Simultaneous video analysis of the kinematics of opercular movement and electromyographic activity during agonistic display in Siamese fighting fish.

Neuroethology seeks to uncover the neural mechanisms underlying natural behaviour. One of the major challenges in this field is the need to correlate directly neural activity and behavioural output. In most cases, recording of neural activity in freely moving animals is extremely difficult. However, electromyographic recording can often be used in lieu of neural recording to gain an understanding of the motor output program underlying a well-defined behaviour. Electromyographic recording is less invasive than most other recording methods, and does not impede the performance of most natural tasks. Using the opercular display of the Siamese fighting fish as a model, we developed a protocol for correlating directly electromyographic activity and kinematics of opercular movement: electromyographic activity was recorded in the audio channel of a video cassette recorder while video taping the display behaviour. By combining computer-assisted, quantitative video analysis and spike analysis, the kinematics of opercular movement are linked to the motor output program. Since the muscle that mediates opercular abduction in this fish, the dilator operculi, is a relatively small muscle with several subdivisions, we also describe methods for recording from small muscles and marking the precise recording site with electrolytic corrosion. The protocol described here is applicable to studies of a variety of natural behaviour that can be performed in a relatively confined space. It is also useful for analyzing complex or rapidly changing behaviour in which a precise correlation between kinematics and electromyography is required.

Catecholaminergic systems in the zebrafish. III. Organization and projection pattern of medullary dopaminergic and noradrenergic neurons.

To characterize the catecholaminergic systems in the zebrafish medulla, immunocytological studies were performed by using antibodies directed against tyrosine hydroxylase and dopamine-beta-hydroxylase. Catecholaminergic neurons could be categorized into three populations based on location, dendritic morphology, axonal projection pattern, and targets: an interfascicular group, a vagal area group, and an area postrema group. All groups contained both dopaminergic and noradrenergic neurons. Interfascicular neurons formed a loose longitudinal column of approximately 16-20 multipolar neurons on each side of the medulla, whose rostrocaudal extension coincided roughly with the vagal lobe. These neurons were relatively large and had dendrites that arborized throughout the reticular formation and in the vagal lobe. They also contributed axonal processes to the longitudinal catecholamine bundle. Neurons associated with structures in the vagal area were mostly dopaminergic. Some cells had a short, thin apical process that arborized into a dense plexus near the ventricular surface, and all cells had a basal dendrite that divided into two main branches: one extended caudally to terminate in the commissural nucleus of Cajal and among the postobecular catecholaminergic cell group; the other extended laterally and joined the longitudinal catecholamine bundle. The caudal extent of this cell group reached the medullospinal junction. The area postrema cell group consisted of densely packed, bipolar neurons. One process of these neurons contacted the ventricular surface in the area postrema, and one terminated in the commissural nucleus of Cajal. Collaterals from the latter innervated the superficial laminae of the vagal lobe and joined the longitudinal catecholamine bundle. The longitudinal catecholamine bundle ascended through the medulla ventral to the secondary gustatory tract. Whether some fibers extended more rostrally is not known. The majority of the terminal fields of medullary catecholaminergic neurons appeared to be restricted to the medulla and were strongly associated with sensory systems. With the exception of some cells in the vagal area, catecholamine-containing neurons in the zebrafish medulla were not obviously homologous to those in the mammalian brainstem.

On the agonistic display of the Siamese fighting fish. I. The frontal display apparatus.

The agonistic behavior of the Siamese fighting fish has long been a popular subject for ethologists. While this behavior is well documented, its physiological basis is still poorly understood. One of the most important components of this behavior is the frontal display, in which a fish faces its opponent directly with tonically extended gill covers. As a first step towards establishing this display as a model for behavioral physiology, the musculoskeletal structure of the frontal display apparatus is examined. The opercular bones and the opercular abductor, the opercular dilator muscle, appear to have undergone adaptive modifications that facilitate the display. The opercular bone rotates around the hyomandibular bone through a ball-and-socket joint. Due partly to a reduction of its articulation with the preopercular bone, the operculum can rotate as much as 90 degrees around this joint. The opercular dilator muscle consists of three parts: a deep belly--DO alpha, and two superficial bellies--DO beta and DO gamma. The three portions have different origins, but all three insert on the lateral surface of the opercular bone just dorsal to the spheroidal joint. In comparison, the opercular dilator muscle of the sunfish and the goldfish lacks the superficial bellies. Innervation of this muscle is derived from the maxillary division of the trigeminal nerve, all three portions are innervated by axons from the same fascicle, suggesting that they are embryologically related. The superficial bellies consist of uniform, large muscle fibers. The deep belly consists of an external group of large fibers around a central tendon, and an internal group of small fibers. Enzyme histochemistry shows that the external group of fibers consists mostly of fast-twitch fibers, whereas the internal group consists of slow, oxidative fibers. Direct stimulation demonstrates that all bellies can mediate opercular extension. The architectural and biochemical differences among the three portions suggest that they are functionally not equivalent. The fast-fatiguing muscles may mediate the initiation, while the large fibers of the superficial bellies and the small oxidative fibers of the deep belly may be involved in the maintenance of the display.

On the agonistic display of the Siamese fighting fish. II. The distribution, number and morphology of opercular display motoneurons.

The agonistic display of the Siamese fighting fish is characterized by tonic extension of the gill opercula, which is mediated by a single abductor, the opercular dilator muscle. This muscle consists of three bellies, which have different origins and biochemical properties. As part of an effort to define in detail the neural substrate for agonistic display, the distribution, number, and morphology of opercular dilator motoneurons in the trigeminal motor nucleus were examined by retrograde horseradish peroxidase labelling and Golgi-impregnation. The trigeminal motor nucleus consists of three subnuclei: rostral, caudomedial and caudolateral. Neuron numbers among these three subnuclei are relatively constant among animals, and between males and females. Innervation of the opercular dilator muscle originates predominantly from the caudolateral subnucleus; a smaller contingent of motoneurons originates from the caudomedial subnucleus. Labelling of the caudolateral subnucleus is usually intense, whereas caudomedial neurons are weakly labelled. These observations suggest that neurons in the caudomedial subnucleus provide either sparse and widespread innervation, or innervate only a small, circumscribed area of the opercular dilator muscle complex, whereas caudolateral neurons probably provide extensive and dense innervations. The dendritic morphology of trigeminal motoneurons is independent of their targets and of the subnuclei in which they are located. All neurons observed are similar in shape. Primary dendrites of neurons in the rostral and caudomedial subnuclei are long, whereas those in the caudolateral subnucleus are short or absent. Regardless of the position of the somata, dendrites arborize only in the lateral half of the reticular formation in the vicinity of the descending tectobulbal tract, the descending trigeminal tract, and the ascending secondary gustatory tract. The morphological differences suggest that inputs to terminal dendrites have stronger effects on neurons in the caudolateral than those in the other two subnuclei.

Catecholaminergic systems in the zebrafish. I. Number, morphology, and histochemical characteristics of neurons in the locus coeruleus.

The locus coeruleus is a noradrenergic nucleus located in the isthmal tegmentum. In mammals, it contains several thousand neurons that have diverse projection patterns and contain various neuropeptides. In fishes, this nucleus contains few neurons. This study attempts to define and quantify morphological types of locus coeruleus neurons, and search for neurochemical subpopulations in the zebrafish. In this fish, the locus coeruleus contains between 3 and 10 neurons, and most nuclei contain between 5 and 8 cells. Nuclei in more inbred lines of fish have a narrower range of neurons. The difference in neuron number between the two sides of the same brain is small, but only 24% of the brains have identical numbers on both sides. These observations suggest that there is a two-step control of neuron number: the genetic constitution of the fish determines the approximate number of cells, while epigenetic factors determine the final number. Based on dendritic orientation, three types of cells are identified: (1) V type, neurons with only ventrally projecting dendrites; (2) L type, neurons with only laterally projecting dendrites; and (3) VL type, neurons with both ventrally and laterally projecting dendrites. Over 65% of the neurons are of the V type; some nuclei have V type cells only. There is a correlation between the total number of neurons and the ratio of each cell type. In nuclei with five cells or fewer, over 80% of the neurons are V type; higher percentages of the other two types are seen in nuclei with 6 or more neurons. The dendritic morphology and orientation suggest that various types of neurons may receive different inputs. Cholinesterases are not detectable in locus coeruleus neurons. Immunocytochemical staining for a number of neuropeptides also fails to demonstrate detectable levels. Neuropeptide Y is present in some cells abutting the locus coeruleus, but these are probably not catecholamine-containing neurons. Some neurons contain choline-acetyltransferase. These observations suggest that locus coeruleus neurons of the zebrafish may be morphologically and neurochemically heterogeneous.

Catecholaminergic systems in the zebrafish. II. Projection pathways and pattern of termination of the locus coeruleus.

The locus coeruleus is a widely projecting isthmal noradrenergic nucleus. In the zebrafish, it consists of between three and ten neurons, most of which have multiple, bilaterally projecting axons. Immunohodological studies show that the locus coeruleus provides most, if not all, of the noradrenergic innervation of the brain rostral to the isthmus. The pathways and targets in the zebrafish are similar to ascending coeruleal projections of other vertebrates. Axons ascend through two main pathways: the longitudinal catecholamine bundle and the periventricular catecholamine pathway. The former is a dense meshwork of varicosity-bearing axons which ascends along the lateral longitudinal fasciculus into the mesencephalon. In the posterior tuberal area, this bundle dives ventrally and assumes a lateral position. In the diencephalon, it takes up a position ventral to the medial forebrain bundle, and follows this bundle into the telencephalon, where it joins the medial olfactory tract to enter the olfactory bulb. The periventricular catecholamine pathway is a diffuse pathway consisting of thick, smooth axons. It is associated with the medial longitudinal fasciculus. Rostral to the nucleus of the medial longitudinal fasciculus, this pathway joins the longitudinal catecholamine bundle around the medial forebrain bundle. The periventricular pathway gives rise to coarse terminal arbors with large but sparse varicosities, whereas the longitudinal catecholamine bundle gives rise to terminal plexuses with fine and dense fibers and varicosities. Among the more densely innervated regions are the raphé nucleus, the interpeduncular nucleus, the torus semicircularis, parts of the hypothalamus, and the suprachiasmatic and preoptic areas. The torus longitudinalis, optic tectum, cerebellum, habenular complex, the dorsomedial zone of area dorsalis telencephali, and the olfactory bulb are moderately innervated. The nucleus glomerulosus, the torus lateralis and lateral subnuclei of the nucleus diffusus, and the anterior tuberal nucleus are devoid of noradrenergic innervation.

Biotin staining in the giant fiber systems of the lobster.

The avidin-biotin-complex method is a popular immunocytochemical technique. This method labels consistently a group of neurons in the lobster ventral nerve cord in the absence of primary antibodies. The specific staining is due to a relatively high level of endogenous biotin (or biocytin) in these neurons. These biotin-positive neurons are located in the supraesophageal, thoracic, and abdominal ganglia. Intraaxonal injection of Lucifer yellow followed by Texas red-conjugated streptavidin staining reveals that the neurons are members of the medial giant (MG) and lateral giant (LG) systems, which are important in mediating rapid tail flipping during escape maneuvers. In neuronal somata, staining is restricted to the cytoplasm. Within MG axons, staining appears as punctate, subaxolemmal structures. Preincubating nerve cords in biocytin or direct intraaxonal injection of biocytin enhances staining of these punctate organelles. In LG axons, staining is localized to fragments of braided filamentous structures that also appear to be associated with the axolemma. Preincubation of ventral nerve cords in various concentrations of biocytin results in the appearance of additional groups of stained neurons, suggesting that there are subsets of neurons with specific biocytin-uptake or -retention mechanisms. In the crayfish, biotin-positive staining is confined to the MG neurons; the LG neurons are not stained. In the earthworm, no staining is observed in the MG and LG axon escape systems. In the goldfish, no biotin-staining is seen in the Mauthner neurons and their axons. The significance of specific localization of biotin or biocytin to subsets of neurons is unclear. It may reflect the presence of high levels of biocytin moieties on biotin-dependent enzymes. Biotin is an important cofactor in the catalytic functions of several decarboxylases crucial in energy production and lipogenesis. Axons of the giant fiber systems in lobsters and crayfish may have high energy and fatty acid synthesis requirements. Increased levels of biotin accumulation may also be related to other functions of the giant axon systems, such as the formation of electrical synapses among themselves and with phasic motoneurons.

Tanycytes in the sunfish brain: NADPH-diaphorase histochemistry and regional distribution.

NADPH-diaphorase histochemistry has been shown to be a useful method for identifying cells that synthesize and release nitric oxide, which is implicated in the modulation of a variety of neural functions, including synaptic transmission, cerebral blood flow, and excitotoxicity. In the sunfish brain, NADPH-diaphorase histochemistry stains tanycytes specifically and almost exclusively, allowing for a thorough examination of the morphology and distribution of this type of cell. Tanycytes are nonciliated, process-bearing ependymal and extraependymal cells that contact the ventricular surface via apical processes, and the pial surface via basal processes. Ependymal tanycytes are located at the ventricular surface, and project basal processes into the parenchyma of the brain. Extraependymal tanycytes are found away from the ventricular matrix. Some extraependymal tanycytes are small, bipolar, and tend to be associated with bundles of basal processes. Isolated extraependymal tanycytes are larger, darkly stained, and multipolar. Their basal processes terminate in specialized endfeet on blood vessels, neuronal somata, or the pial surface. Specialized types of tanycytes are found in the optic tectum, the epineurial septum between axonal bundles along the midline in the medulla, and in restricted regions on the pial surface in the medulla. The only NADPH-diaphorase-positive neurons are found in the commissural nucleus of area ventralis telencephali. Injection of horseradish peroxidase into the ventricles shows that tanycytes lining the third and fourth ventricles are capable of taking up the tracer and transporting it into their basal processes. Tanycytes are unevenly distributed in the brain. There is a rough rostrocaudal gradient of cell density: tanycytes are sparse in the telencephalon and dense in the isthmus and medulla, although cell density is low in the spinal cord. Not all ventricular linings contain tanycytes: cell density is low in the medial ventricle of the telencephalon and in the infundibular recess, and high along the fourth ventricle. The function of tanycytes in the sunfish is not known. The association of tanycytes with both the ventricles and blood vessels raises the possibility that they play some role in sampling the biochemical constituents of both compartments and communicating the information to neural elements. It is proposed that tanycytes react to the biochemical composition in the ventricle and plasma by increasing or decreasing nitric oxide synthesis and release, which in turn influence neuronal activity or cerebral blood flow.

Localization of NADPH diaphorase activity in monoaminergic neurons of the rat brain.

Nitric oxide has recently been implicated as a neurotransmitter, and may modulate synaptic transmission, cerebral blood flow, and neurotoxicity. NADPH diaphorase histochemistry has been shown to be a reliable marker for nitric oxide synthase, the enzyme that synthesizes nitric oxide, in the nervous system. Because monoaminergic neurons frequently contain co-transmitters, we examined whether these cells also exhibit NADPH diaphorase activity. Frozen sections from postnatal and adult rat brains were stained for NADPH diaphorase activity and either serotonin-like immunoreactivity or tyrosine hydroxylase-like immunoreactivity. Numerous neurons in the mesopontine serotoninergic cell groups (including the caudal linear, dorsal, median, supralemniscal, and pontine raphe nuclei) contained both serotonin-like immunoreactivity and NADPH diaphorase activity. Within the dorsal raphe nucleus, approximately 70% of the serotoninergic neurons in the medial subnuclei displayed NADPH diaphorase activity, while less than 10% of the serotoninergic neurons in the lateral subnuclei were doubly labeled. Retrograde labeling with fluorescent microspheres indicated that many raphe-cortical neurons contained NADPH diaphorase activity. No NADPH diaphorase activity was detected in serotoninergic neurons in the medullary nuclei (including the raphe magnus, raphe pallidum, and raphe obscurus). Only a small proportion of tyrosine hydroxylase-like immunoreactive neurons in the periaqueductal gray, rostral linear nucleus, and rostrodorsal ventral tegmental area contained NADPH diaphorase activity. Tyrosine hydroxylase-like immunoreactive neurons in the substantia nigra, locus coeruleus, hypothalamus, olfactory bulb, and dorsal raphe nucleus did not contain detectable NADPH diaphorase activity. The observation that many mesopontine (but not medullary) serotoninergic neurons contain NADPH diaphorase activity suggests that these neurons may release both serotonin and nitric oxide.

Serotonin-containing neurons in lobsters: the actions of gamma-aminobutyric acid, octopamine, serotonin, and proctolin on activity of a pair of identified neurons in the first abdominal ganglion.

1. Serotonin has been shown to be an important neurohormone that modulates behavioral output in lobsters. This study explores the neurochemical identity of excitatory and inhibitory inputs to a pair of identified serotonin-containing neurons in the first abdominal ganglion (A1) of the lobster that also contain the pentapeptide proctolin. These neurons are spontaneously active, appear to be driven by an endogenous pacemaking mechanism, and have been shown to play a role in circuits that control behaviorally relevant postures. 2. To explore the exogenous control of neuronal activity, a number of putative neuroactive compounds were superfused over the A1 serotonin-containing neurons in isolated ventral nerve cords. Three amines, gamma-aminobutyric acid (GABA), octopamine, and serotonin, were found to be potent inhibitors in a dose-dependent manner. GABA inhibition had a rapid onset and termination and yielded a transient postinhibitory rebound activity immediately after amine washout; activity eventually returned to the preinhibition level. Octopamine inhibition had a less rapid onset and termination, and the steady-state activity after inhibition was higher than preinhibition firing in most cells (22% of which were statistically significantly increased). Serotonin inhibition had a relatively slow onset and termination, and the steady-state activity after inhibition remained low in most cells (19% significantly decreased). 3. Repeated or prolonged exposure to these three amines reduced the efficacy of inhibition. Three types of desensitization have been empirically defined: 1) rapid desensitization, which tended to dampen the initial inhibition; this was particularly strong for GABA inhibition; 2) low-dose desensitization, in which the A1 serotonin containing neurons became less sensitive to inhibition by a particular concentration of amine after prior exposure to a lower concentration, although the lower concentration may not by itself have had inhibitory effects; this was seen with octopamine inhibition; and 3) long-term desensitization, in which the efficacy of inhibition of a particular amine concentration dwindled with repeated applications (even with up to 160 min washout between applications); this was seen with octopamine and serotonin inhibition, although not for every A1 serotonin-containing neuron analyzed. 4. Bath application of these three amines was still capable of inhibiting the A1 serotonin-containing neurons when inhibitory synaptic transmission was blocked by use of low Ca2+/high Mg2+ solutions. 5. The pentapeptide proctolin excited the A1 serotonin-containing neurons: it activated silent neurons and increased the firing rate of spontaneously active neurons. The excitatory effect outlasted the presence of the peptide.(ABSTRACT TRUNCATED AT 400 WORDS)

Serotonin-containing neurons in lobsters: origins and characterization of inhibitory postsynaptic potentials.

1. The serotonin-containing neurons in the A1 ganglion of the lobster have been shown to act as "gain setters" in neuronal circuits that control the adoption of behaviorally relevant postures. These neurons are subject to tonic inhibition, which has been proposed as an important regulator of their activity. This study explores the pharmacological nature and anatomic location of the neurons responsible for inhibition of these A1 cells; the role played by inhibitory inputs in controlling the firing rates of these neurons is also examined. 2. Three classes of inhibitory postsynaptic potentials (IPSPs) are distinguished in the somata of A1 serotonin-containing neurons. The most common (type I) has amplitudes ranging from 0.4 to 1.5 mV; types II (2-5 mV) and III (< 0.4 mV) are less often seen. 3. Type I IPSPs are reversibly blocked by picrotoxin, a gamma-aminobutyric acid antagonist, but not by serotonin or octopamine antagonists known to act at other lobster synapses. 4. Elimination of type I IPSPs by reversible or irreversible blockage of conduction from the A3 ganglion results in a firing rate increase of approximately 50% in A1 serotonin-containing neurons; IPSP recovery results in a firing rate decrease of corresponding magnitude. Connective transections that do not affect IPSPs do not cause a firing rate increase. 5. Lesions studies suggest that type I IPSPs originate in neurons whose somata are located near the midline of the A3 ganglion; a cell impaled in this region showed action potentials that correlated with IPSPs in an A1 serotonin-containing neuron.

Barrelettes--architectonic vibrissal representations in the brainstem trigeminal complex of the mouse. II. Normal post-natal development.

Vibrissal representations in the brainstem trigeminal complex (BTC) of rodents are manifested as architectural sub-units called barrelettes. The development of barrelettes was studied by using Nissl staining, cytochrome oxidase histochemistry, and Golgi-impregnation methods. On the day of birth (PND-1), barrelettes are manifested as longitudinal, histochemical cylinders in sub-nuclei principalis, interpolaris and caudalis of the BTC. One day later (PND-2), fully formed histochemical barrelette formations are seen in the three sub-nuclei. The development of cytoarchitectural barrelettes lags behind histochemical barrelettes by about two days. Between PND-2 and PND-3, longitudinal cytoarchitectonic cylinders begin to appear. By PND-3, BTC neurons segregate into five rows of barrelettes in the coronal plane. Segmentation of rows into individual barrelettes begins on PND-4, and complete cytoarchitectonic barrelette formations are seen by PND-5. Golgi-impregnation shows that on the day of birth, primary afferent terminals and dendritic arbors of second-order trigeminal neurons within the BTC are short and poorly ramified. Over the next five post-natal days, lengthening of these processes as well as elaboration into secondary and tertiary branches take place. Growth of these processes continues for two additional weeks, contributing to the increase in barrelette neuropils (hollows). As the neuropils expand, neuronal somata are pushed toward barrelette sides. Morphometric measurements show that there is a relatively constant rate of growth of barrelettes over the first three post-natal weeks. The growth rate of the barrelette formations is identical to that of BTC as a whole. Thus, at the time of birth, the volume of neural tissue in the brainstem allotted to vibrissae is fixed relative to that allotted to other sensory receptors. Several features of the early development of barrelettes are identified: (1) Chemoarchitectural barrelettes appear before cytoarchitectural barrelettes, suggesting that terminal arbors of primary trigeminal afferents are organized before their target neurons form barrelettes. (2) Early cytoarchitecture is manifested in the form of unsegmented rows, suggesting that rough, row-based topological maps are first formed, which are then fine-tuned into individual sub-units. Recent evidence shows that other vibrissal representations--thalamic barreloids and cortical barrels--also follow these "afferent-before-target" and "row-before-individual units" sequences of development. This gradual, afferent-dependent fine-tuning of topological organization is analogous to similar events during the early development of the visual system, and may be a general feature of developing sensory systems.(ABSTRACT TRUNCATED AT 400 WORDS)

Serotonin-containing neurons in lobsters: their role as gain-setters in postural control mechanisms.

1. The electrophysiological properties of two pairs of identified serotonin-containing neurons in the fifth thoracic (T5) and first abdominal (A1) ganglia of the lobster, Homarus americanus, were studied with the use of intracellular recording methods. Intracellular dye injection combined with immunocytochemistry verified the neurochemical status of the recorded neurons. 2. The serotonin-containing neurons usually are spontaneously active at 0.5-1.0 Hz and produce large, overshooting action potentials with a prominent after-hyperpolarization. The action potentials appear to be generated by a pacemaking mechanism endogenous to the cells. Extracellular recordings from thoracic connectives and from second thoracic roots show that action potentials from the cells in A1 and T5 are propagated rostrally along their axons and invade axon collaterals that innervate neurohemal organs in the second thoracic roots and the pericardial organs. These observations suggest that these serotonin-containing cells may function in part as important neurosecretory cells in the lobster. 3. Members of the pairs of serotonin-containing cells are not synaptically connected. They receive prominent inhibitory inputs in the form of inhibitory postsynaptic potentials (IPSPs), which exhibit discrete size classes and probably arise from several sources. Most IPSPs are temporally synchronized among the two pairs of serotonin-containing cells. 4. The serotonin-containing cells respond to stimulation of postural command fibers, with flexion command fibers exciting and extension command fibers inhibiting the cells, suggesting that these cells are a part of the postural flexion circuitry. 5. Intracellular activation or inhibition of the serotonin-containing cells has no effect on the spontaneous readout of postural motor programs recorded from motor nerve roots. Coactivation of the serotonin-containing cells and command fibers, or inhibition of the serotonin-containing cells while activating command fibers, however, shows that the cells act as "gain-setters," modulating the interaction between command inputs and motoneuron outputs. 6. About 24% of the motor neuron units analyzed are influenced by the serotonin-containing cells. There is a bias toward facilitation of the readout of flexion motor programs, particularly with stimulation of strong and moderate flexion command fibers. 7. The serotonin-containing cells in T5 and A1 ganglia are hypothesized to serve two functions, one tonic and the other phasic, in modulating behavioral output in lobsters. Tonic firing of the cells should result in a sustained release of serotonin from central and peripheral sets of nerve terminals, which, in turn, could influence peripheral and central targets of the amine.(ABSTRACT TRUNCATED AT 400 WORDS)

The barrelettes--architectonic vibrissal representations in the brainstem trigeminal complex of the mouse. I. Normal structural organization.

The organization of the brainstem trigeminal complex (BTC) of the mouse is described, with emphasis on the normal organization of the vibrissal representations. Thionin staining for Nissal substance was employed to reveal the cytoarchitecture. Cytochrome oxidase histochemistry was used to reveal the chemoarchitecture. Golgi impregnation methods, in combination with thionin staining, were used to examine the neuronal dendritic morphology within a defined cytoarchitectonic context. An in vitro horseradish peroxidase labelling method was used to study the distribution and morphology of primary trigeminal afferent terminals within the BTC. The BTC consists of four distinct subnuclei: principalis (nVp), oralis (nVo), interpolaris (nVi), and caudalis (nVc). The present study shows that these sub-nuclei can be distinguished from each other on the basis of several anatomical criteria, including the distribution and density of neuronal size classes, histochemical staining intensity, morphology and orientation of neuronal dendrites, and size and texture of primary afferent terminal arbors. Anatomical manifestation of vibrissal representations within the BTC can be described in nVp, nVi, and nVc, but not in nVo. Within the three subnuclei where they are found, anatomical vibrissal representations are composed to architectural subunits that form an overall pattern homeomorphic to the pattern of vibrissae on the face of the animal. Each sub-unit forms a cylindrical tube running in a rostrocaudal orientation within the BTC. These sub-units will be called barrelettes. Cytologically, each barrelette consists of cell-dense "sides," surrounding a practically cell-free "hollow." Individual sub-units are separated by narrow, cell-free "septa." Histochemically, each subunit is manifested as a discrete patch of positive-staining reaction products. Differential interference contrast optics shows that these patches correspond precisely to the barrelette hollows. Evidence is presented to show that the barrelettes are the functional units for the processing of vibrissal sensory information. Terminal arborizations of individual primary afferents seem to be confined to the hollow of single barrelettes. The majority of neurons that form the sides of a barrelette have bitufted dendritic arbors, which project predominantly into the barrelette hollow, although a minority of neurons, particularly in nVi and nVc, also extend part of their dendritic arbors into adjacent barrelette hollows. The barrelette hollows are thus the principal neuropil region in which primary afferents and their target neurons interact. Contacts are made mainly between en passant varicosities and terminal boutons on primary afferent collaterals and dendritic spines and shafts of second order neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Optic fibers follow aberrant pathways from rotated eyes in Xenopus laevis.

The rotated eye paradigm has been a major experimental test of the neuronal specificity model for the development of ordered retinotectal connections in amphibians. In most studies, however, no optic fiber pathways were traced from rotated eyes and correlated with visuotectal projections. As an initial approach to this question, optic fibers from eyes rotated at different embryonic stages were traced with 3H-proline autoradiography. Three experimental series were prepared: in situ eye rotations, isochronic transplants of eyes rotated between embryos at the same stage, and heterochronic transplants of eyes rotated between embryos at different stages. Single or multiple optic fiber pathways developing from rotated eyes are identified by their sites of entry and trajectory in the brain. These include a normal chiasmatic (CH) pathway, and three aberrant pathways, identified as trigeminal (TR), diencephalic (DI), and oculomotor (OC). The latter three enter the brain ipsilaterally, some crossing contralaterally via commissural pathways. Depending on stage and type of operation, TR pathways develop in 50-100% of the animals, while CH pathways are more common after rotation at stage 21/22. The surgical procedure affects the initial trajectory of fibers from the retina, perturbs guidance cues in the surrounding orbit, and determines the patterns of optic pathways that develop. In most cases, optic fibers follow motor (oculomotor) or sensory (trigeminal) nerves, usually the first fibers encountered near the orbit by axonal pioneers exiting the retina. Evidently, optic fibers exhibit no pathway selectivity; any axon serves as a guidance cue. Tecta are innervated in about 50% of the cases, usually by fibers following abnormal trajectories from CH and OC pathways. The results suggest that the development of ordered visuotectal projections from rotated eyes is a complex process that may be independent of the trajectory of fiber arrival. Unless pathways and visuotectal maps are directly compared in each animal, however, the question remains open because we still do not know which anomalous pathways, if any, correlate with ordered projections.

In vitro labeling of axonal projections in the mammalian central nervous system.

Horseradish peroxidase crystals or HRP-NP40 detergent chips were directly applied to brain slices from mice to label primary afferent fibers and their terminal arbors in the brainstem trigeminal complex, and neurons of the ventrobasal complex of the thalamus, their axons in the internal capsule, and their terminals in the primary somatosensory cortex. Anterograde and retrograde labeling of fibers, as well as retrograde labeling of somata, were observed. In vitro labeling of selected non-trigeminal structures and fiber pathways was also demonstrated. Experimental variables have been dealt with in some detail, as have specific advantages and disadvantages of the technique. This in vitro HRP labeling method for neuronal fiber systems is a useful adjunct to currently employed in vivo labeling techniques in the mammalian central nervous system.

Cytoarchitectonic correlates of the vibrissae in the medullary trigeminal complex of the mouse.

Cytoarchitectonic patterns in the medullary trigeminal complex of the mouse corresponding to mystacial vibrissae are described. These patterns are found within trigeminal sub-nuclei principalis, interpolaris and caudalis. The patterns are due to differential cell packing and are homeomorphic to the arrangement of the mystacial vibrissae on the face. The cytoarchitecture is similar, but complementary, to patterns of trigeminal afferents previously described using histochemical staining methods. Neonatal cautery of groups of vibrissae produces appropriate, specific and localized cytoarchitectural changes within all 3 trigeminal sub-nuclei.

Choline acetyltransferase and cholinesterases in the developing Xenopus retina.

To understand the developmental regulation of acetylcholine (ACh) synthesis in the Xenopus retina, the properties of choline acetyltransferase (CAT) and cholinesterase (ChE), as well as histochemical localization of ChE in the retina, were studied during development. CAT activity first became detectable in the developing eyecup at stages 35/36. This was followed by a rapid, 50-fold rise in specific activity between stages 35/36 and 44. Since this rapid rise coincided with an almost identical increase in total ACh synthesis in whole retinae found in previous studies, it is suggested that this increase was sufficient to account for the rapid increase in total ACh synthesis. Moreover, it also correlated with increased rates of synaptogenesis in both the inner and the outer plexiform layers. Total ChE was resolved into specific and nonspecific ChE by the use of tetraisopropylpyrophosphoramide. Total ChE activities first became detectable at stages 35/36. Specific ChE [acetylcholinesterase (AChE)] increased from 50% at stage 39 to 95% of total ChE activities at stage 66. Again, the most rapid increase in both total ChE and AChE activities occurred between stages 35/36 and 44. Histochemical studies showed that AChE was localized predominantly in the two plexiform layers, with the inner plexiform layer more heavily stained at all stages. Moreover, a stratified staining pattern, clearly discerned in the inner plexiform layer, also correlated with synaptogenesis during this early period of retinal development.