses to stimulation of the mesothoracic or metathoracic wing nerves, and some respond to tactile stimulation or movements of a hind leg. These latter interneurons thus collate information from both wings and legs. All three types of intersegmental interneurons have branches in the anterior ventral association centre or around the roots of the wing nerve. The responses of the interneurons described here shed new light on both local and intersegmental network function in this model system. J. Comp. Neurol. 444:95–114, 2002. © 2002 Wiley‐Liss, Inc.

The nucleus incertus (NI) is a distinct cell group in caudoventral regions of the pontine periventricular gray, adjacent to the ventromedial border of the caudal dorsal tegmental nucleus. Recent interest in the NI stems from evidence that it represents one of the periventricular sites with the highest expression levels of mRNA encoding the type 1 corticotropin‐releasing hormone (CRH) receptor, which has a high affinity for naturally occurring CRH, perhaps accounting for some of the extrapituitary actions of the peptide on autonomic and behavioral components of the stress response. However, almost nothing is known about NI function and hodological relationships. In this paper, we present the results of a systematic analysis of NI inputs and outputs using cholera toxin B subunit as a retrograde tracer and Phaseolus vulgaris‐leucoagglutinin as an anterograde tracer. Our retrograde tracer experiments indicate that the NI is in a strategic position to integrate information related to behavioral planning (from the prefrontal cortex), lateral habenular processing, hippocampal function, and oculomotor control. Based on its efferent connections, the NI is in a position to exert significant modulating influences on prefrontal and hippocampal cortical activity, and the nucleus is also in a position to influence brain sites known to control locomotor behavior, attentive states, and learning processes. Overall, the present results support the idea that the NI is a distinct region of the pontine periventricular gray, and together with the superior central (median raphé) and interpeduncular nuclei the NI appears to form a midline behavior control network of the brainstem. J. Comp. Neurol. 438:86–122, 2001. © 2001 Wiley‐Liss, Inc.

In capitate hydropolyps, the spherical end‐knobs of the short tentacles present an exceptional concentration of sensory functions in one of the evolutionarily oldest nervous systems. The tentacular spheres are the basis of sensation and discrimination of objects and of capturing of prey‐objects by the discharge of nematocytes (stinging cells). Recent electrophysiological studies of the spheres revealed combined chemo/mechanosensory functioning of the nematocytes and mechanosensitivity of further types of cells. The present electron microscopical study made use of the small size of the spheres of Coryne tubulosato characterize all cells of some spheres. Five types of ectodermal cells were found to have sensory structural features and to be separated by or enclosed in supporting cells: 1) nematocytesof the stenotele type; 2) shortand 3) long ciliated concentric hair cells, which carry a cilium‐stereovilli bundle, similar to the cnidocil apparatus of nematocytes; 4) cells having a recessed cilium‐microvilli complex equipped with a thick cell‐traversing rootlet (rootlet cells); and 5) cells having a recessed short cilium with no microvilli and only a short rootlet and containing, apically as well as basally, aggregations of dense‐core vesicles (vesicle‐rich cells). Types 1–4 vary the configuration of a concentric cilium‐microvilli complex (variations of a concentric hair bundle) and were demonstrated or inferred to be mechanosensitive. Apical exocytotic activity, which is well known for the nematocytes (discharge of their cnidocyst), is indicated by ultrastructure for the nematocyte‐resembling concentric hair cells and for the vesicle‐rich cells. The tentacular spheres are considered an early paradigm of a sensory epithelium. Its synaptic structures and extensive connectivity are the subject of a subsequent paper. J. Comp. Neurol. 432:550–563, 2001. © 2001 Wiley‐Liss, Inc.

Brevican is one of the most abundant extracellular matrix proteoglycans in the mammalian brain. We have previously shown that brevican produced by gray matter astrocytes constitutes a major component of perineuronal extracellular matrix in the adult brain. In this paper, we investigate the expression of brevican in the postnatal hippocampal fimbria to explore the role of the proteoglycan in central nervous system fiber tract development. We demonstrate that brevican is expressed by both oligodendrocytes and white matter astrocytes in the fimbria, but the expression of brevican in these two glial cell types is differently regulated during development. At P14, brevican immunoreactivity was observed throughout the fimbria, with particularly strong immunoreactivity in the developing interfascicular glial rows. In situ hybridization showed that oligodendrocytes in the glial rows strongly express brevican during the second and third postnatal weeks. Expression in oligodendrocytes was then down‐regulated after P21. In the adult fimbria, no brevican expression was observed in oligodendrocytes. The time window of brevican expression coincides with the phase in which immature oligodendrocytes actively extend membrane processes and enwrap axon fibers. In contrast, the expression in astrocytes started around P21 as oligodendrocytes began to down‐regulate the expression. In the adult fimbria, brevican expression was restricted to astrocytes. In situ hybridization with isoform‐specific probes and RNase protection assays showed that the authentic, secreted form of brevican, not the glycosylphosphatidylinositol‐anchored variant, is the predominant species expressed in the developing fimbria. Our results suggest that brevican plays a dual role in developing and adult fiber tracts. J. Comp. Neurol. 432:285–295, 2001. © 2001 Wiley‐Liss, Inc.

In this paper, the authors report that the expression of tau‐based reporter genes causes severe defects in the morphology of sensory neurons in adult Drosophila. Targeted expression of tau‐green fluorescent protein (tau‐GFP) in sensory neurons, using the galactosidase‐4 (GAL4) system, produced a range of characteristic defects in expressing neurons. The defects observed included loss of axons, abnormal axon bundling, reduced sensory arborisations, and axonal swellings (beads). Blind comparisons of adult sensory neurons labelled with tau‐GFP or CD8‐GFP showed that tau‐GFP neurons exhibited many more defects than CD8‐GFP‐expressing neurons. CD8‐GFP was found to induce no significant defects on sensory neuron morphology. Expression of tau‐lacZ and human tau in sensory neurons produced defects comparable to those seen with tau‐GFP. A developmental study showed that tau‐expressing axons grow normally and innervate the correct regions of the neuropil. The absence of these axons later in development suggests that tau‐expressing axons are lost after initial ingrowth. Examination of silver‐stained sections suggests that the absence of axons is due to axon loss rather than failure of the expression system to label the neurons. The results suggest that the expression of tau‐based reporter constructs causes severe defects in sensory neurons, resulting in degeneration. The results also indicate that Drosophilamay provide a useful model system for examining the role of tau in neurodegenerative disorders. J. Comp. Neurol. 428:630–640, 2000. © 2000 Wiley‐Liss, Inc.

Glutamic acid decarboxylase (GAD) is the biosynthetic enzyme for γ‐aminobutyric acid (GABA), the major inhibitory neurotransmitter in the central nervous system (CNS) of vertebrates. In addition to the adult CNS, GABA and GAD also have been detected in embryos, although their precise localization and specific functions in embryonic development have not been elucidated. In this paper, the authors studied the cellular distribution of two GAD isoforms, GAD65 and GAD67, in midgestation mouse embryos by in situ hybridization histochemistry. With few exceptions, it was found that GAD65 and GAD67 mRNAs are localized in overlapping cellular domains of the embryonic CNS that later develop into regions with a strong GABAergic contribution. The GAD‐expressing cells are situated in the differentiating zone of the embryonic day 10.5 (E10.5) through E11.5 CNS and in the subventricular zone and the mantle zone of the E12.5 CNS, which suggests that they are committed neuronal precursors. By using a specific serum for GABA, a similar pattern of distribution was obtained, indicating that GAD mRNAs are translated efficiently into enzymatically active GAD, which produces embryonic GABA. The expression domains of GAD overlap with those of genes that are known to be involved in the patterning of the embryonic CNS. The two GAD mRNAs also are detected outside of the embryonic CNS in various cell types, mainly those of placodal and neural crest origin. This pattern of expression is consistent with the notion that GAD and its product, GABA, play a signaling role during development. J. Comp. Neurol. 424:607–627, 2000. © 2000 Wiley‐Liss, Inc.

The isolated abdominal central nervous system of Manduca sextaundergoes an increase in cyclic GMP (cGMP) when exposed to the insect peptide eclosion hormone (EH) before pupal ecdysis. Previously, cGMP immunocytochemistry revealed that the EH‐stimulated increase in cGMP was contained in numerous filamentous processes within the transverse nerve associated with each abdominal ganglion. These processes seemed to be the axons of neurosecretory cells projecting to this neurohemal organ. In the present paper, we now show that the EH‐stimulated cGMP is not present in neurosecretory terminals. There is no colocalization of the EH‐stimulated cGMP with immunoreactivity of two peptides, known to be present in axons in the transverse nerves. Furthermore, there is no colocalization of EH‐stimulated cGMP with the synaptic vesicle protein, synaptotagmin. The neurosecretory axons are localized to a narrow band at the anterior margin of the transverse nerve, whereas the cellular elements showing an EH‐stimulated cGMP increase are primarily present in the posterior region. There are two cell types in this region: a granular and a nongranular type. The cGMP immunoreactivity seems to be contained within the nongranular type. During adult development, the cells of the posterior compartment spread in a thin layer between the transverse and dorsal nerves, become positive for myosin immunoreactivity between pupal stages 5 and 8, and seem to form the adult ventral diaphragm muscles. We conclude that the EH‐sensitive filaments in the transverse nerves of Manducaare most likely to be intrinsic cells that subsequently develop into the ventral diaphragm muscles of the adult. J. Comp. Neurol. 424:339–355, 2000. © 2000 Wiley‐Liss, Inc.

Visual stimuli representing looming or receding objects can be decomposed into four parameters: change in luminance; increase or decrease of area; increase or decrease of object perimeter length; and motion of the object's perimeter or edge. This paper describes intracellular recordings from visual neurons in the optic lobes of Manduca sextathat are selectively activated by certain of these parameters. Two classes of wide‐field neurons have been identified that respond selectively to looming and receding stimuli. Class 1 cells respond to parameters of the image other than motion stimuli. They discriminate an approaching or receding disc from an outwardly or inwardly rotating spiral, being activated only by the disc and not by the spiral. Class 2 neurons respond to moving edges. They respond both to movement of the spiral and to an approaching or receding disc. These two classes are further subdivided into neurons that are excited by image expansion (looming) and are inhibited by image contraction (antilooming). Class 2 neurons also respond to horizontal and vertical movement of gratings over the retina. Stimulating class 1 and 2 neurons with white discs against a dark background results in the same activation as stimulation with dark discs against a white background, demonstrating that changes in luminance play no role in the detection of looming or antilooming. The present results show that the two types of looming‐sensitive neurons in M. sextause different mechanisms to detect the approach or retreat of an object. It is proposed that cardinal parameters for this are change of perimeter length detected by class 1 neurons and expansion or contraction visual flow fields detected by class 2 neurons. These two classes also differ with respect to their polarity, the former comprising centripetal cells from the optic lobes to the midbrain, the latter comprising centrifugal neurons from the midbrain to the optic lobes. The significance of these arrangements with respect to hovering flight is discussed. J. Comp. Neurol. 424:356–376, 2000. © 2000 Wiley‐Liss, Inc.

The primate red nucleus consists of three main neuron subpopulations, namely, rubrospinal neurons in the magnocellular nucleus, rubroolivary cells in the parvocellular nucleus, and local circuit neurons in both subnuclei: Each subpopulation has unique cerebellar and neocortical inputs. The structural framework for the interactions of these rubral subpopulations remains poorly defined and was the focus of this study in six macaques. Somata of rubrospinal neurons, dorsolateral‐spinal (DL‐spinal) neurons, as defined in the accompanying paper (Burman et al. [2000] J. Comp. Neurol., this issue), and rubroolivary neurons were labeled retrogradely first with Fast Blue injected either into the cervical spinal cord or the inferior olive. The soma/dendrite profiles of selected cells (53 rubrospinal, 19 DL‐spinal, and 17 rubroolivary cells) were visualized by the intracellular injection of Lucifer Yellow/biocytin in fixed slices (400 μm thick) of midbrain. The descriptive statistics of the somata and the dendritic arborization of each rubral neuron type were established. Projection neuron subpopulations had similar but differentiable soma/dendrite profiles, with four to six slender, spine‐bearing dendritic trees radiating out ≈400 μm from the soma. Twelve presumed interneurons, all in the parvocellular nucleus, differed from projection neurons in that they had smaller somata and many slender, spine‐bearing segments that constituted the multibranching dendrite profile that radiated out ≈250 μm from the soma. A tentative model of the macaque rubral microcircuitry was developed, and its functional implications were explored. It incorporated 1) the known topography of the nucleus and its connections, 2) our data specifying the soma/dendrite morphology of the three main rubral neuron types, and 3) the ultrastructure reported by other laboratories of intrarubral synaptic connections. J. Comp. Neurol. 423:197–219, 2000. © 2000 Wiley‐Liss, Inc.

The cerebellar, spinal, bulbar, and cortical connections of the mammalian red nucleus imply a motor role. However, what information the red nucleus receives, processes, and distributes is poorly understood, partly because the rubral microcircuitry, especially in primates, remains incompletely defined. Multiple retrogradely transported fluorescent tracers were injected into the spinal cord and inferior olive of the macaque to label rubrospinal and rubroolivary neuron populations, respectively. Anterograde dextran amines were used to label the terminals of corticorubral neurons. These data provided the topographic framework for examining the morphology of rubral neurons in the accompanying paper (Burman et al. [2000]). Soma profiles of rubrospinal and rubro‐olivary neurons were respectively segregated in the magnocellular and parvocellular nuclei. A subpopulation of neurons (DL‐spinal cells) with their somas immediately dorsolateral to the rostral magnocellular nucleus and its capsule, also projected to the spinal cord, as did clusters of neurons in the periaqueductal grey matter. Terminals of corticorubral axons originating from ipsilateral primary motor area 4 (the densest projection), the supplementary motor area, cingulate area 24, area 8, and posterior parietal area 5, were each mapped in the parvocellular red nucleus. Only area 4 projected to the magnocellular red nucleus, and this projection as small. DL‐spinal neurons had no cortical input. The somatotopic organization of rubral connections was examined only in (a) the corticorubral input from motor area 4, and (b) the rubrospinal and DL‐spinal projections. These connections and their somatotopic alignment, were mapped in a 3‐dimensional reconstruction of the red nucleus. J. Comp. Neurol. 423:179–196, 2000. © 2000 Wiley‐Liss, Inc.

Acetylcholine and γ ‐aminobutyric acid (GABA) are putative neurotransmitters in the adult vertebrate retina. In this study, cells that coexpress choline acetyltransferase (ChAT) and GABA or glutamic acid decarboxylase (GAD) were investigated in turtle retinas from stage 14 (S14) to adulthood by using a double‐labeling immunofluorescence technique. ChAT immunoreactivity was observed at S15 and included not only the presumptive starburst cholinergic amacrine cells but also a population in the ganglion cell layer (GCL) that expressed ChAT transiently during the embryonic stages (see the accompanying paper: Nguyen et al. [2000] J. Comp. Neurol. 420:512–526). J. Comp. Neurol. 420:527–538, 2000. © 2000 Wiley‐Liss, Inc.

Except during blinks, movements of the upper eyelid are tightly coupled to vertical eye movements. The premotor source for the coordination of lid and eye movements is unknown. The present paper provides the anatomical identification of a new premotor cell group in the rostral mesencephalon of the monkey and human, which lies in close proximity to the premotor center for vertical saccades and is thought to participate in lid‐eye coordination. After injections of a retrograde transsynaptic tracer (tetanus toxin fragment C or BIIb) into the levator palpebrae (LP), the superior rectus (SR), or the inferior oblique (IO) muscle of macaque monkeys, a small circumscribed group of premotor neurons was labeled in the central gray of the rostral mesencephalon, but not after superior oblique or inferior rectus muscle injections. This group lies immediately rostral to the interstitial nucleus of Cajal and medial to the rostral interstitial nucleus of the medial longitudinal fasciculus, each of which contain premotor neurons for vertical saccades, and was termed the M‐group. Injections of tritiated leucine into the M‐group led to afferent labeling primarily over LP motoneurons. In addition, label was present over the SR‐ and IO‐motoneuron subgroups in the oculomotor nucleus and frontalis muscle motoneurons in the facial nucleus. This projection pattern of the M‐group suggests a role in the coordination of the upper eyelid and eyes during upgaze. Double‐labeling experiments in macaque monkeys revealed that the M‐group is strongly parvalbumin immunoreactive and contains high levels of cytochrome oxidase activity. With these two histochemical markers, the homologue of the M‐group was identified in the human brain as well. J. Comp. Neurol. 420:19–34, 2000. © 2000 Wiley‐Liss, Inc.

This paper re‐examines a previously published segmental map of the frog diencephalon (Puelles et al. [1996] Brain Behav.Evol. 47:279–310) by means of immunocytochemical mapping of calretinin, calbindin, and tyrosine hydroxylase. The distribution of neuronal populations, axon tracts, and neuropils immunoreactive for these markers was studied in adult specimens of Rana pereziand Xenopus laevissectioned sagittally or horizontally. Emphasis was placed on study of the relationship of observed chemoarchitectural boundaries with the postulated overall prosomeric organization and the schema of nuclear subdivisions we reported previously, based on acetylcholinesterase histochemistry and Nissl pattern in Rana. The data reveal a large‐scale correspondence with the segmental map in both species, although some differences were noted between Ranaand Xenopus. Notably, retinorecipient neuropils were generally immunoreactive for calretinin only in Rana. Importantly, calretinin immunostaining underlines particularly well the transverse prosomeric boundaries of the dorsal thalamus. A number of nuclear subdivisions noted before with AChE were corroborated, and some novel subdivisions became apparent, particularly in the anterior nucleus of the dorsal thalamus and in the habenular complex. The mapping of tyrosine hydroxylase clarified the segmental distribution of the catecholaminergic cell groups in the frog forebrain, which is comparable to that observed in other vertebrates. J. Comp. Neurol. 419:96–121, 2000. © 2000 Wiley‐Liss, Inc.

Mechanisms underlying axonal pathfinding have been investigated for decades, and numerous molecules have been shown to play roles in this process, including members of the cadherin family of cell adhesion molecules. We showed in the companion paper that a member of the cadherin family (zebrafish R‐cadherin) is expressed in retinal ganglion cells, and in presumptive visual structures in zebrafish brain, during periods when the axons were actively extending toward their targets. The present study extends the earlier work by using 1,1′‐dioctadecyl‐3,3,3′,3′, tetramethylindocarbocyanine perchlorate (DiI) anterograde tracing techniques to label retinal ganglion cell axons combined with R‐cadherin in situ hybridization to explicitly examine the association of retinal axons and brain regions expressing R‐cadherin message. We found that in zebrafish embryos at 46–54 hours postfertilization, DiI‐labeled retinal axons were closely associated with cells expressing R‐cadherin message in the hypothalamus, the pretectum, and the anterolateral optic tectum. These results demonstrate that R‐cadherin is appropriately distributed to play a role in regulating development of the zebrafish visual system, and in particular, pathfinding and synaptogenesis of retinal ganglion cell axons. J. Comp. Neurol. 410:290–302, 1999. © 1999 Wiley‐Liss, Inc.

In the first step toward identifying the neurotransmitter released from spiking interneurons of both local and intersegmental groups in the crayfish terminal abdominal ganglion, the authors examined whether spiking local interneurons and ascending intersegmental interneurons contain the transmitter γ‐aminobutyric acid (GABA). In this paper, 17 identified ascending interneurons and three spiking local interneurons were stained by intracellular injection of Lucifer yellow and subsequently treated for immunocytochemical staining against GABA. Double‐labeling experiments revealed that six identified ascending interneurons are GABAergic, but no spiking local interneurons show GABA‐like immunoreactivity. Four ascending interneurons with GABA‐like immunoreactivity (reciprocal closing ascending neuron 5 [RC‐5], reciprocal opening ascending neuron 6 [RO‐6], variable‐effect ascending interneuron 1 [VE‐1], and no‐effect ascending interneuron 4[NE‐4]) had cell bodies that formed a cluster on the ventral surface of the rostral edge of the ganglion, whereas two GABAergic interneurons (coinhibiting ascending interneuron 2 [CI‐2] and NE‐2) had cell bodies in a caudal region around the cell body of the seventh flexor inhibitor (FI) motor neuron. Another four rostral interneurons (RC‐2, RC‐3, RC‐4, and NE‐3) and seven caudal interneurons (CI‐3, RC‐7, RO‐1, RO‐2, RO‐3, RO‐4, and NE‐1) had no GABA‐like immunoreactivity. Because VE‐1 is known to make direct inhibitory connections with other ascending interneurons, whereas RC‐3 and RO‐1 are known to make direct excitatory connections, the immunocytochemical results from this study are consistent with previous physiological studies. Although many spiking local interneurons (including spiking local interneuron 1 of the anterior group [sp‐ant1]) made direct inhibitory connections with nonspiking local interneurons, three spiking local interneurons (sp‐ant1, spiking local interneuron 6 of the medial group [sp‐med6], and spiking interneuron 5 of the posterior group [sp‐post]) do not show GABA‐like immunoreactivity. These results suggest that the inhibitory transmitter released from spiking local interneurons is not GABA but that another substance mediates the inhibitory action of these interneurons. J. Comp. Neurol. 410:677–688. © 1999 Wiley‐Liss, Inc.

The primary focus of this paper is the abrupt transition that occurs midway through the rhesus lateral geniculate nucleus (LGN) from six layers posteriorly (conventionally numbered 1–6, ventral to dorsal) to four layers anteriorly. At this transition, layers 4 and 6 fuse into a single layer, as do layers 3 and 5, requiring an inversion of the stacking order of the cell categories making up layers 4 and 5. To understand the topology of this transition and its relationship to geniculate retinotopy, we have created a functional atlas of a rhesus LGN that affords three‐dimensional views of morphology and retinotopy at a resolution of 25 μm. The projection of the path of the transition into visual space is highly biased toward lower visual fields, intersecting the upper vertical meridian at 6.4°, the horizonal meridian at 15.4°, and the lower vertical meridian at 25.0°. Between inclinations of −31° and 55°, layers 3 and 5 merge through an elongated tear in layer 4 that subsumes the optic disk gap and extends medially and laterally; elsewhere, layers 4 and 6 merge through a tear in layer 5. These tears cause substantial violations of retinotopy and laminar integrity, so the inversion of layers 4 and 5 requires that the forces establishing retinotopy and grouping by cell class be locally overcome during morphogenesis. The transition and associated tears are evaluated in the context of recent computational models of geniculate morphogenesis. We have also used the atlas to estimate the borders of the binocular (55 ≈ 62°) and monocular (91 ≈ 97°) visual fields. Files containing the atlas are made publicly available on a website. J. Comp. Neurol. 407:92–102, 1999. © 1999 Wiley‐Liss, Inc.

During the first month of postnatal life, the dendritic arborizations of cat retinal ganglion cells continue to develop and undergo a substantial remodeling. Mechanical and pharmacological interferences with the normal development induce, during this period of time, substantial modifications in ganglion cell morphology. Specifically, the degeneration of those neurons whose axons were severed by a neonatal retinal lesion leads to a zone depleted of ganglion cells. Neurons at the border of the depleted area develop an abnormal elongation of the dendritic trees toward the empty space. In the present paper, we report data showing that this dendritic reorganization can be prevented by blocking the electrical activity with repeated tetrodotoxin injections into the eye during the whole critical period. Our analysis was performed on neurons filled with horseradish peroxidase. J. Comp. Neurol. 405:262–270, 1999. © 1999 Wiley‐Liss, Inc.

The electrosensory lobe (ELL) of mormyrid electric fish is a cerebellum‐like brainstem structure that receives the primary afferent fibers from electroreceptors in the skin. The ELL and similar sensory structures in other fish receive extensive input from other central sources in addition to the peripheral input. The responses to some of these central inputs are adaptive and serve to minimize the effects of predictable sensory inputs. Understanding the interaction between peripheral and central inputs to the mormyrid ELL requires knowledge of its functional circuitry, and this paper examines this circuitry in the in vitro slice preparation and describes the axonal and dendritic morphology of major ELL cell types based on intracellular labeling with biocytin. The cells described include medium ganglion cells, large ganglion cells, large fusiform cells, thick‐smooth dendrite cells, small fusiform cells, granule cells, and primary afferent fibers. The medium ganglion cells are Purkinje‐like interneurons that terminate on the two types of efferent cells, i.e., large ganglion and large fusiform cells, as well as on each other. These medium ganglion cells fall into two morphologically distinct types based on the distributions of basal dendrites and axons. These distributions suggest hypotheses about the basic circuit of the ELL that have important functional consequences, such as enhancement of contrast between “on” elements that are excited by increased afferent activity and “off” elements that are inhibited. J. Comp. Neurol. 404:359–374, 1999. © 1999 Wiley‐Liss, Inc.

Production of prostaglandins is a critical step in transducing immune stimuli into central nervous system (CNS) responses, but the cellular source of prostaglandins responsible for CNS signalling is unknown. Cyclooxygenase catalyzes the rate‐limiting step in the synthesis of prostaglandins and exists in two isoforms. Regulation of the inducible isoform, cyclooxygenase 2, is thought to play a key role in the brain's response to acute inflammatory stimuli. In this paper, we report that intravenous lipopolysaccharide (LPS or endotoxin) induces cyclooxygenase 2‐like immunoreactivity in cells closely associated with brain blood vessels and in cells in the meninges. Neuronal staining was not noticeably altered or induced in any brain region by endotoxin challenge. Furthermore, many of the cells also were stained with a perivascular microglial/macrophage‐specific antibody, indicating that intravenous LPS induces cyclooxygenase in perivascular microglia along blood vessels and in meningeal macrophages at the edge of the brain. These findings suggest that perivascular microglia and meningeal macrophages throughout the brain may be the cellular source of prostaglandins following systemic immune challenge. We hypothesize that distinct components of the CNS response to immune system activation may be mediated by prostaglandins produced at specific intracranial sites such as the preoptic area (altered sleep and thermoregulation), medulla (adrenal corticosteroid response), and cerebral cortex (headache and encephalopathy). J. Comp. Neurol. 381:119‐129, 1997. © 1997 Wiley‐Liss, Inc.

This paper is an account of the afferent and efferent projections of the nucleus sphericus (NS), which is the major secondary vomeronasal structure in the brain of the snake Thamnophis sirtalis.There are four major efferent pathways from the NS: 1) a bilateral projection that courses, surrounding the accessory olfactory tract, and innervates several amygdaloid nuclei (nucleus of the accessory olfactory tract, dorsolateral amygdala, external amygdala, and ventral anterior amygdala), the rostral parts of the dorsal and lateral cortices, and the accessory olfactory bulb; 2) a bilateral projection that courses through the medial forebrain bundle and innervates the olfactostriatum (rostral and ventral striatum); 3) a commissural projection that courses through the anterior commissure and innervates mainly the contralateral NS; and 4) a meager bilateral projection to the lateral hypothalamus. On the other hand, important afferent projections to the NS arise solely in the accessory olfactory bulb, the nucleus of the accessory olfactory tract, and the contralateral NS.

The patterns and extent of vagal preganglionic divergence and convergence within the gastrointestinal tract of the rat were characterized with the anterograde tracer Phaseolus vulgaris‐leucoagglutinin (PHA‐L). Three weeks after tracer was iontophoretically injected into two to four sites within the dorsal motor nucleus of the vagus, wholemounts of perfused gut organs (stomach, duodenum, cecum) were prepared, counterstained with Cuprolinic blue, and processed for PHA‐L using the avidin biotin complex with diaminobenzidine. Controls included animals injected with PHA‐L after intracranial deafferentations. Well‐positioned injections labeled an extremely dense and intricate network of varicose efferent axons throughout the gastric myenteric plexus (including that of the fundus). Individual fibers collateralized extensively, forming a variety of pericellular arborizations and terminal complexes made up of both en passant and end swellings. Single axons frequently innervated subsets of neurons within ganglia. Most enteric neurons were contacted by varicosities of more than one vagal fiber. The patterns of vagal preganglionic fibers in the duodenal and cecal myenteric plexuses resembled the organization in the stomach in many aspects, but the projections in each organ had distinctive characteristics, and label was less dense in the intestines than in the stomach. Vagal preganglionic fibers directly innervated submucosal ganglia, although sparsely. Brainstem injections of PHA‐L retrogradely labeled a few myenteric neurons in the corpus, fundus, and duodenum: These “gastrobulbar” and “duodenobulbar” neurons received reciprocal vagal preganglionic innervation. Finally, the PHA‐L that spread to the nucleus of the solitary tract occasionally produced transganglionic labeling of afferent intramuscular arrays (gastric fundus). The results of this paper provide strong evidence that the traditional “command neuron” or “mother cell” hypotheses of vagal‐enteric organization should be abandoned for an integrative neural network model. J. Comp. Neurol. 381:81‐100, 1997. © 1997 Wiley‐Liss, Inc.

In this paper, we extend our previous light microscopic (LM) study of substance P (SP)‐containing amacrine and ganglion cell types of the human retina (Cuenca et al. [1995] J. Comp. Neurol. 356:491–504) to an electron microscopic (EM) and confocal‐imaging study in order to reveal synaptic circuitry and putative input and output neurons. SP‐immunoreactive (‐IR) amacrine cells in primate retina are typically wide‐field cells with large cell bodies occurring in normal or displaced positions relative to the inner plexiform layer (IPL). Their main dendrites bear many spines and are monostratified in stratum 3 (S3) of the IPL. Axon‐like processes arise from dendrites close to the cell body and run for hundreds of microns at the same level as the dendrites, thus forming a relatively dense plexus in S3 of the IPL. SP‐IR axon processes also climb to S1 to surround some amacrine cell bodies, and others pass into the outer plexiform layer (OPL). Still other axons run down to the ganglion cell layer, where they encircle SP‐IR ganglion cells and pass on to end in the nerve fiber layer. The SP‐IR ganglion cell types have large cell bodies (20–22 μm diameter) and dendrites that costratify in S3 among the SP‐IR amacrine cell processes.

The giant fiber system (GFS) mediates the startle response of Drosophila.This response includes an activation of the dorsal longitudinal wing‐depressor muscles (DLMs). However, the morphology of the motor neurons innervating these muscles has not been well studied. Even the location of the somata of these motor neurons has been a source of controversy. This paper identifies the somata and provides a morphological description of these motoneurons.

In this paper, we have mapped the cellular localization of various transmitters onto the central neurons which are involved in male copulation behavior in Lymnaea stagnalis,by combining retrograde tracing with immunocytochemistry and in situ hybridization. Evidence is provided that neurons which were backfilled from the penis nerve, the sole nerve to innervate the male copulatory organ, synthesize a multitude of neuropeptides (APGWamide, Lymnaeaneuropeptide tyrosin [LNPY], conopressin, pedal peptide, SEEPLY, DEILSR, myomodulin, and Lymnaeainhibitory peptide [LIP]) as well as the classical neurotransmitter, serotonin. In the anterior lobe, the backfilled neurons mainly contain the tetrapeptide APGWamide and conopressin, and not LNPY or pedal peptide. The results suggest a central role in the regulation of copulation activity for the anterior lobe neurons that produce APGWamide and conopressin. Immunostainings of backfilled nervous systems revealed immunopositive axons originating from these neurons to form varicosities on the cell somata of neurons in the other clusters contributing to the innervation of the male sexual system. Neurons from the right parietal ganglion projecting into the penis nerve were electrophysiologically and morphologically identified by simultaneously recording from the cell body intracellularly and the penis nerve extracellularly and subsequently filling them with an anterograde tracer and subjecting them to immunocytochemistry. This method has provided links between morphology, physiology, and the transmitter contents of these neurons. J. Comp. Neurol. 395:440–449, 1998. © 1998 Wiley‐Liss, Inc.

We present a description of the last half of embryonic development in the European medicinal leech, Hirudo medicinalis, based entirely on externally visible morphological features, and establish reliably observable stages during that development. Embryogenesis, from the time fertilized eggs are deposited in an eggcase (called a cocoon) to the emergence of juveniles from the cocoon, takes approximately 4 weeks at room temperature. The stages described in this paper extend from the completion of segmentation to the appearance of the final bands of pigmentation. Developmental stages are expressed as percentages of total embryonic developmental time. This staging table was constructed for embryos kept at 20°C. In addition, the development of animals kept at 17°C or at 24°C was compared with those held at 20°C. Development proceeds more quickly at higher temperatures. Because development in embryos held at higher or lower temperatures was linearly related to the stages determined for embryos held at 20°C, the rate of development at any intermediate temperature can be predicted from the staging table at 20°C by simple multiplication. J. Comp. Neurol. 402:155–167, 1998. © 1998 Wiley‐Liss, Inc.

In a study on descending pathways from the nucleus retroambiguus (NRA) to hindlimb motoneurons (see accompanying paper), it appeared impossible, using data from the literature, to precisely determine which muscles were innervated by the motoneurons receiving the NRA fibers. This lack of data made it necessary to produce a detailed map of the lumbosacral motoneuronal cell groups in the cat. Therefore, 50 different muscles or muscle compartments of hindlimb, pelvic floor and lower back were injected with horseradish peroxidase (HRP) in 135 cases. The respective muscles were divided into ten groups: I, sartorius and iliopsoas; II, quadriceps; III, adductors; IV, hamstrings; V, gluteal and other proximal muscles of the hip; VI, posterior compartment of the distal hindlimb; VII, anterior compartment of the distal hindlimb; VIII, long flexors and intrinsic muscles of the foot; IX, pelvic floor muscles; and X, extensors of the lower back and tail. The L4‐S2 segments were cut and incubated, and labeled motoneurons were counted and plotted. A new method was developed that made it possible, despite variations in size and segmental organization between the different cases, to compare the results of different cases. The results show that the spatial interrelationship between the hindlimb and pelvic floor lumbosacral motoneuronal cell groups remains constant. This finding enabled the authors to compose an accurate overall map of the location of lumbosacral motoneuronal cell groups. The general distribution of the motoneuronal cell groups is also discussed in respect to their dorsoventral, mediolateral, and rostrocaudal position within the lumbosacral ventral horn. J. Comp. Neurol. 382:46‐76, 1997. © 1997 Wiley‐Liss Inc.

Little is known about the neural substrates controlling circadian rhythms in day‐active compared to night‐active mammals primarily because of the lack of a suitable diurnal rodent with which to address the issue. The murid rodent, Arvicanthis niloticus, was recently shown to exhibit a predominantly diurnal pattern of activity and body temperature, and may be suitable for research on the neural mechanisms underlying circadian rhythms. This paper describes, in A. niloticus, the anatomy of two neural structures that play important roles in the control of circadian rhythms, the suprachiasmatic nucleus (SCN) and the intergeniculate leaflet (IGL). Immunohistochemical techniques were used to examine the distribution of neuroactive peptides in the SCN and IGL, and retinal projections to these structures were traced with anterograde transport of the beta subunit of cholera toxin. In A. niloticus, distinct subdivisions of the SCN contained cell bodies with immunoreactive (IR) vasopressin, vasoactive intestinal polypeptide, gastrin‐releasing peptide, and corticotropin‐releasing factor. The SCN did not contain cell bodies with met‐enkephalin‐IR and substance P‐IR, but did contain fibers with substance P‐IR and neuropeptide Y‐IR. Retinal fibers were present throughout the SCN, but were most densely concentrated along its ventral edge, particularly in the contralateral SCN. Retinal fibers also extended to a variety of hypothalamic regions outside the SCN, including the supraoptic nucleus and the subparaventricular region. The IGL contained cells with neuropeptide Y‐IR and enkephalin‐IR cells. Retinal fibers projected to both the ipsilateral and contralateral IGL. The anatomy of the SCN and IGL were compared and contrasted with that previously described for other nocturnal and diurnal species. J. Comp. Neurol. 403:190–208, 1999. © 1999 Wiley‐Liss, Inc.

This paper describes the regional and cellular distribution of serotonin 5‐hydroxytryptamine2a(5‐HT2a) receptor mRNA in (sub)regions of the rat striatum by using in situ hybridization. Our results indicate that 5‐HT2amRNA is distributed heterogeneously in this brain region. Regional densitometry of autoradiograms from striatal sections hybridized with isotope‐labeled cRNA probes showed that mRNA levels were highest in the olfactory tubercle, lower in the nucleus accumbens, and lowest in the caudate‐putamen. In the nucleus accumbens, the average mRNA levels in the shell were higher than those in the core. These data suggest a particular relevance for the 5‐HT2areceptor for olfactory tubercle‐ and shell‐related functions. Therefore, in the nucleus accumbens and the olfactory tubercle, the cellular localization of 5‐HT2amRNA was investigated by determining the colocalization of 5‐HT2amRNA with enkephalin mRNA or dynorphin mRNA. 5‐HT2amRNA was found in enkephalinergic as well as dynorphinergic neurons. Thus, there does not seem to be a differential distribution of this receptor in the output routes of the ventral striatum. In all of the subregions investigated (core, medial shell, and lateral shell of the nucleus accumbens and the olfactory tubercle), only subpopulations of the total enkephalinergic and dynorphinergic populations were found to contain 5‐HT2amRNA. For enkephalin, the percentage colocalization was higher in the lateral shell (61%) compared with the other subregions (38–45%). For dynorphin, the percentage colocalization was higher in the olfactory tubercle (68%) than in the other subregions (34–43%). The differences in (sub)regional mRNA levels and in colocalization with opioids suggest a considerable regional differentiation in the effects of 5‐HT2a‐mediated neurotransmission in the striatum. J. Comp. Neurol. 389:1–11, 1997. © 1997 Wiley‐Liss, Inc.

This paper is a light microscopical study describing the detailed morphology and quantitative distribution of local circuit neurones in areas 25, 32, and 24b of the medial prefrontal cortex (mPFC) in the rat. Cortical interneurones were identified immunocytochemically by their expression of calretinin (CR), parvalbumin (PV), and calbindin D‐28k (CB) immunoreactivity. Neurones immunoreactive for γ‐aminobutyric acid (GABA) were also investigated, as were interneurones containing reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity. Several distinct classes of CR+, PV+, and CB+ neurones were identified; the most frequent were: bipolar/bitufted CR+ cells in upper layer 3; multipolar PV+ neurones in layers 3 and 5; and bitufted/multipolar CB+ neurones in lower layer 3. CB+ neurones resembling Martinotti and neurogliaform cells were also present in layers 5/6. The morphologies and depth distributions of each cell type were consistent across the three areas of mPFC studied.

Acetylcholine (ACh) is synthesized in nerve terminals from choline and acetyl coenzyme A by the cytoplasmic enzyme choline acetyltransferase (ChAT). The neurotransmitter is thereafter transported into synaptic vesicles, where it is stored until release. cDNA clones encoding a vesicular ACh transporter (VAChT) were recently isolated. In this paper, we report on the generation of highly specific goat polyclonal antisera to the rat VAChT protein by using a synthetic carboxy‐terminal 20‐amino‐acid peptide sequence as an immunogen. Characterization of the antisera revealed recognition of VAChT, but not vesicular monoamine transporter (VMAT) protein, in transfected CV‐1 cells. VAChT immunoreactivity was also detected in cells that endogenously express the protein, such as in PC12 cells and in primary cultures of spinal motoneurons. Absorption controls showed that the VAChT antisera could be completely blocked at the 10 5M concentration by cognate peptide used for immunization. The antisera cross‐reacted with the VAChT protein in rat and mouse but not in guinea pig, rabbit, or cat. Immunohistochemistry and confocal laser microscopy, using the goat VAChT antisera, showed strong immunoreactivity in discrete fibers and neuronal cell bodies of the central and peripheral nervous systems. Within cell bodies and axonal nerve terminals, as well as in dendrites, the staining appeared granular, presumably representing labeling of synaptic vesicles containing ACh. In the rat central nervous system, VAChT‐positive cell bodies were demonstrated in the cerebral cortex, striatum, septum, nucleus basalis, medial habenula, mesopontine complex, cranial, and autonomic and spinal motor nuclei and in the intermediomedial region near the central canal. High densities of VAChT‐immunoreactive axonal fibers were encountered in areas such as the olfactory bulb, cerebral cortex, striatum, basal forebrain, amygdala, thalamus, hypothalamus including median eminence, hippocampal formation, superior colliculus, interpeduncular nucleus, and pedunculopontine and laterodorsal tegmental nuclei. In cranial and spinal motor nuclei, particularly large varicosities were seen in close proximity to the motoneuron cell somata and their proximal dendrites. In the peripheral nervous system, VAChT immunoreactivity was also detected in motor endplates of skeletal muscle as well as in fibers of sympathetic and parasympathetic abdominal ganglia, heart atrium, respiratory tract, gastrointestinal tract, pancreas, adrenal medulla, male genitourinary tract, and salivary and lacrimal glands. Direct double labeling revealed colocalization of VAChT and ChAT immunoreactivity in neurons. The results show that VAChT antisera represent novel and unique tools for the study of cholinergic neurons in the central and peripheral nervous systems. J. Comp. Neurol. 378:454–467, 1997. © 1997 Wiley‐Liss, Inc.