Afferent and efferent connections of the diencephalic prepacemaker nucleus in the weakly electric fish Eigenmannia virescens interactions between the electromotor system and the neuroendocrine axisкод для вставкиСкачать
THE JOURNAL OF COMPARATIVE NEUROLOGY 383:18–41 (1997) Afferent and Efferent Connections of the Diencephalic Prepacemaker Nucleus in the Weakly Electric Fish, Eigenmannia virescens: Interactions Between the Electromotor System and the Neuroendocrine Axis CALVIN J.H. WONG* The Neurobiology Unit, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA 92093-0201 ABSTRACT The afferent and efferent connections of the gymnotiform central posterior nucleus of the dorsal thalamus and prepacemaker nucleus (CP/PPn) were examined by retrograde and anterograde transport of the small molecular weight tracer, Neurobiotin. The CP/PPn was identified by physiological assay and received a local iontophoretic injection of Neurobiotin. Retrogradely labeled somata were observed in the ventral telencephalon, hypothalamus, and the pretectal nucleus electrosensorius. Anterogradely labeled fibers were traced from the CP/PPn to the ventral telencephalon, the hypothalamus, the neuropil immediately adjacent to the most rostral subdivision of the nucleus electrosensorius, the optic tectum, and the pacemaker nucleus. Retrograde transport of tracer following injections into the ventral telencephalon, preoptic area, lateral hypothalamus, tectum, and pacemaker nucleus confirmed these efferent targets. A rostromedial subarea of the CP/PPn can be identified that projects to basal forebrain regions and to a lateral region of the CP/PPn that contains afferents to the pacemaker. Many of the targets, which are connected with the CP/PPn, have been linked to reproductive behavior or neuroendocrine control in other fishes. A comparative analysis reveals that the efferent pathways of the CP/PPn appear similar and may be homologous to efferent pathways of some components of the auditory thalamus among tetrapods. J. Comp. Neurol. 383:18–41, 1997. r 1997 Wiley-Liss, Inc. Indexing terms: communication; dorsal thalamus; forebrain; pacemaker; electroreception The glass knifefish, Eigenmannia virescens, generates a weak (ca. 10 µV/cm) quasi-sinusoidal electric field in the water by means of an electric organ, located in its tail. The electric organ, which is derived from muscle tissue, is innervated by spinal motoneurons, termed electromotoneurons (see Bass, 1986, for review). Generally, the frequency of the electric organ discharge (EOD) is fixed, driven by the medullary pacemaker nucleus. Under certain contexts, such as the jamming avoidance response (JAR) or novelty response, the fish can raise or lower its EOD frequency (see Heiligenberg, 1991, for review). The fish may also produce interruptions in the EOD during courtship or agonistic encounters (Hopkins, 1974). These interruptions in E. virescens are termed chirps (Hagedorn and Heiligenberg, 1985). Both gradual rises in frequency and chirps in r 1997 WILEY-LISS, INC. E. virescens are controlled by a nucleus situated in the thalamus. This bilaterally situated nucleus projects to the midline pacemaker nucleus and is termed the prepacemaker nucleus (PPn; Heiligenberg et al., 1981; Kawasaki and Heiligenberg, 1988; Kawasaki et al., 1988; Zupanc and Heiligenberg, 1989, 1992). Grant sponsor: NIMH; Grant number: R37 MH26149-18; Grant sponsor: NINCDS; Grant number: RO1 NS22244-08; Grant sponsor: NSF; Grant number: BNS-9106705. *Correspondence to: Calvin J.H. Wong, The Neurobiology Unit, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA 92093-0201. E-mail: firstname.lastname@example.org Received 20 September 1996; Revised 17 December 1996; Accepted 7 January 1997 CONNECTIONS OF THE ELECTROCOMMUNICATORY SYSTEM There appear to be at least two anatomical and functional subdivisions to the PPn: a lateral magnocellular region and a medial parvocellular region. Stimulation of the multipolar cells of the lateral region elicits chirps and is therefore designated the chirp subdivision (PPn-C). Stimulation of the ovoid cells of the medial subdivision elicits gradual rises in EOD frequency and is therefore designated the gradual rise subdivision (PPn-G). Medially adjacent to the PPn-G is the central posterior nucleus of the dorsal thalamus (CP). As the cells of the CP and the cells of the PPn-G cannot be distinguished from each other, by using cytoarchitectonic, immunohistochemical, or ultrastructural criteria, the term CP/PPn was developed to encompass both those cell populations within the region 19 that specifically project to the pacemaker nucleus (i.e., the PPn) and those that do not (Zupanc and Zupanc, 1992; Zupanc and Heiligenberg, 1992). The only other known input to the pacemaker nucleus is a mesencephalic cell group, the sublemniscal prepacemaker nucleus (SPPn; Keller et al., 1991). This nucleus is responsible for gradual reductions in EOD frequency in E. virescens (Metzner, 1993). In other gymnotiforms, the SPPn appears to control a variety of other electrocommunicatory behaviors (Kawasaki and Heiligenberg, 1989; Keller et al., 1991; Spiro, 1994; Heiligenberg et al., 1996). One cell group that projects to the CP/PPn is the pretectal complex of the nucleus electrosensorius (nE; Bastian and Yuthas, 1984; Keller et al., 1990). The nE Abbreviations A AC ALLG ATh BDA cANS CE CP CP/PPn cT Dc Dd DFl Dl Dld Dlp DLTh Dlv Dm Dm2c Dm2d Dm2r Dm2v DMTh Dp DPn DTn dtP-Cb Ec EGm EGp ELL EO EOD Er eTS FR H Ha Hc Hd Hl Hv IPn JAR LFB LL MA MFB MgT MLF MOB nAPv nE nE> nE< nEAR nEb pretectal nucleus A anterior commissure anterior lateral line ganglion anterior thalamic nucleus biotinylated dextran amine ansulate commissure central nucleus of the inferior lobe central posterior nucleus (thalamus) complex of the central posterior nucleus (thalamus) and the prepacemaker nucleus tectal commissure central division of the dorsal telencephalon dorsal division of the dorsal telencephalon lateral diffuse nucleus of the inferior lobe lateral division of the dorsal telencephalon dorsolateral telencephalon, dorsal subdivision dorsolateral telencephalon, posterior subdivision dorsolateral thalamic nucleus dorsolateral telencephalon, ventral subdivision medial division of the dorsal telencephalon dorsomedial telencephalon, subdivision 2, caudal dorsomedial telencephalon, subdivision 2, dorsal dorsomedial telencephalon, subdivision 2, rostral dorsomedial telencephalon, subdivision 2, ventral dorsomedial thalamic nucleus posterior division of the dorsal telencephalon dorsal posterior nucleus (thalamus) dorsal tegemental nucleus decussation of the tractus praeminentialis-cerebellaris caudal enteropeduncular nucleus eminentia granularis, pars medialis eminentia granularis, pars posterior electrosensory lateral line lobe efferent octavolateral nucleus electric organ discharge rostral enteropeduncular nucleus torus semicircularis efferents fasciculus retroflexus habenula anterior hypothalamic nucleus caudal hypothalamic nucleus dorsal hypothalamic nucleus lateral hypothalamic nucleus ventral hypothalamic nucleus interopeduncular nucleus jamming avoidance response lateral forebrain bundle lateral lemniscus Mauthner cell axons medial forebrain bundle magnocellular tegmental nucleus medial longitudinal fasciculus medial olfactory bulb anterior periventricular nucleus nucleus electrosensorius nucleus electrosensorius, ‘‘up’’ subdivision nucleus electrosensorius, ‘‘down’’ subdivision acousticolateralis region of the nucleus electrosensorius beat subdivision of the nucleus electrosensorius nI nLV nM nPPv nRLl nT OC P PC PeG PG PGl PGm Pn POC PPa PPn PPn-C PPn-G PPp PT PTh PTl PTPv R Rc Rd RF Sc SE SPPn TA TAd TAv Tel TeO TL tODM TP TPP TSd TSv V Vc VCbm Vd Vi Vl VLTh VMTh Vp VP Vs VT Vv Vv-d Vv-v isthmic nucleus nucleus of the lateral valvula nucleus medialis posterior periventricular nucleus lateral nucleus of the lateral recess nucleus taenia optic commissure pacemaker cell posterior commissure periglomerular nucleus preglomerular complex preglomerular complex, lateral subdivision preglomerular complex, medial subdivision pacemaker nucleus postoptic commissure anterior periventricular preoptic nucleus prepacemaker nucleus prepacemaker nucleus, ‘‘chirp’’ subdivision prepacemaker nucleus, ‘‘gradual rise’’ subdivision posterior periventricular preoptic nucleus pretectal nucleus nucleus prethalamicus lateral pretectal nucleus periventricular pretectal nucleus red nucleus central raphé nucleus dorsal raphé nucleus reticular formation suprachiasmatic nucleus subelectrosensorius sublemniscal prepacemaker nucleus anterior tuberal nucleus anterior tuberal nucleus, dorsal subdivision anterior tuberal nucleus, ventral subdivision telencephalon optic tectum torus longitudinalis dorsomedial optic tract posterior tuberal nucleus periventricular nucleus of the posterior tuberculum torus semicircularis, dorsal subdivision torus semicircularis, ventral subdivision ventricle ventral telencephalon, central subdivision valvula of the cerebellum, medial subdivision ventral telencephalon, dorsal subdivision ventral telencephalon, intermediate subdivision ventral telencephalon, lateral subdivision ventrolateral thalamic nucleus ventromedial thalamic nucleus ventral telencephalon, posterior subdivision valvular peduncle ventral telencephalon, supracommisural subdivision ventral dendritic territory ventral telencephalon, ventral subdivision ventral telencephalon, dorsal aspect of the ventral subdivision ventral telencephalon, ventral aspect of the ventral subdivision 20 C.J.H. WONG receives ascending electrosensory, mechanosensory lateral line, and octaval information from the midbrain torus semicircularis (Carr et al., 1981; Keller et al., 1990), the homologue of the inferior colliculus. Previous studies have shown that some neurons in the nE, which appear to fire preferentially to playbacks of chirps, project to the hypothalamus (Heiligenberg et al., 1991). It has been suggested that this might provide a substrate for conspecific sensory signals to influence the motivational and reproductive state of the animal. Further support for the presence of interactions between the electrosensory/motor system and the neuroendocrine system is suggested by immunohistochemical studies in a closely related gymnotiform, Apteronotus leptorhynchus. These studies demonstrate that CP/PPn receives monoaminergic and peptide-ergic innervation which may originate from the hypothalamus (see Johnston and Maler, 1992; Zupanc and Maler, 1997, for review). Of particular interest is the presence of a sexual dimorphism in substance-P innervation of the CP/PPn (Weld and Maler, 1992). In addition, the extent of substance-P innervation is correlated with propensity to chirp in testosteroneimplanted female fish (Dulka and Maler, 1994; Dulka et al., 1995). Other evidence for a functional role of the CP/PPn in reproductive behavior is the presence of androgen receptors in cells of CP/PPn as well as the nE. These results suggest that the cells that detect or produce electrocommunicatory behaviors may be subject to hormonal influence (Zakon, 1993; Gustavson et al., 1994). Indeed, the PPn-C cells in E. virescens show seasonal variation in their dendritic structure (Zupanc and Heiligenberg, 1989). The CP/PPn is a key link of the electrocommunicatory circuit, residing at the interface of ascending electrosensory information and the eventual transformation of this information into modulations of the EOD. In spite of this, only preliminary mapping studies of the connections of CP/PPn have been published (Keller et al., 1990; Stroh and Zupanc, 1995). This study was undertaken to further clarify the organization of this sensory-motor interface by mapping the afferent and efferent microcircuitry of CP/ PPn. Preliminary results have been published previously in abstract form (Wong, 1995a,b). MATERIALS AND METHODS Adult fish (15–20 cm) of both sexes of E. virescens were used. Injections into the CP/PPn in other gymnotiforms, A. leptorhynchus and Hypopomus pinnicaudatus revealed generally similar results, though I will restrict the results to E. virescens. Some species-specific differences in connectivity were observed between A. leptorhynchus and E. virescens, particularly with respect to the SPPn, and these have been presented elsewhere (Heiligenberg et al., 1996). Fish were obtained from commercial suppliers (Bailey’s Tropical Fish, San Diego, CA) and maintained in aquaria containing water of 20 to 30 kOhms/cm resistivity and neutral pH at a temperature of 26–28°C. Fish were kept on a diurnal cycle of 12 hours light /12 hours dark and fed a diet of live blackworms. Animal care, anesthesia, surgery, and euthanasia were carried out in compliance with guidelines set forth by the Animal Subjects Committee for the University of California, San Diego. Anesthesia consisted of placing the fish in a 1:15,000 solution of tricaine methane sulfonate (MS-222; Sigma Chemical Co., St. Louis, MO) until somatic reactions stopped. This was followed by immobilization with an intramuscular injection of 2 µl of 2 mg/ml gallamine triethiodide (Sigma Chemical Co., St. Louis, MO) in saline. The fish was then placed in a fish holder with aerated water perfusing the gills. The surgical site was prepared with topical application of 20 mg/ml lidocaine, and the skull was opened to expose the rostral pole of the brain. The tail was placed in a plastic tube and a wire inserted in the tube so that the EOD could be amplified and monitored. The PPn was localized by physiological assay. A single-barrel glass micropipette was filled with L-glutamate (0.1 M, pH 8.0), which was iontophoresed with negatively biased current (100 nA). L-glutamate selectively stimulates cell bodies and dendrites but not fibers of passage. As the electrode was advanced, the PPn could be readily identified by modulations in the EOD. Once the depth and location of the PPn was ascertained (approx. 1,500–2,000 µm in depth), the single-barrel electrode was replaced with a triple-barrel electrode consisting of two barrels filled with glutamate and one with the tracer, Neurobiotin (Vector Laboratories, Burlingame, CA). The Neurobiotin was made up at 2% in 0.2 µm filtered 1 M KCl. Tip resistances for the Neurobiotin electrodes were approximately 5 MOhms. Upon successful reidentification of the site, the tracer was iontophoresed with positive DC current (ca. 2–5 µA) for 20 minutes to 1 hour. Following iontophoresis of the tracer, the electrode was left in place for 5 to 10 minutes, and then slowly withdrawn to avoid leakage. The hole in the skull was patched with gelfoam, and the site was sealed with Vetbond (3M). Survival times were 8 to 30 hours at 27°C for retrograde and anterograde transport of tracer. Although Neurobiotin was the primary tracer used in these studies, biotinylated dextran amines (BDA; Molecular Probes, Eugene, OR) were also used (Veenman et al., 1992). One animal received an iontophoretic injection of 3,000 molecular weight (3 kDa) BDA. Four animals received pressure injections of 10 kDa BDA. These other cases revealed qualitatively similar results, although the injection sites tended to be larger. Fish were euthanised in MS-222 and perfused transcardially with 0.9% NaCl followed by fixative for half an hour (approximately 30 ml of fixative). The fixative was composed of 4% paraformaldehyde and 0.25% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). Brains were then removed and postfixed overnight for tissue processing the next day. Tissue processing Free floating Vibratome sections were processed by using the avidin-biotin method (ABC Standard Elite Kit; Vector Laboratories, Burlingame, CA) with a nickel intensified peroxidase-3,3’ diaminobenzidine (DAB) reaction. Brain sections were processed according to a protocol modified from previous procedures (Kita and Armstrong, 1991; Lapper and Bolam, 1991; Vaney, 1991; Huang et al., 1992). The following solutions were used: phosphate buffer saline (PBS; 0.02 M phosphatebuffer, pH 7.6 and 0.9% NaCl), 0.1% Triton X-100 in PBS (PBST), and Tris buffer (TB; 0.1 M Tris buffer, pH 7.2). Vibratome sections were cut at 50 µm thickness into PBS. They were then ‘‘prebleached’’ by soaking for 10 minutes in 0.5% H2O2 in PBS to inhibit endogenous peroxidases in the tissue. CONNECTIONS OF THE ELECTROCOMMUNICATORY SYSTEM By using a Vectastain ABC kit (Vector Laboratories), an ABC solution was prepared (4 drops A 1 4 drops B in 12 ml PBST) and stored for 30 minutes. Following prebleaching, sections were washed three times for 10 minutes in PBS and incubated in the ABC solution in small, covered dishes at 4°C for 4–12 hours. Sections were then washed three times for 10 minutes in PBS and once for 10 minutes in TB, and then processed by the DAB procedure. Sections were presoaked for 15 minutes in a solution of TB, 0.064% nickel ammonium sulfate, and 0.04% DAB (Sigma Chemical Co., St. Louis, MO). Following presoak, H2O2 was added to a final concentration of 0.0018%. The reaction was allowed to proceed for 5 to 15 minutes, depending on the intensity of the background label. The reaction was stopped by washes in TB. Sections were washed at least three times for 10 minutes in TB and mounted on chrom-alum gelatin coated slides. Sections were counterstained with neutral red (Sigma Chemical Co., St. Louis, MO) and coverslipped with Permount (Fisher Scientific, Rockville, MD). Sections were analyzed and compared with reference cresyl violet and Klüver-Berrera stained paraffin series of E. virescens as well as the brain atlas of A. leptorhynchus (Maler et al., 1991), which uses as its basis the nomenclature of a variety of investigators. Generally, the nomenclature of the A. leptorhynchus atlas has been adopted for E. virescens. Chartings of representative cases were made by using a camera lucida. Photomicrographs were taken by using TMAX-100 black and white film (Kodak, Rochester, NY) with a Wratten 44 filter to enhance the counterstain. Methodological considerations Neurobiotin, when iontophoresed, appeared to be taken up very locally by neurons, with little if any glial label unless there was ventricular spread. In such instances, a non-specific gray background reaction product developed with labeled radial glia. Owing to the low molecular weight of the tracer, any excess that was not immediately taken up by cells or terminals in the vicinity of the injection site appeared to be metabolized or washed out. Fortunately, the center of the injection was relatively easy to assess, as there was much heavier cell labeling at the injection site as well as local tissue damage arising from the iontophoretic current. Due to the sensitivity of the technique, anterograde label of axon collaterals from retrogradely labeled cell bodies would cause confusion, by suggesting false efferent targets of the CP/PPn. In cases in which it was not possible to trace fibers directly to the CP/PPn due to labeling of such collaterals, reciprocal injections were placed to determine the site of originating cell bodies. Many connections observed appeared to be reciprocal with both labeled fibers and terminals. Such patterns of connections might be explained by transneuronal transport of tracer. There have been some reports that Neurobiotin is transported transneuronally, particularly in cases where neurons are electrotonically coupled (Vaney, 1991; Huang et al., 1992). Controls for transneuronal transport were performed three ways. First, preliminary experiments using BDA, which is not transported transneuronally, retrogradely labeled the same cell groups as Neurobiotin. As BDA injections tended to be larger than Neurobiotin injections and the rate of transport of BDA was much slower than that of Neurobiotin, Neurobiotin was preferable as a tracer in E. virescens. 21 Second, Neurobiotin injections were made into structures whose connectivity had been extensively documented by previous investigators (i.e., pacemaker, tectum, torus semicircularis, and cerebellum). Transneuronal transport was observed following injections into lamina VI of the dorsal torus and following injections into the auditory torus. With the injections made into lamina VI, retrograde transport through electrotonic gap junctions extended from the spherical cells afferent to layer VI, to the cells of the anterior lateral line ganglion, which electrotonically synapse onto spherical cells (Carr et al., 1986; Mathieson et al., 1987). Additionally, massive injections of Neurobiotin into the auditory torus revealed transneuronal transport to fibers that could be traced to the eighth nerve root. It is uncertain if there is electrotonic coupling in the central auditory pathways of teleost fishes. Otherwise, injections into the tectum, torus, and pacemaker nucleus revealed no connections other than those established by previous investigators using other tracers. Finally, in a few fish, longer survival times were used (.30 hours). In these cases, the extent of label was actually reduced. This decrease in label is probably due to metabolism of Neurobiotin (Kita and Armstrong, 1991). In the fish that received injections of Neurobiotin into CP/PPn, various iontophoretic protocols were used. Electrode resistance, total injection time, and magnitude of the injection current had the greatest effects on the discreteness of the injection site. The protocol that was most commonly used and which yielded discrete injections was a 12 µA square wave pulse, 10 seconds on /5 seconds off for 45 minutes. Although iontophoretic injections with small diameter pipette tips (,10 µm) yielded discrete injections (,100 µm in diameter), uptake by damaged fibers of passage as of concern due to the small size and complexity of the teleost diencephalon. To further account for fibers of passage, hypotheses of connectivity were tested by placing injections reciprocally, and by comparisons of labeled fiber tracts from over 40 additional injections into other structures from other experiments. RESULTS Cytoarchitecture of the CP/PPn A prominent nuclear complex in the dorsal thalamus of E. virescens, termed the CP/PPn, can be identified adjacent to a sulcus in the third ventricle. It is most simply recognized as a band of cells appearing to extend in a ventrolateral direction. Rostrally (Fig. 1A), the CP/PPn is sandwiched between the third ventricle and the medioventral edge of the horseshoe-shaped anterior thalamic nucleus (ATh). The CP/PPn caps the dorsal edge of the ventromedial thalamic nucleus. At slightly more caudal levels, CP/PPn displaces the ATh ventrolaterally and forms several lamina parallel to the ventricular surface (Fig. 1C). At these more caudal levels, the rostral pole of the periventricular nucleus of the posterior tuberculum (TPP) flanks the ventral aspect of the CP/PPn. In addition, it is clear that at this level, the CP/PPn possesses slightly larger and more diffusely scattered cells along its ventrolateral wing. Further caudally, the lamina of cell bodies in the CP/PPn appear to fuse, with the cells appearing as a continuous stream with somata oriented perpendicular to the ventricular surface (Fig. 1E). Cells of the CP/PPn at this transverse level are typically Fig. 1. Rostral to caudal series of photomicrographs through the diencephalon of E. virescens from a normal reference series (left-hand column of each page) and a typical injection site (right-hand column) centered on and generally confined to the complex of the central posterior nucleus/ prepacemaker nucleus (CP/PPn). A,C,E,G: Rostral to caudal reference series of transverse sections through the dorsal thalamus of E. virescens (15 µm paraffin sections, cresyl-violet counterstain). B,D,F,H: Rostral to caudal Vibratome series of injection site in a single individual corresponding to the same transverse level as the micrographs to the left (50 µm Vibratome sections, neutral red counterstain). Center of injection is in F. Alternate sections are shown. See List of Abbreviations. Scale bars 5 200 µm. CONNECTIONS OF THE ELECTROCOMMUNICATORY SYSTEM Figure 1 (Continued.) 23 24 C.J.H. WONG clustered with a cell free boundary separating the smaller, medial cells from the larger, lateral cells. At the most caudal extent of the CP/PPn (Fig. 1G), the dorsal posterior nucleus of the thalamus (DPn) displaces the CP/PPn, except for the large multipolar cells, which can be observed for approximately 100 µm caudal to the center of the CP/PPn. Analysis of injection site Twenty E. virescens received an injection centered on the CP/PPn. Of these, four animals received a pressure injection of 10 kDa biotinylated dextran amine (BDA). One animal received an iontophoretic injection of 3 kDa BDA. The BDA pressure injections tended to be much larger, encompassing the entire dorsal thalamus. In the remaining 15 cases, iontophoresis of Neurobiotin was used. Of these, one injection extended rostrally into the ATh and one injection was centered more ventrally, encroaching on the TPP and the ventral thalamus. The rest generally appeared to be confined to the CP/PPn. In two cases, the injections appeared to be confined to the rostral, medial portion of the CP/PPn. In these cases, there were no anterogradely labeled terminals within the pacemaker nucleus. A series of photomicrographs of a confined, laterally centered injection is presented in Figure 1. From this case, although the cells right at the center of the injection are heavily labeled (Fig. 1F), cells located in nuclei which lie adjacent to the injection site are only sparsely labeled (ATh, TPP, and DPn). Subsequent uptake from more medially situated cells delineates the boundary of the CP/PPn and is most evident at the rostral-most level where the CP/PPn envelops the ventromedial edge of the ATh (Fig. 1B). A few cells within the caudal aspect of the ventrolateral thalamic nucleus (VLTh) showed uptake of tracer (Fig. 1D) and it is unclear as to whether this is due to interruption of fibers of passage or represents an actual afferent projection. A rostral to caudal charting of a global injection of Neurobiotin (200 µm diameter) centered on, but not confined to the CP/PPn, is presented in Figure 2. In this case, the injection was centered in the caudal aspect of the CP/PPn (Fig. 2K) yet encroached extensively onto rostral and ventral areas. This injection may have interrupted fibers originating from the TPP and the ventral thalamus, as revealed by extensive cell labeling in these nuclei. The number of labeled somata in these nuclei was considerably reduced following more restricted injections into the CP/ PPn (e.g., Fig. 1). Label in the habenula (H), anterior periventricular nucleus (nAPv), and anterior hypothalamic nucleus (Ha), which was observed in the global injection of Figure 2, was not observed with more confined injections. All other labels charted in Figure 2 were present with injections that were restricted to the CP/PPn. This suggests that this case represents the total extent of connections of this nucleus. There were three exceptions. The optic tectum was observed to be very sparsely labeled in this case. Labeled somata within the dorsorostral region of the anterior tuberal nucleus (TA) and within the beat-sensitive subdivision of the nucleus electrosensorius (nEb) were observed only following medially centered injections but were not observed in this case. Afferents to the CP/PPn Following injections into the CP/PPn, retrogradely labeled cells were most prominent along the neuraxis in cell groups associated with the medial forebrain bundle. Typically, these areas were reciprocally connected with the CP/PPn and contained both retrogradely labeled cell bodies and anterogradely labeled terminal fields (see Efferents from the CP/PPn below). These areas include the basal forebrain, the anterior preoptic area, and some nuclei in the hypothalamus. Other diencephalic cell groups projecting to the CP/PPn include the nucleus of the posterior tuberculum and some subdivisions of the pretectal nucleus electrosensorius. A few retrogradely labeled cells were also observed in the nucleus of the lateral valvula, the dorsal and central raphe nuclei, and occasionally within the reticular formation. No projections were observed from the pallium of the telencephalon, the torus semicircularis, or the cerebellum. Ventral telencephalon. The telencephalon of rayfinned fishes is divided into a dorsal part and ventral part (Nieuwenhuys, 1963; Northcutt and Braford, 1980; Northcutt and Davis, 1983; Braford, 1995, for reviews). The dorsal part comprises the pallium, whereas the ventral part comprises the subpallium. The most rostral cell group projecting to the CP/PPn was observed in a continuum between the ventral nucleus of the ventral telencephalon (Vv) and the dorsal nucleus of the ventral telencephalon (Vd). This cigar-shaped cell group contained retrogradely labeled perikarya that were typically medium-small (8 µm) and round with lateral extending dendrites. These cells were clustered in a dorsal subdivision of the Vv (Vv-d: Fig. 3A,B) continuous with the ventral subdivision of the Vd. Slightly larger, round, scattered cells were retrogradely labeled in the ventral subdivision of the Vv (Vv-v). Labeled cells were not consistently observed within the more dorsal laminar region of the Vd. The extent of retrogradely labeled somata within the Vv and the Vd was from approximately 50–100 µm caudal to the olfactory bulb, extending to the caudal pole of the Vv/Vd for a total rostral to caudal extent of approximately 600 µm. These retrograde transport data are corroborated by anterograde transport of tracer following discrete injections placed in Vv/Vd (unpublished observations). Preoptic area. At the level of the anterior commissure lies the anterior periventricular preoptic nucleus (PPa). Retrogradely labeled cells were consistently observed in this region following both global and confined injections of tracer into the CP/PPn. Within the PPa, labeled mediumsized cells (12–15 µm) were observed approximately 20–30 µm from the periventricular zone (Fig. 3C,D). In contrast to the PPa, which was characterized by consistently labeled cells, the posterior periventricular preoptic nucleus (PPp) rarely contained retrogradely labeled cells after confined injections into CP/PPn. As discussed above, cells found within the anterior hypothalamic nucleus (Ha), anterior periventricular nucleus (nAPV), and habenula (H) depicted from this global injection (Fig. 2E–G) were not consistently observed after confined injections into the CP/PPn. Hypothalamus. The anterior tuberal nucleus (TA) is a large complex nucleus, lying ventral to the lateral recess and dorsal to the lateral hypothalamic nucleus (Hl). It is a particularly prominent nucleus in gymnotiforms (Striedter, 1992) and consists of at least two distinctive cytoarchitectonic subdivisions (Heiligenberg et al., 1991). Retrogradely labeled somata were observed only within the dorsal subdivision and generally only from those injections that were centered on the rostromedial regions of the CP/PPn (Fig. 4). These bipolar cells that were labeled were slightly Fig. 2. Rostral to caudal charting of retrogradely labeled cell bodies (large dots) and anterogradely labeled fibers (short lines) and terminals (small dots) following an injection centered on but not confined to the CP/PPn in E. virescens. A-H: Label in the forebrain. Arrow in F indicates the rostromedial fiber tract within the medial forebrain bundle. See text for details. Nomenclature based on the atlas of a related gymnotiform, A. leptorhynchus (Maler et al., 1991). Scale bar 5 500 µm. I–L: Label in the diencephalon and rostral mesencephalon. The center of the injection was in part K. An asterisk in part L demarcates the caudal fiber tract innervating the pacemaker nucleus. See text for details. Scale bar 5 500 µm. M–O: Label in the caudal mesencephalon (part M,N) and medulla (part O). Asterisk indicates caudal fiber tract innervating the pacemaker nucleus. See text for details. See List of Abbreviations. Scale bar 5 500 µm. 26 C.J.H. WONG Figure 2 ovoid in shape (7.5 3 10 µm). Following more laterally placed injections, such as the one charted in Figure 2, retrograde label within the TA was observed to be considerably reduced. Within the lateral hypothalamic nucleus (Hl), retrogradely labeled cells were observed lying in close apposition to the boundary between the Hl and the ventral portion of the TA (Figs. 2J,K, 5A). These retrogradely (Continued.) labeled cell bodies tended to be more rostrally situated within the Hl over a rostral to caudal extent of approximately 150 µm. There appeared to be at least two cell types labeled: ovoid cells (5 3 8 µm) and round cells (approximately 7 µm diameter). Cells were occasionally retrogradely labeled in the ventral hypothalamic nucleus (Hv). However, since injections into both the overlying optic tectum and the nearby CONNECTIONS OF THE ELECTROCOMMUNICATORY SYSTEM Figure 2 nucleus subelectrosensorius heavily retrogradely labeled somata in the Hv, it is unclear whether the Hv truly projects to the CP/PPn or if the observed labeling represents leakage of tracer into adjoining areas. With larger injections of tracer into the CP/PPn, cells were labeled within the vicinity of the lateral nucleus of the lateral recess (nRLl; Fig. 5B). Retrograde label within this nucleus occurred only in a few cases in which the injection site did not appear to be confined to the CP/PPn. Further caudally, retrogradely labeled cell bodies were found within the caudal hypothalamic nucleus (Hc), situated primarily along the lateral edge of the nucleus abutting the basal hypothalamic tract (tBH). These cells in the Hc (Fig. 5C) were generally slightly ovoid in shape (ca. 8 3 10 µm) and adendritic. The central nucleus of the inferior lobe (CE) lies immediately caudal to the lateral recess. A group of medium-large, slightly ovoid cells were observed to be retrogradely labeled within the CE (Fig. 5D). These cells are 10–15 µm across with extensive dendritic arbors that generally remain confined to the CE. Nucleus electrosensorius. Nucleus electrosensorius (nE) is a pretectal nucleus extending from the nucleus prethalamicus caudal to the interstitial reticular area, consisting of several subdivisions (Keller et al., 1990; Heiligenberg et al., 1991). 27 (Continued.) The most rostrally situated subdivision is the acoustic and lateral line region of the nE (nEAR), which receives input from the ventral subdivision of the torus semicircularis (Carr et al., 1981; Keller et al., 1990). Within the nEAR and in the neuropil medial to it, a few labeled cells were observed to be retrogradely labeled. Labeled cells were also observed within the beatsensitive subdivision of the nE (nEb). These cells project into the vicinity of the CP/PPn by means of a tight fascicle of fibers which course immediately dorsal to the preglomerular complex. These relatively large multipolar cells (Fig. 6A) of the nEb (10–15 µm) have dendrites that ramify extensively within the confines of the nucleus. Cells within this region were retrogradely labeled from medially placed injections. The most caudal subdivision of the nucleus electrosensorius is termed the nE-up (nE>) as stimulation of this region with L-glutamate can elicit gradual rises in EOD frequency. Small adendritic ovoid cells (5 3 10 µm) in the nE> were observed to be retrogradely labeled following CP/PPn injections (Fig. 6B). The axons from nE> cells appeared to enter the CP/PPn by a ventromedially directed band of fibers. Other afferents to the CP/PPn. Retrogradely labeled cells were observed in the nucleus of the posterior tuberculum (TP). These small ovoid cells (5 3 10 µm) were Fig. 3. Label in the forebrain following an injection into CP/PPn. A: Retrogradely labeled cells and anterogradely labeled fibers and terminals within the ventral telencephalon ipsilateral to a large injection of Neurobiotin into the CP/PPn. Cytoarchitectonic boundaries indicated on the contralateral side. B: High-power micrograph of labeled cells within the dorsal subregion of the ventral nucleus of the ventral telencephalon (Vv-d) following a more confined injection into the CP/PPn. C: Labeled fibers and cells within the forebrain at the level of the anterior commissure following an injection of 3 kDa biotinylated dextran amine (BDA) into CP/PPn. D: High-power photomicrograph of neurons labeled in the anterior periventricular preoptic nucleus (PPa). Same section as in C. E,F: Labeled fibers within the medial forebrain bundle arising from an injection of 3 kDa BDA centered on but not confined to the CP/PPn. Note that labeled cells in the anterior hypothalamus (Ha) in part E were not observed with smaller injections although labeled fibers were. E is 100 µm rostral to F. Scale bars 5 100 µm in A,C, 20 µm in B, D, 200 µm in E,F. CONNECTIONS OF THE ELECTROCOMMUNICATORY SYSTEM Fig. 4. Photomicrograph of an injection into the rostromedial subdivision of the CP/PPn with labeled cells and terminals within the lateral nucleus of the ventral thalamus (VLTh) and the dorsal subdivision of the anterior tuberal nucleus (TAd). Scale bar 5 100 µm. distributed bilaterally over the entire rostrocaudal extent of the TP. Retrogradely labeled cells were also found in the dorsal and central raphe nuclei. Labeled cells in the dorsal raphe were generally more consistently observed than in the central raphe. As serotonergic fiber bundles are extensive in the diencephalon in gymnotiforms (Johnston et al., Fig. 5. Label cells and fibers in the hypothalamus. A: Labeled cells and fibers in the lateral hypothalamus (Hl). B: Heavily labeled fibers and cells within the lateral nucleus of the lateral recess (nRLl) from a global injection into the CP/PPn. Uncounterstained section. C: Retrogradely labeled neurons and anterogradely labeled fibers in the caudal hypothalamus. D: Retrogradely labeled neurons in the central nucleus of the inferior lobe. Differential interference contrast optics. See List of Abbreviations. Scale bar 5 100 µm in A–C, 50 µm in D. 29 30 C.J.H. WONG Fig. 6. Retrograde label in the nucleus of the electrosensorius (nE). A: Retrogradely labeled cells in the beat sensitive subdivision (nEb). B: Retrogradely labeled neurons in the ‘‘up’’ subdivision of the nucleus electrosensorius (nE>). Scale bar 5 20 µm for both A,B. 1990), it is unclear which of these cell groups provides serotonergic innervation to the CP/PPn. Following CP/PPn injections, retrogradely labeled cells were consistently observed within the nucleus of the lateral valvula (nLV). However, the number of cells within this very large nucleus are few and may represent uptake by fibers of passage. Following injections of tracer into the nLV, extensive retrograde and anterograde label were observed in the diencephalon, with particularly prominent terminal fields in nuclei adjacent to the CP/PPn. Only a few sparsely anterogradely labeled fibers were observed in the CP/PPn. Efferents from the CP/PPn Anterogradely labeled fibers and terminals following CP/PPn injections are charted with afferents in Figure 2. However, as Neurobiotin is transported in both retrograde and anterograde directions, observed labeled fibers and terminals may be collaterals of retrogradely labeled cell bodies. Therefore, the description of efferent fibers originating from the CP/PPn will be focused on those that were corroborated by subsequent placement of reciprocating injections (see Retrograde transport below). One exception is the projection into the medial shell of nEAR, which was particularly prominent and consistently observed with discrete CP/PPn injections. Anterograde transport Ventral telencephalon. A rostromedial bundle was observed to emerge from the injection site. This bundle courses in a paraventricular position within the medial forebrain bundle, medial to the rostral-most pole of the dorsal thalamus. A few fibers from this rostromedial bundle could be traced through the postoptic commissure to the contralateral medial forebrain bundle. At the transverse level of the suprachiasmatic nucleus, the fiber bundle courses in a ventrolaterally opening crescent along the dorsomedial aspect of the diencephalon (Figs. 2F, 3E,F). The arms of the crescent divide at the telencephalicdiencephalic junction, giving rise to dorsally coursing fibers that innervate the rostral midline subdivisions of the ventral telencephalon and ventromedially running fibers that innervate the preoptic area (Figs. 2A–F, 3). There was no evidence of any labeled fibers within the lateral forebrain bundle. Anterogradely labeled fibers and terminals, partially coextensive with retrogradely labeled neurons, were found within the midline nuclei of the ventral telencephalon in a continuum of the Vd-v, the Vv-d, extending caudally to the supracommisural nucleus of the ventral telencephalon (Vs). These anterogradely labeled fibers never extended into the dorsal portion of the Vd, even after massive injections into the CP/PPn. Projections from the CP/PPn to the pallium were not observed. Hypothalamus. Injections into the CP/PPn yielded extensively labeled fibers and terminals within the PPa, and the PPp. These putative anterogradely labeled terminals were most prominent within the nucleus proper, with sparser label in the lateral neuropil. Labeled fibers were seen emanating from the center of the injection site, entering the rostral segment of the TA, and innervating primarily the rostral, dorsal region (TAd) coextensive with the retrogradely labeled cell bodies. Anterogradely labeled fibers were only occasionally observed in the ventral portion of the TA. The lateral hypothalamus contained fibers and putative terminals from CP/PPn injections. The fibers innervating the lateral hypothalamic nucleus originate from the injection site and arc ventrolateral around the TA, rostral to the lateral recess. The fiber bundle was observed to turn medially and innervate the lateral hypothalamus over its entire rostrocaudal extent (approximately 600 µm). Extensive fiber labeling was also evident within the nRLl (Fig. 5B). More caudally, within Hc and its surround there were frequently anterogradely labeled fibers with clear en passant terminals (Fig. 5C). The inferior lobe however lacked anterogradely labeled fibers except following injections that did not appear to be confined to the CP/PPn. Nucleus electrosensorius. Following injections into the CP/PPn, a band of labeled fibers was observed to innervate the neuropil immediately medial to the nEAR. This region CONNECTIONS OF THE ELECTROCOMMUNICATORY SYSTEM 31 Fig. 7. A: Fiber and terminal labeling within the neuropil, medial to the acousticolateralis subdivision of the nucleus electrosensorius (nEAR) following a confined injection of Neurobiotin into the CP/PPn. A few cells are also labeled within this region as well. Dashed line indicates the traditionally recognized medial boundary of the nEAR. B: High power of photomicrograph A. Scale bars 5 100 µm in A,B. is relatively cell sparse and is situated between the pretectal nucleus and the ATh. The anterogradely labeled fibers appear to exit from the CP/PPn and arc around the ATh. Labeled fibers and terminals were observed in a dense plexus, enwrapping the nEAR in a rostromedially situated shell (Fig. 7). Pacemaker nucleus. Along the base of the brain, interlaced with the commissural fibers of the decussation of the praeminential-cerebellar tract, run the fibers afferent to the pacemaker nucleus. These descending fibers project primarily ipsilaterally to the level of the pacemaker nucleus where they appear to coalesce at the midline and sweep dorsally in a tight fascicle flanked by the medial longitudinal fasciculus. These descending fibers enter the pacemaker nucleus from the ventral edge, procuring terminals throughout the entire extent of the midline nucleus (Fig. 8A). The pacemaker nucleus consists of two cell types: smaller, intrinsic pacemaker cells and larger relay cells whose thick diameter axons project out of the nucleus down the spinal cord (reviewed in Dye and Meyer, 1986). Both cell types receive dense innervation from the CP/PPn with clear terminal punctata enveloping the cells (Fig. 8B). Other efferents of CP/PPn. In cases where the injection site was not limited to CP/PPn, the intermediate nucleus of the ventral telencephalon (Vi) also contained anterogradely labeled fibers. Cases in which there was no tracer leakage outside of the CP/PPn, the Vi showed only sparse label. Labeled fibers were occasionally observed in the optic tectum following large injections into the CP/PPn. These labeled fibers in the tectum were generally restricted to the stratum albumen centrale (see Sas and Maler, 1986 for details on tectal organization in gymnotiforms). Anterogradely labeled fibers were also observed in the vicinity of the SPPn in E. virescens; however their source could not be traced specifically to the CP/PPn. Retrograde transport. The relative distribution of efferent cells of the CP/PPn was investigated by placing 32 C.J.H. WONG Fig. 8. Anterograde label in the pacemaker nucleus (Pn). A: Anterograde label in the pacemaker nucleus following a large injection of BDA in the CP/PPn. A single cell in the reticular formation adjacent to the pacemaker nucleus is retrogradely labeled (arrow). B: High-power photomicrograph (oil immersion) of putative anterogradely labeled terminals in the pacemaker nucleus. A pacemaker cell (P) is shown. Same case as A. See list of Abbreviations. Scale bars 5 200 µm in A, 25 µm in B. injections into the pacemaker nucleus (n56), ventral telencephalon (n54), preoptic area (n52), hypothalamus (n51), and optic tectum (n51) for retrograde transport of tracer. Cell bodies originating from the CP/PPn and vicinity are charted in Figure 9. An example of retrogradely labeled neurons, following an injection into the pacemaker nucleus, is charted in Figure 9A. Observed labeled cells were situated within the ventrocaudal regions of the CP/PPn (Fig. 9A). No cells were ever found at the most rostral pole of the CP/PPn where the nucleus forms a shell encapsulating the ATh. In contrast, cell bodies within the CP/PPn that project to the Vv and the Vd were consistently observed within this rostral region (Figs. 9B,C, 10A,B) but not within the more caudolateral region of the nucleus. Two injections were placed in the preoptic area, with one centered in the PPa and one centered more caudally at the boundary of the PPa and the PPp. Figure 9D documents the more caudally centered injection. The neurons that were retrogradely labeled within the CP/PPn were indistinguishable in morphology and distribution from those that project to the ventral telencephalon. This suggests that the same neurons within the CP/PPn may project to both the ventral telencephalon and the preoptic area. Following an injection into the optic tectum retrogradely labeled cells were observed in the CP/PPn (Fig. 9E). Many other nuclei in the vicinity of the CP/PPn also project much more heavily to the optic tectum than does the CP/PPn. These include the dorsal posterior thalamic nucleus (DPn) and the periventricular pretectal nucleus (PTPv). Figure 9F documents an injection centered on but not confined to the nucleus of the lateral recess and the lateral hypothalamus. In this case, large, round retrogradely labeled cells were observed in the CP/PPn (Fig. 10C). The CP/PPn cells that project to the hypothalamus appear to overlap extensively with the pacemaker projecting cells. Intrinsic connections of the CP/PPn The placement of the injection into the CP/PPn was determined by iontophoresis of L-glutamate into the CP/ PPn to stimulate the PPn cells and elicit chirps laterally and gradual rises medially. Following injections that were centered and appeared confined to the lateral portion of the CP/PPn, neurons were always observed in medial and rostral regions of the nucleus. In contrast, following confined, medially centered injections, no cells were observed in the lateral portion of the nucleus although extensive anterogradely labeled fibers were present (Fig. 11A). The CONNECTIONS OF THE ELECTROCOMMUNICATORY SYSTEM location of these fibers and putative terminals were in the vicinity of the prepacemaker cells, in particular the ventral dendritic territory (VT) of the PPn-C cells (Fig. 11B). Moreover, there were no terminals observed in the pacemaker, suggesting that a subset of cells within the medial CP/PPn projects laterally to the pacemaker-projecting cells (Fig. 11B). DISCUSSION The present study provides a fine-grained analysis of the connections of the CP/PPn in E. virescens. This confirms and extends previous connectional (Heiligenberg et al., 1981; Kawasaki et al., 1988; Keller et al., 1990; Johnston and Maler, 1992; Stroh and Zupanc, 1995) and immunohistochemical studies on gymnotiform fishes (see Johnston and Maler, 1992; Zupanc and Maler, 1997, for reviews). The results of the present study are summarized in Figures 12 and 13. The dominant sensory input to the CP/PPn appears to be electrosensory and possibly mechanosensory lateral line from the nE, which relays ascending information from the torus semicircularis (Carr et al., 1981; Keller et al., 1990). Three subdivisions of the nE project to the CP/PPn: the nE>, the nEb, and the nEAR, although this latter pathway appears to be relatively weak. Cells of the CP/PPn project through the medial forebrain bundle to the midline nuclei of the ventral telencephalon (Vv and Vd) and to the preoptic area (PPa, and possibly Ha and PPp). Other intrinsic diencephalic projections are primarily to the hypothalamus (Hl, Hc, and nRLl). Many of these areas also project back to the CP/PPn (Vv, Vd, PPa, Hl, and Hc), suggesting that the CP/PPn may be a critical linkage in the medial forebrain bundle circuit of gymnotiforms. The CP/PPn appears to consist of distinctive subregions with unique inputs and outputs. The rostromedial portion appears to be the major recipient of input from the forebrain, the nEb, and the TAd as well as a major source of afferents to the basal forebrain. This rostromedial portion also projects caudolaterally to the PPn cells, which in turn constitute the major descending output of this complex. The projection to the Vs (Fig. 12) was consistently observed; however, it could not be completely corroborated. As it was possible to place injections in the Vv/Vd, which excluded the Vs, afferent innervation to the Vv from the CP/PPn could be confirmed. In contrast, since it was not possible to place injections in the Vs that excluded the Vv/Vd, it remains unclear as to whether the CP/PPn projects to the Vs. Thalamic connections in teleosts The term CP/PPn was developed to encompass both the CP of the dorsal thalamus, which is a nuclear complex recognized in other teleosts, and the PPn (Zupanc and Heiligenberg, 1992). The present cytoarchitectonic description of the CP/PPn is in agreement with previous descriptions of the CP in other closely related teleosts (Braford and Northcutt, 1983; Striedter, 1990a). Similarly, many of the connections identified in the present study have also been described among other teleost fishes. Comparisons between connections identified in this study and those identified on previous studies on the thalamus in other teleosts will be discussed as telencephalic connections, intrinsic diencephalic connections, and mesencephalic connections. 33 Telencephalic connections. Projections from the thalamus to the ventral telencephalon have been reported among a variety of fishes by both retrograde methods (Sloan, 1989; Striedter, 1990b, 1991; Shiga et al., 1985; Murakami et al., 1986) and anterograde methods (Ito et al., 1986). In the common goldfish (C. auratus) and the channel catfish (I. punctatus), the cell bodies of origin were specifically located in the CP (Sloan, 1989; Striedter, 1990b, 1991). In studies on the other teleosts (Shiga et al., 1985; Murakami et al., 1986), although it is clear that the thalamus projects to the ventral telencephalon, which nucleus of the thalamus that actually projects cannot be readily determined as these investigators used the parcellation scheme of Schnitzlein (1962). This scheme does not demarcate the CP. Nonetheless, thalamic projections to the ventral telencephalon are probably shared among teleost fishes. In contrast, only a single retrograde transport study from the telencephalon (Gnathonemus petersii; Wulliman and Northcutt, 1990) has identified a projection from the CP to the dorsal telencephalon or pallium. Such a projection could not be identified in E. virescens. It is unclear if this represents a real species difference, or if the pallial injection sites in G. petersii encroached onto the subpallium, and thus could account for retrograde label in the CP. Further work on other fishes, with more discrete injections, will be necessary to ascertain if there is phylogenetic variation in a projection from the CP to the pallium. No other study has identified descending projections to the thalamus from the ventral telencephalon, although only a single studied employed relatively discrete injections into the thalamus (Ito et al., 1986). Anterograde transport data supports the presence of these descending connection in E. virescens (unpublished observations). Intrinsic diencephalic circuitry. There have been few studies that specifically examine the intrinsic microcircuitry of the teleost diencephalon (Shiga et al., 1985; Sloan, 1989; Keller et al., 1990; Wulliman and Northcutt, 1990; Striedter, 1991, 1992; Lamb and Caprio, 1993; Wulliman and Roth, 1994). Similar to projections to the ventral telencephalon, thalamic projections to the preoptic area have also been described (Shiga et al., 1985; Sloan, 1989) and it is likely that thalamic projections to the preoptic area may also be shared among teleosts. Projections from the CP to the TA have been described previously in the channel catfish (Striedter, 1991) and in E. virescens (Keller et al., 1990). Reciprocal connections between the CP/PPn and other hypothalamic nuclei had also been suggested by previous immuohistochemical studies on both E. virescens and A. leptorhynchus (Sas and Maler, 1991; Zupanc et al., 1991; Weld and Maler, 1992; Yamamato et al., 1992; Dulka et al., 1995; Richards and Maler, in press). It is unknown whether these other hypothalamic nuclei are connected with the thalamus among non-gymnotiform fishes. In contrast to a previous in vitro tracing study on A. leptorhynchus (Striedter, 1992), no connection was observed between the CP/PPn and the preglomerular complex in E. virescens (this study) or A. leptorhynchus (unpublished observations). As the efferents of the CP/PPn that project to the lateral hypothalamus clearly pass near the preglomerular complex, inadvertent labeling might explain Striedter’s findings. Indeed, in the channel catfish, a connection between the CP and the preglomerular complex was not observed (Striedter, 1991, 1992). 34 C.J.H. WONG Fig. 9. A–F: Retrograde confirmation of efferents of the CP/PPn. Left hand side indicates center and approximate extent of injections (black area in left hand transverse section) yielding retrograde labeled cells (open circles) within the CP/PPn and vicinity. Each dot indicates a single cell labeled in that section. Every other section is presented corresponding to approximately the same rostral-caudal levels shown in Figure 1. See text for details. Scale bars 5 500 µm for injection sites and 250 µm for chartings of labeled cells in the CP/PPn. Previous retrograde studies have suggested that the CP may project to the pituitary in both goldfish (Johnston and Fryer, 1990) and A. leptorhynchus (Johnston and Maler, 1992). Such a projection was not observed in the present study, except in a single case: a large injection of BDA to the CP/PPn. However, even after this large injection, only a single fiber was observed in the pituitary. The present study suggests that any direct influence from the CP/PPn on the pituitary in E. virescens is minor. Mesencephalic connections. In A. leptorhynchus, a prominent projection was observed from cells of the CP/ PPn to the SPPn (Heiligenberg et al., 1996). In E. vire- scens, such a projection could not be readily identified. In the present study, a few anterogradely labeled fibers were observed in the vicinity of the SPPn, following injections into the CP/PPn of E. virescens. However, their source of origin could not be readily determined as originating from the CP/PPn. The fiber tract, linking the CP/PPn and the SPPn, which was clearly observed in A. leptorhynchus, was not present in E. virescens and hence this may represent a species-specific difference. A projection was observed from the CP to the optic tectum in the channel catfish (Striedter, 1990b) and from the CP/PPn in E. virescens (this study). However, input from the CP/PPn to the optic tectum would CONNECTIONS OF THE ELECTROCOMMUNICATORY SYSTEM Figure 9 appear to be relatively minor in comparison to projections originating from other diencephalic nuclei. Conspicuously absent in E. virescens was a connection between the CP/PPn and the torus semicircularis. Previous anatomical (Echteler, 1984; Striedter, 1991) and physiological studies (Echteler, 1985; Lu and Fay, 1995) on other teleosts have suggested that there is a direct reciprocal connection between the midbrain auditory nucleus in the torus semicircularis and the CP. The ascending portion of this pathway may be homologous to the auditory pathway through the dorsal thalamus of other vertebrates (see McCormick, 1992, for a review). Large injections of either Neurobiotin or BDA into the CP/PPn failed to label cells or terminals within either the ventral or dorsal subdivisions 35 (Continued.) of the torus semicircularis, except when there were instances of leakage along the electrode tract into the torus. Such leakage was accompanied by labeled fibers in the lateral lemniscus. Similarly, discrete injections of tracer into either the ventral or dorsal subdivisions of the torus semicircularis also failed to anterogradely label terminals within the CP/PPn although other diencephalic fields were clearly labeled (C.J.H. Wong, unpublished observations). These data suggest that linkages between the torus semicircularis and the CP/PPn in gymnotiforms are indirect, mediated through the nE, and are not direct as in other teleosts. The connections of the medial CP/PPn of E. virescens are very similar to those of CP of other teleosts. Based on topology, cytoarchitecture, and connections, these 36 C.J.H. WONG Fig. 10. A: Photomicrograph of the injection site centered at the interface of the ventral telencephalon, ventral subdivision (Vv) and the dorsal nucleus of the ventral telencephalon (Vd), depicted in Figure 9B. B: Example of retrogradely labeled neurons in the CP/PPn from the injection in A. C: Example of retrogradely labeled neurons in the CP/PPn from the injection centered on but not confined to the lateral nucleus of the lateral recess (shown in Fig. 9F). Scale bars 5 200 µm in A, 100 µm for B,C. areas are clearly homologous. One of the major efferent targets of the medial CP/PPn are the PPn cells (this study) that control EOD modulations for conspecific communication (Kawasaki et al., 1988). Previously, it has been hypothesized that the dorsal thalamus may subserve motor circuits for reproductive behaviors in other fishes (Demski and Dulka, 1986; Zupanc and Heiligenberg, 1992). In A. leptorhynchus, most of the CP/PPn cells that actually project to the pacemaker are in fact somatostatinergic (Stroh and Zupanc, 1995). Examination of both the projection patterns and chemical identity of cells in the dorsal thalamus of other teleosts may be instructive in determining if the pacemaker-projecting component of the CP/PPn of gymnotiforms has a homologue in other fishes. E. virescens, although it is not necessary for the JAR of A. leptorhynchus (Heiligenberg et al., 1996). The nEb is also retrogradely labeled from injections into the CP/PPn confirming previous tract tracing (Keller et al., 1990) and intracellular labeling (Heiligenberg et al., 1991) studies. The cells of the nEb project to rostromedial areas of the CP/PPn. Neurons of the nEb have been recorded from that respond to beat modulations in electrosensory input. These would arise from interference of a neighboring fish’s EOD (Keller, 1988; Heiligenberg et al., 1991). Although the nEb is not necessary for the JAR, it may be used for recognition and detection of conspecific EOD’s (Heiligenberg et al., 1991). The nEAR provides a weak projection to the CP/PPn. This region of the nE region receives mechanosensory and ampullary electrosensory input from the ventral torus semicircularis (Keller et al., 1990; Heiligenberg et al., 1991; see Metzner and Viete, 1996, for review). The projection from the nEAR to the CP/PPn would appear to be relatively minor in comparison to inputs from other nE subdivisions to the CP/PPn. The nE may also offer indirect pathways to the CP/PPn. One such pathway may pass through the anterior tuberal nucleus of the hypothalamus. One of the major efferent targets of the nEb is the TAd (Keller et al., 1990; Heiligenberg et al., 1991), which is also reciprocally connected with Functional considerations Parallel pathways from the nucleus electrosensorius to CP/PPn. The present study identifies at least five parallel pathways which may subsequently relay information from the nE to the CP/PPn (see Fig. 13). The most prominent subnucleus projecting to the CP/ PPn is from the most caudal subdivision of the nE, the nE> (Bastian and Yuthas, 1984; Keller, 1988; Keller and Heiligenberg, 1989; Keller et al., 1990). This confirms previous tract-tracing studies by Keller et al. (1990). This pathway appears to be necessary for the JAR (Metzner, 1993) of CONNECTIONS OF THE ELECTROCOMMUNICATORY SYSTEM 37 Fig. 11. A: Anterogradely labeled fibers in the caudal portion of the CP/PPn arising from a medially and rostrally placed injection (same case as depicted in Fig. 4, 150 µm caudal to the section in Fig. 4). B: Retrogradely labeled neurons in the CP/PPn from an injection into the pacemaker nucleus. Note that in A, there are very few labeled neurons though extensive fibers and putative terminals in the lateral region where the prepacemaker neurons typically label. See text for details. See List of Abbreviations. Scale bar 5 100 µm for A,B. the CP/PPn (Keller et al., 1990; this study). Further physiological investigation of the TAd will be necessary to examine the function of this pathway. A second indirect pathway may pass through the inferior lobe. Intracellular recording from cells within the nEAR has shown that there are combination sensitive units that fire preferentially to mimics of chirps (Heiligenberg et al., 1991). Single cell intracellular labeling as well as discrete extracellular injections have revealed that among the efferent targets for some of these chirp-sensitive cells is the central nucleus of the inferior lobe (Keller et al., 1990; Heiligenberg et al., 1991). As cells within the CE retrogradely label from the CP/PPn, this may represent another link, relaying sensory chirp information. In addition to the various ascending pathways to the CP/PPn from the nE, there may be a feedback pathway present. The chirp-sensitive cells in the PT/nEAR have extensive dendrites that appear to extend into the neuropil medial to the nEAR (Heiligenberg et al., 1991). As the CP/PPn projects into this area, there might be contact between the cells in the CP/PPn which generate chirps and the nEAR cells which detect them. Links with reproductive centers. The cells of the CP/PPn are reciprocally connected with cell groups of the basal forebrain, preoptic area, and hypothalamus. These regions have been implicated in reproductive behaviors and the neuroendocrine control of reproduction in teleosts as well as other vertebrates (see Demski, 1984, for a review). For example, in goldfish, a direct preopticospinal pathway that mediates gamete release has been identified (Demski and Sloan, 1985; Sloan, 1989). Furthermore, gonadotropin-releasing hormone (GnRH) immunoreactive somata have been identified in the Vv and the PPa among a variety of teleosts (Demski, 1984). In addition, many nuclei that are in receipt of input from CP/PPn (Vv, PPa, PPp, Hl, and Hc) also have cells that project to the pituitary (Johnston and Maler, 1992). Besides receiving input from the CP/PPn, the Vv, Vs and PPa also receive input from the medial olfactory bulb (Bass, 1981; Levine and Dethier, 1985; Sas et al., 1993). In other teleost fishes, there is evidence that the medial olfactory bulb may encode sex pheromonal information (see Dulka, 1993 for review). It will of interest to examine if gymnotiforms utilize chemical cues in addition to electrosensory signals in their reproductive behavior. In addition to anatomical evidence, both behavioral and physiological data support a role for many of these regions in reproductive behaviors. Lesions of the Vs and Vv reduce reproductive behavior in goldfish (Kyle and Peter, 1982; Kyle et al., 1982). Similarly, stimulation of the ventral 38 Fig. 12. Schematic side view of the gymnotiform brain displaying connections with the CP/PPn; those identified in the present study are indicated by bold lines. Double asterisks (**) indicates that this nucleus is an efferent target of the nE. Minor connections (e.g., tectum, raphé, etc.) are not presented in this summary for clarity. Dashed lines indicate connections for which there is physiological evidence (sublem- C.J.H. WONG niscal prepacemaker nucleus, SPPn) or which is suggested (supracommisural nucleus of the ventral telencephalon, Vs) by the presence of anterogradely labeled fibers and terminals yet which could not be confirmed by retrograde transport. The arrow within the CP/PPn demarcates connectional differences between the rostromedial regions and the caudolateral regions of this nucleus. See text for details. telencephalon, preoptic area, and hypothalamus in a variety of teleosts can elicit reproductive behaviors, including gamete release, nest-building, as well as a host of speciesspecific communicatory signals (Demski and Knigge 1971; Satou et al., 1984; Fine and Perini, 1994; reviewed in Demski, 1983). Preliminary results suggest that electrical stimulation of these brain areas in E. virescens elicits EOD modulations (C.J.H. Wong, unpublished observations), implicating a role of these regions in electrocommunication. Comparisons with other vertebrates Fig. 13. Possible parallel pathways through the diencephalon, which may relay ascending electrosensory information from the torus, through the nucleus electrosensorius (thick lines) to the CP/PPn. Lines in grey are those connections primarily associated with the medial portion of the CP/PPn. The thick dashed line indicates a putative feedback connection as the axons originating from the CP/PPn may be contacting medially directed dendrites of the cells of the nEAR. Thin dashed line indicates a very weak connection from the nEAR to the CP/PPn. See List of Abbreviations. Interestingly, the connections identified for the CP/PPn appear to be similar to those identified for the proposed homologue in anuran amphibians: the central thalamic nucleus (Neary and Northcutt, 1983; Northcutt, 1995). Unfortunately, as detailed data is lacking to adequately compare all connections of the CP/PPn with the CP among non-teleost fishes (see Northcutt, 1995), it is too early to determine the primitive condition of the octavolateralis pathways in anamniotes. One may, however, put forward hypotheses based on comparisons between the CP/PPn of E. virescens and the central thalamic nucleus of frogs. These hypotheses will become testable as more data is gathered among other classes of fishes. The ascending electrosensory pathway from the CP/PPn to the telencephalon appears to be similarly organized to auditory pathways in anuran amphibians. The central nucleus of frogs, which receives direct input from the auditory torus, is reciprocally connected with the septum and projects to the striatum (see Neary, 1990; Northcutt, CONNECTIONS OF THE ELECTROCOMMUNICATORY SYSTEM 1995, for reviews). Although still somewhat contentious, homologues for telencephalic areas in receipt of input from the CP/PPn are lateral septum and nucleus accumbens for the Vv and striatum for the Vd (see Northcutt, 1995, for review). Also similar to what has been identified in E. virescens, the hypothalamus and preoptic area may receive input from the central nucleus in anuran amphibians (Hall and Feng, 1987; Allison and Wilczynski, 1991; Neary, 1995). Therefore, pending examination of other fishes, it would appear that connections with septal and striatal areas of the telencephalon, as well as the preoptic area and hypothalamus may be primitive for the dorsal thalamic octavolateralis nucleus in anamniotes. Owing to the tremendous expansion of the dorsal thalamus between amniotic vertebrates versus anamniotic vertebrates (Butler, 1994), direct comparisons between their individual thalamic nuclei are complicated. Nonetheless, it is likely that the central nucleus of anuran amphibians and hence CP/PPn may be homologized to at least some portions of the auditory thalamus in amniotes. Projections to subpallial areas of the telencephalon and the hypothalamus from dorsal thalamic auditory nuclei have also been described in birds (Durand et al., 1992; Cheng and Zuo, 1994) and mammals (LeDoux et al., 1985; see Frost and Masterton, 1992, for review). These may be homologous to the pathways discussed above in frogs and in E. virescens. All these pathways appear similar in serving reproductive/ autonomic functions (LeDoux et al., 1985; Kawasaki et al., 1988; Allison, 1992; Wilczynski et al., 1993; Cheng and Zuo, 1994), suggesting that this might be a primitive functional feature of dorsal thalamic octavolateralis pathways. ACKNOWLEDGMENTS I thank Dr. R.G. Northcutt for helpful guidance and discussions throughout on this project and for comments on an earlier draft of this manuscript. Additional thanks to Dr. T.H. Bullock, Dr. J.B. Graham, the SIO Director’s Office, the NIH, and the NIMH, for allowing the Heiligenberg lab to remain open for the completion of this work. Grace Kennedy provided excellent technical assistance. Dr. L. Maler, Dr. J.C. Prechtl, and Dr. G.K.H. Zupanc provided helpful discussions during their tenure as visiting scholars to the Heiligenberg lab. I gratefully acknowledge C.B. Braun, Dr. T.H. Bullock, Dr. J.G. Dulka, Dr. C.H. Keller, Dr. L. Maler, K.T. Moortgat, M.-S. 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