Distribution of adenylate cyclase in the brain of Apteronotus leptorhynchus as revealed by forskolin binding.

An antibody directed against an isoform of the rat regulatory subunit of protein kinase A and brain dissection was used for immunoblot analysis of this protein in various brain regions of Apteronotus leptorhynchus. Western blots revealed that the antibody labeled a band of the expected molecular mass (approximately 53 kDa) for this enzyme in mammalian cortex and electric fish brain, suggesting that this protein is also found in fish brains. The 53-kDa band was enriched in fish forebrain. [3H]Forskolin binding was used as a marker for the distribution of adenylate cyclase. [3H]Forskolin binding was nearly completely displaced by excess cold forskolin; specific [3H]forskolin binding sites were heterogenously distributed with relatively high densities in some gray matter regions and low densities in fiber tracts. A high density of [3H]forskolin binding sites was found in the dorsal forebrain with lower densities in most ventral forebrain nuclei. Moderate binding densities were observed in the preoptic and hypothalamic areas with the exception of the nucleus tuberis anterior, which had high levels. The thalamus and midbrain had low levels of binding. The cerebellar molecular layer had dense binding, in contrast to the granule cell layer where binding was low. In the electrosensory lateral line lobe (ELL), there was moderate binding in the dorsal and ventral molecular layers, which contain feedback inputs; the cellular layers of the ELL had low binding densities.     

Distribution of calcium/calmodulin-dependent kinase 2 in the brain of Apteronotus leptorhynchus.

Antibodies directed against the mammalian alpha and beta subunits of calcium/calmodulin-dependent kinase 2 (CaMK2) and brain dissection were used for immunoblot analysis of these proteins in various brain regions of Apteronotus leptorhynchus. Western blots revealed that the CaMK2alpha antibody labeled a single band of the expected molecular mass (approximately 50 kDa) for this enzyme in rat cortex and electric fish brain. CaMK2alpha was enriched in fish forebrain and hypothalamus and also strongly expressed in midbrain sensory areas. Western blots revealed that CaMK2beta antibodies labeled bands in an appropriate molecular mass range (approximately 58-64 kDa) for this enzyme in mammalian cortex and electric fish brain. However, a higher molecular mass band (approximately 80 kDa) was also labeled; because all these bands were eliminated by preadsorbtion with the CaMK2-derived peptide antigen, they may all represent CaMK2beta-like isoforms. We mapped the brain distribution of CaMK2 isoforms with emphasis on the electrosensory system. CaMK2alpha was present at high density in dorsal forebrain, hypothalamic nuclei, torus semicircularis, and tectum. It was also enriched in discrete fiber tracts in forebrain, diencephalon, and rhombencephalon. CaMK2beta-like isoforms were enriched in ventral forebrain, hypothalamic nuclei, torus semicircularis and the reticular formation. Unlike CaMK2alpha, CaMK2beta -like isoforms were predominantly present in cell bodies and rarely found in fiber tracts or neuropil. In the electrosensory lateral line lobe, CaMK2alpha was restricted to specific feedback fibers, i.e., tractus stratum fibrosum and its terminal field in the ventral molecular layer. In contrast, CaMK2beta-like isoforms were enriched in somata and dendrites of pyramidal cells and granular interneurons.     

Heterogeneous localization of protein kinase C in rat brain: autoradiographic analysis of phorbol ester receptor binding

Protein kinase C is a calcium- and phospholipid-stimulated enzyme present in high concentration in the brain. Phorbol esters are potent tumor promoters that bind to specific receptors with high affinity. Several lines of evidence indicate that the phorbol ester receptor is identical to protein kinase C. To determine the distribution of protein kinase C, we have localized phorbol ester receptors in the rat brain by autoradiography, using [3H]phorbol 12,13-dibutyrate ([3H]PDBu) and have performed a variety of lesions to assess the nature of the cellular elements possessing the binding sites. The [3H]PDBu binding sites in the rat brain are discretely localized and primarily associated with neurons. Evidence is presented for localization to intrinsic neurons of the cortex and hippocampus, terminals of the striatonigral projection, a projection to the molecular layer of the dentate gyrus, and to dendrites of Purkinje cells.     

Development of phorbol ester (protein kinase C) binding sites in cat visual cortex

Tritiated phorbol-12,13-dibutyrate [( 3H]PDBu), a phorbol ester, was utilized to autoradiographically localize protein kinase C (PKC) in the cat visual cortex. Thin, slide-mounted sections of adult cat brain were used to characterize binding of [3H]PDBu. This was found to be saturable, reversible, and more readily displaced by phorbol ester than by synthetic diacylglycerols. Binding sites displayed a tissue concentration of 20 pmol/mg protein, and a dissociation constant of 8.0 nM. [3H]PDBu was slow to associate with its receptor, requiring 9.5 h to reach equilibrium. Autoradiograph revealed that PKC is heterogeneously distributed in the cat brain, and displays a laminar-specific pattern in the visual cortex. This laminar distribution undergoes marked changes during the first two months of postnatal life. In the visual cortex of neonatal kittens, [3H]PDBu binding is confined to layers I and V. Layer III acquires high levels of binding by postnatal day 15, layer II by 28 days, and layer VI becomes labelled by 40 days of age. Adult animals exhibit high levels of binding in all laminae except layer IV. Age-dependent changes in PKC's laminar distribution do not seem to be correlated with specific anatomical, neurochemical, or behavioural events during development. PKC appears to be associated with cell bodies or processes intrinsic to the visual cortex, and is probably not located on the terminals of cortical afferents.     

Activation of protein kinase C by phorbol dibutyrate modulates GABAA receptor binding in rat brain slices

Effects of protein kinase C (PKC) activation on the function of the GABA/benzodiazepine receptor-chloride complex were analyzed by quantitative autoradiography using [³H]muscimol, [³H]flunitrazepam and [³⁵S]TBPS in rat brain slices. The density of [³H]muscimol binding was highest in cerebellar granular layers and high in both the frontal cortex and thalamus, but binding levels in the hippocampus were low. After activation of PKC by 100 nM phorbol-12,13-dibutyrate (PDBu), [³H]muscimol binding was decreased in the frontal cortex, striatum and thalamus, but binding levels were not changed in the hippocampus or cerebellum. The density of [³H]flunitrazepam binding was high in the cortex, hippocampus and molecular layers of cerebellum but was low in thalamus. PDBu increased the [³H]flunitrazepam binding only in the striatum and in part of the cortex and thalamus after activation of PKC. After activation of PKC by PDBu, [³⁵S]TBPS binding was increased in most areas, but binding levels were not changed in the brainstem or cerebellum. The receptor binding was markedly decreased in almost all areas by the addition of 2.5 mM Mg²⁺. Elevated [³⁵S]TBPS binding produced by PDBu was significantly inhibited by the addition of Mg²⁺. These results suggest that the activation of PKC potentiates benzodiazepine and TBPS binding, but decreases muscimol binding in a region-specific manner in the rat brain.     

Distribution of muscarinic acetylcholine receptor mRNA in the brain of the weakly electric fish Apteronotus leptorhynchus

Various neuromodulators have been shown to be involved in shaping the sensory information available to the brain. Acetylcholine (ACh) modulation, through muscarinic receptors, is a particularly widespread mechanism of controlling sensory information transmission. The precise effects of ACh modulation depend on the subtype of muscarinic ACh receptors that are activated. In weakly electric fish, previous work suggested a role of ACh, via muscarinic receptors, in the modulation of information transmission in the electrosensory lateral line lobe (ELL) of the hindbrain. In this study, we determined which muscarinic receptor (mAChR) subtypes are present in the brain of Apteronotus leptorhynchus as well as their spatial distribution. We partially cloned three subtypes of muscarinic receptors (mAChR2, ?3, and ?4) from brain tissue of A. leptorhynchus and used in situ hybridization in transverse sections of the brain to determine their distributions. Sites labeled for the three muscarinic receptor mRNAs were found in various brain regions devoted to the processing of different sensory modalities. The mRNA probes for the three receptor types showed differential distribution but also overlapping presence of two or more receptors in particular nuclei. In addition to the presence of mAChR3 in the ELL region, electrosensory nuclei including the nucleus praeeminentialis, dorsal torus semicircularis and optic tectum showed expression of one or more mAChRs. Thus, the overall pattern of mAChR expression found is in agreement with mAChR expression in other species, with additional presence evident in specialized regions of the electrosensory system, which suggests an important modulating role of ACh in this sensory modality. J. Comp. Neurol. 521:1054–1072, 2013. © 2012 Wiley Periodicals, Inc.     

Activation of protein kinase C by phorbol dibutyrate modulates GABAA receptor binding in rat brain slices

Effects of protein kinase C (PKC) activation on the function of the GABA/benzodiazepine receptor-chloride complex were analyzed by quantitative autoradiography using [³H]muscimol, [³H]flunitrazepam and [³⁵S]TBPS in rat brain slices. The density of [³H]muscimol binding was highest in cerebellar granular layers and high in both the frontal cortex and thalamus, but binding levels in the hippocampus were low. After activation of PKC by 100 nM phorbol-12,13-dibutyrate (PDBu), [³H]muscimol binding was decreased in the frontal cortex, striatum and thalamus, but binding levels were not changed in the hippocampus or cerebellum. The density of [³H]flunitrazepam binding was high in the cortex, hippocampus and molecular layers of cerebellum but was low in thalamus. PDBu increased the [³H]flunitrazepam binding only in the striatum and in part of the cortex and thalamus after activation of PKC. After activation of PKC by PDBu, [³⁵S]TBPS binding was increased in most areas, but binding levels were not changed in the brainstem or cerebellum. The receptor binding was markedly decreased in almost all areas by the addition of 2.5 mM Mg²⁺. Elevated [³⁵S]TBPS binding produced by PDBu was significantly inhibited by the addition of Mg²⁺. These results suggest that the activation of PKC potentiates benzodiazepine and TBPS binding, but decreases muscimol binding in a region-specific manner in the rat brain.     

Decreased phorbol ester binding in the parahippocampal gyrus from subjects with schizophrenia is not associated with changes in protein kinase C

Combining in situ radioligand binding with autoradiography, we previously identified a reduction of [(3)H]phorbol 12,13-dibutyrate binding in the parahippocampal gyrus from schizophrenic subjects. To determine whether these changes were due to decreases in the level of protein kinase C, we measured [(3)H]phorbol 12,13-dibutyrate binding, levels of the protein kinase C isoforms alpha, beta, delta, epsilon, gamma, eta and theta, as well as protein kinase C activity in crude particulate membranes from parahippocampal gyri of 15 schizophrenic and 15 control subjects. There was a significant decrease in the density (mean +/- SEM: 6.56 +/- 0.73 pmol mg(-1) vs 9.68 +/- 1.22 pmol mg(-1); P < 0.05) and affinity (mean K(D) +/- SEM: 4.64 +/- 0.34 nM vs 2.95 +/- 0.35 nM; P < 0.005) of [(3)H]phorbol 12,13-dibutyrate binding in homogenates from schizophrenic subjects. There were no significant changes in levels of the protein kinase C isoforms which are known to bind phorbol esters or in the activity of protein kinase C in membranes from schizophrenic subjects. These results suggest that there are changes in molecules capable of binding [(3)H]phorbol 12,13-dibutyrate, other than protein kinase C, in the parahippocampal gyrus from subjects with schizophrenia.     

Differential distribution of SK channel subtypes in the brain of the weakly electric fish Apteronotus leptorhynchus

Calcium signals in vertebrate neurons can induce hyperpolarizing membrane responses through the activation of Ca(2+)-activated potassium channels. Of these, small conductance (SK) channels regulate neuronal responses through the generation of the medium after-hyperpolarization (mAHP). We have previously shown that an SK channel (AptSK2) contributes to signal processing in the electrosensory system of Apteronotus leptorhynchus. It was shown that for pyramidal neurons in the electrosensory lateral line lobe (ELL), AptSK2 expression selectively decreases responses to low-frequency signals. The localization of all the SK subunits throughout the brain of Apteronotus then became of substantial interest. We have now cloned two additional SK channel subunits from Apteronotus and determined the expression patterns of all three AptSK subunits throughout the brain. In situ hybridization experiments have revealed that, as in mammalian systems, the AptSK1 and 2 channels showed a partially overlapping expression pattern, whereas the AptSK3 channel was expressed in different brain areas. The AptSK1 and 2 channels were the primary subunits found in the major electrosensory processing areas. Immunohistochemistry further revealed distinct compartmentalization of the AptSK1 and 2 channels in the ELL. AptSK1 was localized to the apical dendrites of pyramidal neurons, whereas AptSK2 channels are primarily somatic. The distinct expression patterns of all three AptSK channels may reflect subtype-specific contributions to neuronal function, and the high homology between subtypes from a number of species suggests that the functional roles for each channel subtype are conserved from early vertebrate evolution.     

Distribution of protein kinase Mzeta and the complete protein kinase C isoform family in rat brain

Protein kinase C (PKC) is a multigene family of at least ten isoforms, nine of which are expressed in brain (alpha, betaI, betaII, gamma, delta, straightepsilon, eta, zeta, iota/lambda). Our previous studies have shown that many of these PKCs participate in synaptic plasticity in the CA1 region of the hippocampus. Multiple isoforms are transiently activated in the induction phase of long-term potentiation (LTP). In contrast, a single species, zeta, is persistently activated during the maintenance phase of LTP through the formation of an independent, constitutively active catalytic domain, protein kinase Mzeta (PKMzeta). In this study, we used immunoblot and immunocytochemical techniques with isoform-specific antisera to examine the distribution of the complete family of PKC isozymes and PKMzeta in rat brain. Each form of PKC showed a widespread distribution in the brain with a distinct regional pattern of high and low levels of expression. PKMzeta, the predominant form of PKM in brain, had high levels in hippocampus, frontal and occipital cortex, striatum, and hypothalamus. In the hippocampus, each isoform was expressed in a characteristic pattern, with zeta prominent in the CA1 stratum radiatum. These results suggest that the compartmentalization of PKC isoforms in neurons may contribute to their function, with the location of PKMzeta prominent in areas notable for long-term synaptic plasticity.     

Anatomical organization of the hypophysiotrophic systems in the electric fish, Apteronotus leptorhynchus.

The organization of afferents to the pituitary was investigated by applying DiI crystals to the pituitary or pituitary stalk of the gymnotiform electric fish, Apteronotus leptorhynchus. Most hypophysiotrophic cells were found in the hypothalamus and were distributed throughout its rostrocaudal extent: nucleus preopticus periventricularis, pars anterior and posterior; suprachiasmatic nucleus; anterior, dorsal, ventral, lateral, and caudal hypothalamic nuclei; and nucleus tuberis lateralis, pars anterior and posterior. In addition a small number of retrogradely labeled cells were found in the ventral telencephalon (area ventralis, pars ventralis) and, most surprisingly, in a thalamic nucleus (nucleus centralis posterioris). The nucleus preopticus periventricularis pars posterior and the anterior hypothalamic nucleus appear to correspond to the parvicellular and magnocellular divisions of the nucleus preopticus of other teleosts. Integration of these results with immunohistochemical localization of monoamines and neuropeptides in the apteronotid brain suggests many homologies between the hypophysiotrophic nuclei of teleosts and other vertebrates, including mammals. Apteronotus communicates electrically during agonistic and sexual interactions. There are numerous anatomical links between the hypophysiotrophic systems and the brain areas related to electrocommunication.     

Differential growth in the brain of the weakly electric fish, Apteronotus leptorhynchus (Gymnotiformes), during ontogenesis

Growth characteristics of the total brain and of several individual brain regions are described in Apteronotus leptorhynchus. The increase of brain volume relative to total length growth results in a sigmoid growth curve, in which three phases--proportional growth, positive-allometric and negative-allometric growth--could be distinguished. This type of enlargement of the total brain is due to the differential volume increase of individual brain regions and to the increase in the volume of the rhombencephalic structures in particular. Pros- and mesencephalic regions mainly grow in accordance with the overall enlargement of the total brain throughout development, whereas rhombencephalic and certain nuclear structures show such isometric growth only with the onset of the juvenile period. The results are interpreted in the view of functionality of the individual brain regions.     

Stimulation of spontaneous transmitter release by the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, an activator of protein kinase C

Treatment of frog neuromuscular preparations with the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA) (100 nM) resulted in a 40% rise in the frequency of miniature endplate potentials (MEPPs) (P less than 0.05). When these experiments were repeated on preparations bathed in saline containing 10 mM K+ (a procedure that raises MEPP frequency by approximately 10-fold due to the elevated level of [Ca2+]i), TPA caused a 70-75% increase in MEPP frequency (P less than 0.02), although the data did not differ significantly from those recorded in standard frog saline due to the variability between preparations. It is concluded that transmitter release mechanisms may be modulated by protein kinase C and that such modulation probably acts synergistically with [Ca2+]i.     

Odor increases [3H]phorbol dibutyrate binding to protein kinase C in olfactory structures of rat brain. Effect of entorhinal cortex lesion

Since protein kinase C (PKC) is known to be activated in the olfactory bulb and in several limbic areas related to odor processing, we determined whether an olfactory stimulus was able to modulate the activity of PKC in animals with bilateral entorhinal cortex lesion. The translocation of PKC from the cytosol to the membrane was studied using the phorbol ester 12,13-dibutyrate ([3H]PDBu) binding in control and bilateral entorhinal cortex (EC) lesioned rats. The lesion of EC per se did not significantly affect [3H]PDBu binding in any of the brain structures analyzed, while odor stimulation induced it in both control and EC-lesioned groups in the external plexiform layer of the olfactory bulb. In contrast, an odor-induced increase of [3H]PDBu binding in internal glomerular layer of the olfactory bulb was only observed in EC lesioned animals. Similar results were obtained in the piriform cortex. In both CA1 and CA3 hippocampal subfields, odor stimulation induced an increase of [3H]PDBu binding in both control and EC-lesioned animals, the increase being potentiated only in CA1 of lesioned rats. The dentate gyrus and the amygdala exhibited a similar pattern of [3H]PDBu binding, showing a significant increase exclusively in EC-lesioned animals after odor stimulation. The results strongly suggest that the EC plays a key role in odor processing. PKC appears to play an important role in responding to the activation of lipid second messengers, which have been described to be involved in the processing of odor stimuli in several structures of the olfactory pathway.     

Tyrosine phosphorylation of platelet protein induced by phorbol ester

The presence of phosphotyrosine in proteins from washed rabbit platelets was demonstrated by electrophoresis and by alkali stability of phosphorylated polypeptides. The phosphotyrosine content increased about twofold by treating the platelets with PMA in spite of the fact that the specific activity of labeled ATP was unchanged. It was shown that phosphorylated proteins with apparent molecular weight of 48k, 53k and 250k were alkali-stable. Although phosphorylation of 48k protein in platelets was stimulated by 1 U thrombin as well as by 100 ng PMA, only the phosphorylated samples from PMA-treated platelets were alkali-stable. The 48k protein from platelets stimulated by PMA showed the fivefold increase of 32P-tyrosine content as compared with the control. Relatively lower increase of 32P-serine or 32P-threonine, 2- and 1.7-fold respectively, was thereby observed. The differences in time-sequence between 48k and 250k protein of total and alkali-stable phosphorylations suggested that tyrosine phosphorylation of 48k protein is the earliest event after the PMA-stimulation.     

Protein kinase C mediates potentiation of synaptic transmission by phorbol ester at parallel fibers in the dorsal cochlear nucleus

Many cells in the outer two layers of the dorsal cochlear nucleus (DCN) express high levels of the phospholipid-activated, calcium dependent kinase, protein kinase C (PKC), an enzyme that can phosphorylate numerous proteins involved in neurotransmission and postsynaptic signaling. We investigated the effects of stimulating PKC with phorbol esters (phorbol 12-13 diacetate; PDAc) on parallel fiber synaptic transmission in brain slices of the guinea pig DCN. Phorbol esters increased the amplitude of the postsynaptic components of the field potential, including the excitatory post-synaptic field potential (fEPSP) and the population spike following electric stimulation of parallel fibers. Phorbol esters simultaneously decreased paired-pulse facilitation, suggesting that transmitter release mechanisms were affected. Potentiation of synaptic transmission and diminished paired-pulse potentiation were also observed in intracellular recordings of DCN neurons. The effects of phorbol esters were antagonized by the specific PKC blockers bisindolylmaleimide and calphostin C. Although modulation of the synaptic potentials appears to be mediated by presynaptic PKC, the differential effects of PDAc on the fEPSP and the population spike also suggest the involvement of postsynaptic PKC and postsynaptic targets. These experiments demonstrate that protein kinase C is capable of profoundly modulating synaptic transmission at parallel fiber synapses in the DCN.     

Distribution of the protein kinase C substrates MARCKS and MRP in the postnatal developing rat brain

The myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP) are both membrane-associated phosphoproteins that interact with calmodulin and filamentous actin in a protein kinase C phosphorylation-dependent manner. In the present study, we examined MARCKS and MRP gene expression in the postnatal (P) rat brain (1, 7, 14, 21, and 90 days after birth) by using quantitative in situ hybridization. At P1, MRP expression was high in neocortex, striatum, thalamus, cerebellar cortex, and hippocampus (CA1–CA3, hilus, and granule cell layer) but low in brainstem and, between P7 and P14, exhibited a dramatic decline in each of these regions except hippocampal CA1 and granule cell layers. Between P14 and P21, MRP expression increased in white matter regions including the corpus callosum, fimbria/fornix, and cerebellar deep white matter. At P90 (adult), MRP remained strongly expressed in the olfactory bulb, medial habenula, hippocampal CA1, and the inner two-thirds of granule cell layer, temporal, and entorhinal cortices, the corpus callosum and fimbria/fornix, and cerebellar white matter. At P1, MARCKS was strongly expressed in the majority of brain regions except the brainstem, which subsequently declined gradually to approximate adult levels by P14. Between P14 and P21, MARCKS expression declined gradually in the hilus, remained elevated in hippocampal CA1, CA3, and granule cell layers, and increased dramatically in the corpus callosum and fimbria/fornix. At P90, MARCKS expression declined in hippocampal CA3 and hilus and remained strongly expressed in hippocampal CA1 and granule cell layers, regions of the olfactory bulb, the medial habenula, temporal cortex, and cerebellar granule and Purkinje cells. Expression of both MARCKS and MRP in regions undergoing neuronal proliferation, migration, and neurite outgrowth suggest a common role in these developmental events, whereas differences in expression during development and in the adult brain provide evidence of differential regulation. J. Comp. Neurol. 397:337–356, 1998. © 1998 Wiley-Liss, Inc.     

Ontogeny of phorbol ester receptors in rat brain studied by in vitro autoradiography

Summary The ontogeny of phorbol ester receptors, which have been considered to correspond to protein kinase C, in the rat brain was studied through in vitro autoradiography with³H-phorbol 12,13-dibutyrate (³H-PDBu). The distribution of³H-PDBu binding sites in the adult rat brain was similar to the previous reports by other researchers. The developmental pattern of³H-PDBu binding sites varied with brain region.³H-PDBu binding sites in the amygdala, thalamus, stratum pyramidale of CA 1 of the hippocampus, dentate gyrus, superior colliculus, substantia nigra, interpeduncular nucleus and cerebellar molecular layer were postnatally increased to adult levels and after that they remained constant. On the other hand, in the stratum oriens and stratum radiatum of CA 1 of the hippocampus, and in the lateral and medial geniculate bodies,³H-PDBu binding sites reached peaks at 21 or 28 days of postnatal age and after that they declined to adult levels. The cerebellar granular layer showed a low level of³H-PDBu binding sites throughout all the ontogenetic stages. A distinct ontogenetic pattern of phorbol ester receptors in various regions of the brain may reflect a role of protein kinase C in the neural development of each discrete area.     

Identification of a nucleus isthmi in the weakly electric fish Apteronotus leptorhynchus (Gymnotiformes)

The nucleus isthmi of teleost fish, amphibians, reptiles and birds, and its probable homologue, the nucleus parabigeminalis of mammals, share in common certain features such as location in the dorsal tegmentum and reciprocal connectivity with the optic tectum. In gymnotid fish the nucleus isthmi is located dorsolaterally in the brainstem tegmentum, ventral to the torus semicircularis and the lateral mesencephalic reticular area and dorsal to the rostral nucleus praeeminentialis. The nucleus isthmi has an ovoid shape, with a compact cellular part on its dorsal, medial and ventral aspects surrounding a hilar region with a sparse population of larger cells. Following wheat germ agglutinin-conjugated horseradish peroxidase injections into the optic tectum, anterogradely labeled fine terminals were observed leaving the tectobulbar tract and entering the ipsilateral nucleus isthmi via its laterally facing hilar region. Retrogradely labeled cells were present in the nucleus isthmi on both sides, indicating the presence of a bilateral isthmotectal projection similar to that reported in amphibians. The putative isthmal nucleus stains densely for acetylcholinesterase. Based on the similarity of its location, shape, cholinesterase histochemistry and reciprocal connectivity with the optic tectum, we identified this structure as the nucleus isthmi of gymnotids. An interesting observation of this study was that the nucleus isthmi, in addition to receiving fine terminals from the optic tectum, is also the recipient of a sparser population of thicker-caliber afferent fibers which terminate not only in the large-celled hilar region but also within the smaller-celled component of the nucleus; this projection appears to emanate from the torus semicircularis dorsalis.