J. Meek

Distribution and quantification of corticotropin-releasing hormone (CRH) in the brain of the teleost fish Oreochromis mossambicus (tilapia)

The recent characterization of the corticotropin‐releasing hormone (CRH) prehormone of the fish tilapia (Oreochromis mossambicus) showed that more variation exists between vertebrate CRH amino acid sequences than recognized before. The present study investigates whether the deviating composition of tilapia CRH coincides with an atypical distribution of CRH in the brain. For this purpose we applied immunohistochemistry, as well as radioimmunoassay (RIA) quantification in brain slices. The results are plotted in a new atlas and reconstruction of the tilapia brain. The largest population of CRH‐immunoreactive (ir) neurons is present in the lateral part of the ventral telencephalon (Vl). Approximately tenfold less CRH‐ir neurons are observed in the preoptic and tuberal region. The CRH‐ir neurons observed in the preoptic region are parvocellular and do not, or hardly, display arginine‐vasotocin (AVT) immunoreactivity. CRH‐ir neurons are also present in the glomerular layer of the olfactory bulb, in the periventricular layer of the optic tectum, and caudal to the glomerular nucleus. A very dense plexus of CRH‐ir terminals is located in the most rostral part of the dorsal telencephalon. This region has not been described in other teleosts and is in the present study subdivided into the anterior part of the dorsal telencephalon (Da) and the anterior part of the laterodorsal telencephalon (Dla). High densities of CRH‐ir terminals were observed in and around Vl, in the tuberal region, around the rostral part of the lateral recess, and in the caudal part of the vagal lobe. In the pituitary, CRH‐ir terminals are concentrated in the neuro‐intermediate lobe. Overall, the immunohistochemical and quantitative data correlated well, as the RIA CRH profile in serial 160‐μm slices revealed four peaks, which corresponded with major ir‐cell groups and terminal fields. Our results strongly suggest that the CRH‐ir cells of Vl project to the rostro‐dorsal telencephalon. Consequently, they may not be primarily involved in regulation of pituitary cell types but may subserve other functions. The presence of a CRH‐containing Vl‐Da/Dla projection seems to be restricted to the most modern group of teleosts, i.e., the Acanthopterygians. Further anatomic indications for non‐pituitary‐related functions of CRH are found in the vagal lobe and the optic tectum of tilapia. Although the low CRH content of the preoptic region reported here for tilapia may be typical for unstressed fish, the fact remains that remarkably few CRH‐ir neurons are involved in regulating the pituitary. Overall, the CRH distribution in the brain of tilapia is more widespread than previously reported for other teleosts. J. Comp. Neurol. 453:247–268, 2002.

Afferent and efferent connections of cerebellar lobe C3 of the mormyrid fish Gnathonemus petersi: An HRP study

The present paper is devoted to the extrinsic connections of lobe C3 of the highly differentiated corpus cerebelli of the electric fish Gnathonemus petersi. For this purpose, HRP injections or gels were placed in distinct parts of lobe C3 or its peduncle, in the pretectal region, and in the eye. Moreover, the presence of serotonin and tyrosine‐hydroxylase was studied with immunohistochemical methods.

Interneurons of the ganglionic layer in the mormyrid electrosensory lateral line lobe: Morphology, immunohistochemistry, and synaptology

This is the second paper in a series that describes 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 of the fish are detected on the basis of changes in the reafferent electrosensory signals that are generated by the animals own electric organ discharge. The present paper describes interneurons in the superficial (molecular, ganglionic, and plexiform) layers of the ELL cortex that were analyzed in the light and electron microscopes after Golgi impregnation, intracellular labeling, neuroanatomical tracing, and γ‐aminobutyric acid (GABA) immunohistochemistry.

Projection neurons of the mormyrid electrosensory lateral line lobe: Morphology immunohistochemistry, and synaptology

This paper describes the morphological, immunohistochemical, and synaptic properties of projection neurons in the highly laminated medial and dorsolateral zones of the mormyrid electrosensory lateral line lobe (ELL). These structures are involved in active electrolocation, i.e., the detection and localization of objects in the nearby environment of the fish on the basis of changes in the reafferent electrosensory signal generated by the animals own electric organ discharge. Electrosensory, corollary electromotor command‐associated signals (corollary discharges), and a variety of other inputs are integrated within the ELL microcircuit. The organization of ELL projection neurons is analyzed at the light and electron microscopic levels based on Golgi impregnations, intracellular labeling, neuroanatomical tracer techniques, and γ‐aminobutyric acid (GABA), γ‐aminobutyric acid decarboxylase (GAD), and glutamate immunohistochemistry.

Immunocytochemical identification of cell types in the mormyrid electrosensory lobe

The electrosensory lobes (ELLs) of mormyrid and gymnotid fish are useful sites for studying plasticity and descending control of sensory processing. This study used immunocytochemistry to examine the functional circuitry of the mormyrid ELL. We used antibodies against the following proteins and amino acids: the neurotransmitters glutamate and γ‐aminobutyric acid (GABA); the GABA‐synthesizing enzyme glutamic acid decarboxylase (GAD); GABA transporter 1; the anchoring protein for GABA and glycine receptors, gephyrin; the calcium binding proteins calbindin and calretinin; the NR1 subunit of the N‐methyl‐D‐aspartate glutamate receptor; the metabotropic glutamate receptors mGluR1α, mGluR2/3, and mGluR5; and the intracellular signaling molecules calcineurin, calcium calmodulin kinase IIα (CAMKIIα) and the receptor for inositol triphosphate (IP3R1α). Selective staining allowed for identification of new cell types including a deep granular layer cell that relays sensory information from primary afferent fibers to higher order cells of ELLS. Selective staining also allowed for estimates of relative numbers of different cell types. Dendritic staining of Purkinje‐like medium ganglion cells with antibodies against metabotropic glutamate receptors and calcineurin suggests hypotheses concerning mechanisms of the previously demonstrated synaptic plasticity in these cells. Finally, several cell types including the above‐mentioned granular cells, thick‐smooth dendrite cells, and large multipolar cells of the intermediate layer were present in the two zones of ELL that receive input from mormyromast electroreceptors but were absent in the zone of ELL that receives input from ampullary electroreceptors, indicating markedly different processing for these two types of input. J. Comp. Neurol. 483:124–142, 2005.

Tyrosine hydroxylase-immunoreactive cell groups in the brain of the teleost fishGnathonemus petersii

Different antibodies against tyrosine hydroxylase (TH) were used to obtain detailed information about the distribution, morphology and chemical differentiation of catecholaminergic neurons in the highly differentiated brain of the electric mormyrid fish Gnathonemus petersii. The results show that the distribution of catecholaminergic neurons is much more widespread than was previously thought on the basis of dopamine and noradrenaline immunohistochemistry. Tyrosine hydroxylase-immunoreactive neurons were observed not only in clearly dopaminergic regions (the suprachiasmatic nucleus, the magnocellular hypothalamic nucleus and the area postrema) and noradrenergic cell groups (the locus coeruleus and inferior reticular cell group), but also in regions that do not, or only fragmentarily, display dopamine or noradrenaline immunoreactivity, including the ventral and intermediate telencephalon, the anterior and posterior preoptic cell group, the ventromedial thalamus, the pretectal region and the nucleus of the solitary tract, suggesting that they either represent depleted dopaminergic cell groups or L-dihydroxy phenylalanine-producing nuclei. Most TH-immunoreactive neurons are rather small (< 10 microns) and have only a few slender processes, but neurons in the magnocellular hypothalamic nucleus and the inferior reticular formation are multipolar and larger (10-20 microns), while those of the locus coeruleus are even more than 20 microns in diameter. The hypothalamic paraventricular organ, which is strongly dopamine and noradrenaline immunoreactive, displays minimal TH immunoreactivity, suggesting that its cerebrospinal fluid-contacting neurons do not synthesize catecholamines, but acquire them from external sources. Comparison with other teleosts shows that the catecholaminergic system in the brain of Gnathonemus is similarly organized as in Carassius, Gasterosteus, Anguilla and Aperonotus, with some variations that may partly be due to technical reasons, and partly reflect true species differences. However, TH-immunoreactive neurons in the midbrain tegmentum were not observed, confirming previous conclusions that a major difference between teleosts and mammals concerns the absence of dopaminergic midbrain groups and correlated mesencephalo-telencephalic projections in teleosts.

Myelinated dendrites in the mormyrid electrosensory lobe.

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 animals 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.

Anatomy of the posterior caudal lobe of the cerebellum and the eminentia granularis posterior in a mormyrid fish

The cerebellum of mormyrid fish is of interest for its large size and unusual histology. The mormyrid cerebellum, as in all ray‐finned fishes, has three subdivisions—valvula, corpus, and caudal lobe. The structures of the mormyrid valvula and corpus have been examined previously, but the structure of the mormyrid caudal lobe has not been studied. The mormyrid caudal lobe includes a posterior caudal lobe associated with the electrosense and an anterior caudal lobe associated with lateral line and eighth nerve senses. In this article we describe cellular elements of the posterior caudal lobe and of the eminentia granularis posterior (EGp) in the mormyrid fish Gnathonemus petersii. The EGp gives rise to the parallel fibers of the posterior caudal lobe. We used intracellular injection of biocytin, extracellular injection of biotinylated dextran amine, and immunohistochemistry with antibodies to gamma‐aminobutyric acid, inositol triphosphate receptor I, calretinin, and Zebrin II. The histological structure of the posterior caudal lobe is markedly irregular in comparison to that of the corpus and the valvula, and a tight modular organization of cerebellar elements is less apparent here. Most Purkinje cell bodies are in the middle of the molecular region. Their dendrites are only roughly oriented in the sagittal plane, extend both ventrally and dorsally, and branch irregularly. Climbing fibers terminate only on smooth dendrites near the soma. Most Purkinje cell axons terminate locally on eurydendroid cells that project outside the cortex. The results provide an additional variant to the already large set of different cerebellar and cerebellum‐like structures. J. Comp. Neurol. 502:714–735, 2007.

Cell morphology and circuitry in the central lobes of the mormyrid cerebellum

The cerebellum of mormyrid electric fish is large and unusually regular in its histological structure. We have examined the morphology of cellular elements in the central lobes of the mormyrid cerebellum. We have used intracellular injection of biocytin to determine the morphology of cells with somas in the cortex, and we have used extracellular placement of anterograde tracers in the inferior olive to label climbing fibers. Our results confirm previous Golgi studies and extend them by providing a more complete description of axonal trajectories. Most Purkinje cells in mormyrids and other actinopterygian fishes are interneurons that terminate locally in the cortex on efferent neurons that are equivalent to cerebellar nucleus cells in mammals. We confirm the markedly sagittal distribution of the fan‐like dendrites of Purkinje cells, efferent cells, and molecular layer interneurons. We show that Purkinje cell axons extend further than was previously thought in the sagittal plane. We show that climbing fibers are distributed in narrow sagittal strips and that these fibers terminate exclusively in the ganglionic layer below the molecular layer where parallel fibers terminate. Our results together with those of others show that the central lobes of the mormyrid cerebellum, similar to the mammalian cerebellum, are composed of sagittally oriented modules made up of Purkinje cells, climbing fibers, molecular layer interneurons, and cerebellar efferent cells (cerebellar nucleus cells in mammals) that Purkinje cells inhibit. This modular organization is more apparent and more sharply defined in the mormyrid than in the mammal. J. Comp. Neurol. 497:309–325, 2006.

Dye coupling without gap junctions suggests excitatory connections of γ-aminobutyric acidergic neurons

After injections of the low‐molecular‐weight tracer neurobiotin into the preeminential nucleus of the brain of the mormyrid fish Gnathonemus petersii, we observed that retrogradely labeled, large fusiform projection neurons (LFd cells) of the deep granular layer of the electrosensory lobe (ELL) were surrounded by 30–50 labeled satellite granular cells. More superficially located projection cells, including large fusiform cells in the superficial granular layer (LFs) and large ganglionic (LG) cells in the ganglionic layer, were never surrounded by labeled satellites. LFd‐satellite cells have a small soma (diameter 5–8 μm), a few small dendrites, and an apical axon that terminates in the plexiform and ganglionic layers of the ELL. They contact LFd projection neurons with dendrodendritic, dendrosomatic, and somatodendritic puncta adhaerentia‐like appositions, designated here as “neurapses.” In the electron microscope, these contacts resemble synapses without presynaptic vesicles. Because no gap junctions were found between LFd and satellite granule cells, we suggest that the neurapses allow the passage of neurobiotin, though not biocytin or biotinylated dextran amine. These contacts may provide the intermediate substrate for the postulated, but so far unknown, excitatory connection between primary afferent input and LFd projection neurons, via γ‐aminobutyric acid (GABA)‐ergic granular cells. We suggest that certain puncta adhaerentia‐like contacts might not be only adhesive structures and that LFd‐satellite granular cells might both excite LFd projection cells via neuraptic contacts of their dendrites and cell bodies and inhibit more superficial LF and LG cells via their GABAergic axonal synapses. Our results suggest that puncta adhaerentia‐like contacts could be responsible in some cases for the electrical coupling found electrophysiologically in local inhibitory circuits. J. Comp. Neurol. 468:151–164, 2004.