Neural coding of difference frequencies in the midbrain of the electric fishEigenmannia: Reading the sense of rotation in an amplitude-phase plane

Eigenmannia is able to discriminate the sign of the difference, Df, between the frequency of a neighbor's electric organ discharge (EOD) and that of its own EOD. This discrimination can be demonstrated at the level of individual neurons of the midbrain. Intracellular and extracellular recordings of such sign-selective cells revealed the following: Units preferring positive Dfs and units preferring negative Dfs were found with equal frequency. The degree of selectivity was also similar for these two classes of neurons. All sign-selective units were sensitive to the magnitude of the frequency difference, i.e. the beat rate. Most units responded best to beat rates in the 4-8 Hz range. Sign-selectivity was observed only when the jamming signal (S2) was presented through electrodes other than those used to deliver the mimic (S1) of the fish's EOD, i.e. only when amplitude modulations were accompanied by modulations of differential phase. Intracellular studies suggest that most sign-selective neurons of the tectum are large, multipolar cells in the stratum album centrale. These cells send projections to the reticular formation, to lamina 9 of the torus semicircularis and to the N. electrosensorius.     

Sex recognition and neuronal coding of electric organ discharge waveform in the pulse-type weakly electric fish, Hypopomus occidentalis

1. Hypopomus occidentalis, a weakly electric gymnotiform fish with a pulse-type discharge, has a sexually dimorphic electric organ discharge (Hagedorn 1983). The electric organ discharges (EODs) of males in the breeding season are longer in duration and have a lower peak-power frequency than the EODs of females. We tested reproductively mature fish in the field by presenting electronically generated stimuli in which the only cue for sex recognition was the waveshape of individual EOD-like pulses in a train. We found that gravid females could readily discriminate male-like from female-like EOD waveshapes, and we conclude that this feature of the electric signal is sufficient for sex recognition. 2. To understand the possible neural bases for discrimination of male and female EODs by H . occidentalis, we conducted a neurophysiological examination of both peripheral and central neurons. Our studies show that there are sets of neurons in this species which can discriminate male or female EODs by coding either temporal or spectral features of the EOD. 3. Temporal encoding of stimulus duration was observed in evoked field potential recordings from the magnocellular nucleus of the midbrain torus semicircularis. This nucleus indirectly receives pulse marker electroreceptor information. The field potentials suggest that comparison is possible between pulse marker activity on opposite sides of the body. 4. From standard frequency-threshold curves, spectral encoding of stimulus peak-power frequency was measured in burst duration coder electroreceptor afferents. In both male and female fish, the best frequencies of the narrow-band population of electroreceptors were lower than the peak-power frequency of the EOD. Based on this observation, and the presence of a population of wide-band receptors which can serve as a frequency-independent amplitude reference, a slope-detection model of frequency discrimination is advanced. 5. Spectral discrimination of EOD peak-power frequency was also shown to be possible in a more natural situation similar to that present during behavioral discrimination. As the fish's EOD mimic slowly scanned through and temporally coincided with the neighbor's EOD mimic, peak spike rate in burst duration coder afferents was measured. Spike rate at the moment of coincidence changed predictably as a function of the neighbor's EOD peak-power frequency. 6. Single-unit threshold measurements were made on afferents from peripheral burst duration coder receptors in the amplitude-coding pathway, and midbrain giant cells in the time-coding pathway.(ABSTRACT TRUNCATED AT 400 WORDS)     

‘Recognition units’ at the top of a neuronal hierarchy?

The electric fish, Eigenmannia, is able to discriminate the sign of the frequency difference, Df, between a neighbor's electric organ discharges (EODs) and its own. The fish lowers its EOD frequency for positive Dfs and raises its frequency for negative Dfs to minimize jamming of its electrolocation ability by a neighbor's EODs of similar frequency. This jamming avoidance response (JAR) is controlled by a group of 'sign-selective' neurons in the prepacemaker nucleus (PPN) that is located at the boundary of the midbrain and the diencephalon (Fig. 1). Extracellular recordings from a total of 35 neurons revealed a great similarity between behavioral and neuronal response properties: 1. All neurons fired vigorously for negative Dfs and were almost silent for positive Dfs, regardless of the orientation of the jamming stimulus, and thus discriminated the sign of Df unambiguously (Fig. 2). 2. In accordance with behavioral observations, individual neurons failed to discriminate the sign of Df when the jamming stimulus had the same field geometry as the signal mimicking the animal's own EOD (Fig. 3). 3. Df magnitudes which evoke strongest JARs, usually 4 to 8 Hz, also induced most vigorous responses in sign-selective neurons (Fig. 5). 4. Behavioral and neuronal thresholds for the detection of small jamming signals were similar. Threshold for sign selectivity was reached when the amplitude ratio of the jamming signal to the EOD mimic, measured near the head surface, was 0.001. This value corresponds to a maximal temporal disparity (a necessary cue for performing a correct JAR) of 1 to 2 microseconds for signals received by the two sides of the body in a transverse jamming field (Fig. 7). 5. The effects of two jamming fields, offered orthogonally to each other, may interact nonlinearly at the behavioral as well as at the neuronal level. A positive Df presented in one field may suppress behavioral and neuronal responses to modulations of the sign of Df in the other field (Fig. 8c).     

The coding of signals in the electric communication of the gymnotiform fish Eigenmannia: From electroreceptors to neurons in the torus semicircularis of the midbrain

Summary In the context of aggression and courtship, Eigenmannia repeatedly interrupts its electric organ discharges (EODs) These interruptions (Fig. 1) contain low-frequency components as well as high-frequency transients and, therefore, stimulate ampullary and tuberous electroreceptors, respectively (Figs. 2, 3). Information provided by these two classes of receptors is relayed along separate pathways, via the electrosensory lateral line lobe (ELL) of the hindbrain, to the dorsal torus semicircularis (TSd) of the midbrain. Some neurons of the torus receive inputs from both types of receptors (Figs. 14, 15), and some respond predominantly to EOD interruptions while being rather insensitive to other forms of signal modulations (Figs. 12, 13). This high selectivity appears to result from convergence and gating of inputs from individually less selective neurons.Abbreviations CP central posterior thalamic nucleus - Df frequency difference between neighbor's EOD and fish's own - DPn dorsal posterior nucleus (thalamus) - EOD electric organ discharge - ELL electrosensory lateral line lobe - JAR jamming avoidance response - LMR lateral mesencephalic reticular formation - nE nucleus electrosensorius - nEb nucleus electrosensorius, beat-related area - nE nucleus electrosensorius, area causing rise of EOD frequency - nE nucleus electrosensorius, area causing fall of EOD frequency - nEar nucleus electrosensorius-acusticolateralis area - NPd nucleus praeeminentialis, pars dorsalis - PPn prepacemaker nucleus - PT pretectal nucleus - SE nucleus subelectrosensorius - TeO optic tectum - TSd dorsal (electrosensory) torus semicircularis - TSv ventral (mechano-sensory and auditory) torus semicircularis     

Differential distribution of ampullary and tuberous processing in the torus semicircularis of Eigenmannia

Summary Gymnotiform electric fish sense low-and high frequency electric signals with ampullary and tuberous electroreceptors, respectively. We employed intracellular recording and labeling methods to investigate ampullary and tuberous information processing in laminae 1–5 of the dorsal torus semicircularis of Eigenmannia. Ampullary afferents arborized extensively in laminae 1–3 and, in some cases, lamina 7. Unlike tuberous afferents to the torus, ampullary afferents had numerous varicosities along their finest-diameter branches. Neurons that were primarily ampullary were found in lamina 3. Neurons primarily excited by tuberous stimuli were found in lamina 5 and, more rarely, in lamina 4. Cells that had dendrites in lamina 1–3 and 5 could be recruited by both ampullary and tuberous stimuli. These bimodal cells were found in lamina 4. During courtship, Eigenmannia produces interruptions of its electric organ discharges. These interruptions stimulate ampullary and tuberous receptors. The integration of ampullary and tuberous information may be important in the processing of these communication signals.Abbreviations JAR jamming avoidance response - EOD electric organ discharge - S1 sinusoidal signal mimicking fish's EOD - S2 jamming signal - Df frequency difference (S2-S1) or between a neighbor's EODs and fish's own EODs - CNS central nervous system     

Comparison of calbindin D 28K and cytochrome c oxidase in electrosensory nuclei of high- and low-frequency weakly electric fish (Gymnotiformes)

Summary Weakly electric fish (Gymnotiformes) emit quasi-sinusoidal electric organ discharges within speciesspecific frequency ranges. The electrosensory system is organized into 2 parallel pathways which convey either the amplitude or the timing of each electric organ discharge cycle. Two putative metabolic activity markers, calbindin D 28K and cytochrome c oxidase, and their relationship with the electrosensory nuclei of high- and low-frequency species were studied. Calbindin is found in the somata of the spherical neurons in the first-order electrosensory recipient nucleus, the electrosensory lateral-line lobe, and in layer VI of the midbrain's torus semicircularis, in Eigenmannia virescens, an intermediate-frequency species, and Apteronotus leptorhynchus, a high-frequency species. Calbindin immunoreactivity was completely absent in these nuclei in Sternopygus macrurus, a closely related, low-frequency species. Cytochrome c oxidase levels were inversely related to calbindin immunoreactivity since relatively high levels were observed in the electrosensory lateral-line lobe and torus semicircularis of S. macrurus but were absent in these nuclei in A. leptorhynchus. Our studies indicate that calbindin immunoreactivity is present in tonic, repetitively firing neurons with high frequencies.     

A role of burst firings in encoding of spatiotemporally-varying stimulus

To investigate a role of burst firings of neurons in encoding of spatiotemporally-varying stimulus, we focus on electrosensory system of a weakly electric fish. Weakly electric fish generates electric field around its body using electric organ discharge and can accurately detect the location of an object using the modulation of electric field induced by the object. We developed a model of fish body by which we numerically describe the spatiotemporal patterns of electric field around the fish body. We also made neural models of electroreceptor distributed on the fish body and of electrosensory lateral-line lobe (ELL) to investigate what kinds of information of electric field distorted by an object they detect. Here we show that the spatiotemporal features of electric field around the fish body are encoded by the timing of burst firings of ELL neurons. The information of object distance is extracted by the area of synchronous firings of neurons in a higher nucleus, torus semicircularis.     

GABAergic inhibition shapes frequency tuning and modifies response properties in the auditory midbrain of the leopard frog

The functional role of GABAergic inhibition in shaping the frequency tuning of 96 neurons in the torus semicircularis of the leopard frog, Rana pipiens, was studied using microiontophoresis of the GABA_A receptor antagonist, bicuculline methiodide. Bicuculline application abolished, or reduced in size, the inhibitory tuning curves of 72 neurons. In each case, there was a concommitant broadening of the excitatory tuning curve such that frequency-intensity combinations that were inhibitory under control conditions, became excitatory during disinhibition with bicuculline methiodide. These effects were observed irrespective of the excitatory tuning curve configuration prior to bicuculline methiodide application. Results indicate an important role for GABA-mediated inhibition in shaping the frequency selectivity of neurons in the torus semicircularis of the leopard frog. Bicuculline application also affected several other response properties of neurons in the leopard frog torus. Disinhibition with bicuculline methiodide increased both the spontaneous firing rate (18 cells) and stimulus-evoked discharge rate (81 cells) of torus neurons, decreased the minimum excitatory threshold for 18 cells, and altered the temporal discharge pattern of 47 neurons. Additional roles for GABAergic inhibition in monaural signal analysis are discussed. Accepted: 25 August 1999     

Responses of auditory brainstem neurons in the grassfrog to clicks

Single unit recordings were made in the dorsal medullary nucleus and in the torus semicircularis of the immobilized grassfrog. The natural calls have a periodic pulsatile structure. To investigate the coding of pulse repetition rate periodic click trains with varying pulse repetition rate and an ensemble of clicks distributed randomly in time were used as stimuli. In the dorsal medullary nucleus strong time-locking to clicks was found. Most units showed an activation followed by suppression response. Some units showed a preference for pulse repetition rates matching their low-frequency sensitivity. In the torus semicircularis part of the units showed responses similar to dorsal medullary nucleus units. Other response types were activation irrespective of pulse repetition rate, and suppression followed by activation. The responses to the two stimulus ensembles were more compatible in the dorsal medullary nucleus than in the torus semicircularis.     

Influence of descending forebrain projections on processing of acoustic signals and audiomotor integration in the anuran midbrain

We tested the role of descending projections for auditory processing and audiomotor integration in the anuran torus semicircularis. Intracellular recordings were made from isolated brain preparations, impaled neurons were stained. Auditory neurons responded to electrical stimulation of striatum and/or dorsal thalamus, they integrated forebrain and auditory nerve inputs. High frequency stimulation in striatum or thalamus changed the auditory response of torus neurons located in the laminar subnucleus. Our results suggest that the laminar nucleus is the primary target of forebrain projections, which provides a basis for modulation of acoustically guided behaviour.     

The torus semicircularis in a gekkonid lizard

The cytoarchitecture and neuromorphology of the torus semicircularis in the tokay gecko, Gekko gecko, were examined in Nissl-stained, fiber-stained, and Golgi-impregnated tissues. From a superficial position, the torus semicircularis extends rostrally under the caudal half of the optic tectum. Caudally, the two tori abut upon one another; rostrally, they diverge. The torus semicircularis consists of central, laminar, and superficial nuclei. The central nucleus consists of fusiform, spherical and triangular neurons. Their dendrites are highly branched, with numerous dendritic spines, and are oriented mediolaterally, dorsoventrally, and rostrocaudally. Fusiform and spherical neurons display two dendritic patterns: “single axis,” ramifying in one axis, and “dual axis,” exhibiting higher-order branches perpendicular to the primary dendrites. Triangular neurons exhibit a “radiate” dendritic pattern. In the rostral half of the torus semicircularis, the laminar nucleus caps the central nucleus. The laminar nucleus encircles the central nucleus in the caudal torus semicircularis. The neurons of the laminar nucleus have dendritic arrays oriented parallel to the border of the central nucleus. These dendrites exhibit a paucity of dendritic spines and higher-order branches. Fusiform and spherical neurons exhibit “single axis” and “dual axis” dendritic patterns. Triangular neurons display “radiate” patterns. The caudal superficial nucleus lies dorsal and dorsolateral to the central nucleus. The superficial nucleus is sparsely populated by small fusiform and spherical neurons with moderately branched dendrites and moderate numbers of dendritic spines. These neurons display “single axis” (fusiform neurons) as well as “dual axis” and “radiate” (spherical neurons) dendritic patterns. They are oriented either parallel to or perpendicular to the boundary of the laminar nucleus.     

Seasonal changes in frequency tuning and temporal processing in single neurons in the frog auditory midbrain

Frogs rely on acoustic signaling to detect, discriminate, and localize mates. In the temperate zone, reproduction occurs in the spring, when frogs emerge from hibernation and engage in acoustically guided behaviors. In response to the species mating call, males typically show evoked vocal responses or other territorial behaviors, and females show phonotactic responses. Because of their strong seasonal behavior, it is possible that the frog auditory system also displays seasonal variation, as evidenced in their vocal control system. This hypothesis was tested in male Northern leopard frogs by evaluating the response characteristics of single neurons in the torus semicircularis (TS; a homolog of the inferior colliculus) to a synthetic mating call at different times of the year. We found that TS neurons displayed a seasonal change in frequency tuning and temporal properties. Frequency tuning shifted from a predominance of TS units sensitive to intermediate frequencies (700-1200 Hz) in the winter, to low frequencies (100-600 Hz) in the summer. In winter and early spring, most TS neurons showed poor, or weak, time locking to the envelope of the amplitude-modulated synthetic call, whereas in late spring and early summer the majority of TS neurons showed robust time-locked responses. These seasonal differences indicate that neural coding by auditory midbrain neurons in the Northern leopard frog is subject to seasonal fluctuation.     

The jamming avoidance response ofEigenmannia: properties of a diencephalic link between sensory processing and motor output

Summary Brain regions participating in the control ofEigenmannia's electric organ discharge frequency were localized by electrical microstimulation and anatomically identified by means of horseradish peroxidase deposition. A diencephalic region was found which, when stimulated, caused electric organ discharge (EOD) frequency increases of similar magnitude and time course as the frequency increases seen during the jamming avoidance response. Single unit recordings from this region revealed one cell type which preferentially responded to stimuli that cause the acceleration phase of the jamming avoidance response (electric organ discharge frequency increase). A second cell type responded preferentially to stimuli which cause EOD frequency decrease, and both cell types were tuned to stimuli which evoked maximal jamming avoidance behaviors.The results of the horseradish peroxidase experiments showed that the recording and stimulation sites correspond to the previously described nucleus electrosensorius. Our results confirm the earlier finding that this nucleus receives output from the torus semicircularis and we also found that the N. electrosensorius projects to the mesencephalic prepacemaker nucleus. The prepacemaker projects to the medullary pacemaker nucleus which generates the commands that evoke electric organ discharges.The anatomical and physiological results described here establish this diencephalic region as a link between the major sensory processing region for the jamming avoidance response, the torus semicircularis, and a mesencephalic pre-motor region, the prepacemaker nucleus.Abbreviations AM amplitude modulation - DF Delta F - ELLL electrosensory lateral line lobe - EOD electric organ discharge - JAR jamming avoidance response - NE nucleus electrosensorius - PPN prepacemaker nucleus - PN pacemaker nucleus     

Temporal selectivity by single neurons in the torus semicircularis of Batrachyla antartandica (Amphibia: Leptodactylidae)

We investigated the response selectivities of single auditory neurons in the torus semicircularis of Batrachyla antartandica (a leptodactylid from southern Chile) to synthetic stimuli having diverse temporal structures. The advertisement call for this species is characterized by a long sequence of brief sound pulses having a dominant frequency of about 2000 Hz. We constructed five different series of synthetic stimuli in which the following acoustic parameters were systematically modified, one at a time: pulse rate, pulse duration, pulse rise time, pulse fall time, and train duration. The carrier frequency of these stimuli was fixed at the characteristic frequency of the units under study (n=44). Response patterns of TS units to these synthetic call variants revealed different degrees of selectivity for each of the temporal variables. A substantial number of neurons showed preference for pulse rates below 2 pulses s(-1), approximating the values found in natural advertisement calls. Tonic neurons generally showed preferences for long pulse durations, long rise and fall times, and long train durations. In contrast, phasic and phasic-burst neurons preferred stimuli with short duration, short rise and fall times and short train durations.     

Stimulus discrimination in the diencephalon ofEigenmannia: the emergence and sharpening of a sensory filter

Summary Neuronal reliability and sensitivity to behaviorally relevant stimulus patterns were investigated in a higher-order nucleus of the diencephalon believed to participate in the jamming avoidance response (JAR) of the weakly electric fish,Eigenmannia. The fish raises or lowers its frequency of electric organ discharge (EOD) to minimize interference from a neighboring fish's EOD. Proper JARs require determination of the sign of the difference frequency (Df) between the neighboring fish's EOD and the fish's own EOD. Bastian and Yuthas (1984) recently described diencephalic neurons within the nucleus electrosensorius that are able to make this determination. In the present study, response properties of such neurons were compared with those of lower-level sign-selective cells found in the torus semicircularis and the optic tectum (Heiligenberg and Rose 1985) as well as with properties of the intact behavior.Most sign-selective cells within the nucleus electrosensorius show a high degree of selectivity for one sign of the difference frequency; cells with either sign preference were found in approximately equal numbers. The sign preference and the degree of sign selectivity is most often independent of the spatial orientation of the jamming stimulus. In contrast, the responses of toral and tectal cells are less robust and consistent and are often highly dependent on the geometry of the jamming stimulus.Determination of the sign of the difference frequency requires the analysis of amplitude modulations coupled with modulations in phase (timing) differences between pairs of areas of the body surface. The most sensitive cells recorded in the nucleus electrosensorius can determine the sign of the difference frequency with timing differences of 1 s or less, roughly comparable to the behavioral threshold of 400 ns (Carr et al. 1986). The best toral/tectal response required at least a 16 s modulation.Cells within the nucleus electrosensorius thus code the sign of Df with a high degree of reliability and sensitivity. Ambiguities persist, however, which suggest that single cells at this level cannot completely account for the behavioral discrimination. Additional processing may be necessary to transform a still primarily sensory code into a motor program for control of the JAR (Rose et al. 1988).Abbreviations EOD electric organ discharge - JAR jammning avoidance response - Df difference frequency between jamming signal and fish's own EOD - S ₁ sinusoidal EOD mimic of subject fish - S ₂ sinusoidal EOD mimic of neighbor     

Adaptation of differential sensitivity of auditory system neurons to amplitude modulation after abrupt change of signal level

Impulse activity of neurons of brainstem auditory nuclei (medulla dorsal nucleus and midbrain torus semicircularis) of the grass frog (Rana temporaria) was recorded under action of long amplitude-modulated tonal signals. After adaptation of neuronal response to acting stimulus (30–60 s after its onset), we performed a sharp change (by 20–40 dB) of the mean signal level with preservation of unchanged frequency and depth of modulation. We also recorded a change of density impulsation and of degree of its synchronization with the modulation period as well as the phase of maximum reaction at the modulation period and phase of the response every 2 or 4 s. In the adapted state, the sharp change of the mean level had been provided, while maintaining frequency and depth unchanged. During the adaptation to long signals with small modulation indexes the firing rate continuously decreased, but synchronization with envelope usually increased considerably. A sharp rise in the mean level resulted in an increase of firing rate, which could be accompanied either by a continuation of synchronization growth (the effect is more typical of the dorsal nucleus) or by a sharp fall in synchrony with its subsequent slow recovery (the effect is more typical of the torus semicircularis). Nature of the changes following the change of the intensity of the reaction could depend on the signal parameters (initial level, magnitude of the jump, frequency and depth of modulation). The connection between the observed physiological data and the psychophysics of differential intensity coding is discussed.     

Gonadotropin-releasing hormone (GnRH) modulates auditory processing in the fish brain

Gonadotropin-releasing hormone 1 (GnRH1) neurons control reproductive activity, but GnRH2 and GnRH3 neurons have widespread projections and function as neuromodulators in the vertebrate brain. While these extra-hypothalamic GnRH forms function as olfactory and visual neuromodulators, their potential effect on processing of auditory information is unknown. To test the hypothesis that GnRH modulates the processing of auditory information in the brain, we used immunohistochemistry to determine seasonal variations in these neuropeptide systems, and in vivo single-neuron recordings to identify neuromodulation in the midbrain torus semicircularis of the soniferous damselfish Abudefduf abdominalis. Our results show abundant GnRH-immunoreactive (-ir) axons in auditory processing regions of the midbrain and hindbrain. The number of extra-hypothalamic GnRH somata and the density of GnRH-ir axons within the auditory torus semicircularis also varied across the year, suggesting seasonal changes in GnRH influence of auditory processing. Exogenous application of GnRH (sGnRH and cGnRHII) caused a primarily inhibitory effect on auditory-evoked single neuron responses in the torus semicircularis. In the majority of neurons, GnRH caused a long-lasting decrease in spike rate in response to both tone bursts and playbacks of complex natural sounds. GnRH also decreased response latency and increased auditory thresholds in a frequency and stimulus type-dependent manner. To our knowledge, these results show for the first time in any vertebrate that GnRH can influence context-specific auditory processing in vivo in the brain, and may function to modulate seasonal auditory-mediated social behaviors.     

Time disparity sensitive behavior and its neural substrates of a pulse-type gymnotiform electric fish, Brachyhypopomus gauderio

Roles of the time coding electrosensory system in the novelty responses of a pulse-type gymnotiform electric fish, Brachyhypopomus, were examined behaviorally, physiologically, and anatomically. Brachyhypopomus responded with the novelty responses to small changes (100 μs) in time difference between electrosensory stimulus pulses applied to different parts of the body, as long as these pulses were given within a time period of ~500 μs. Physiological recording revealed neurons in the hindbrain and midbrain that fire action potentials time-locked to stimulus pulses with short latency (500–900 μs). These time-locked neurons, along with other types of neurons, were labeled with intracellular and extracellular marker injection techniques. Light and electron microscopy of the labeled materials revealed neural connectivity within the time coding system. Two types of time-locked neurons, the pear-shaped cells and the large cells converge onto the small cells in a hypertrophied structure, the mesencephalic magnocellular nucleus. The small cells receive a calyx synapse from a large cell at their somata and an input from a pear-shaped cell at the tip of their dendrites via synaptic islands. The small cells project to the torus semicircularis. We hypothesized that the time-locked neural signals conveyed by the pear-shaped cells and the large cells are decoded by the small cells for detection of time shifts occurring across body areas.     

FMRFamide-like immunoreactivity in the brain and pituitary of the teleost. Eigenmannia lineata (Gymnotiformes)

The distribution of cells immunoreactive for the molluscan tetrapeptide FMRFamide in the brain and the pituitary of Eigenmannia was investigated immunohistochemically by the use of the peroxidase-antiperoxidase (PAP) technique and unlabelled antibodies. FMRFi neurons were located in the ganglion of the nervus terminalis at the rostroventral side of the bulbus olfactorius. FMRFi perikarya were also found in a dorsomedial diencephalic nucleus, in the nucleus ventromedialis, in some liquor-contacting neurons of the nucleus lateralis tuberis and of the nucleus recessus lateralis and posterior. The perikarya of the midbrain pre-pacemaker nucleus were only weakly immunoreactive for FMRFamide while large FMRFi neurons (T-cells) occurred in lamina VI of the torus semicircularis, in the brain stem, in dorsal and medial layers of the lobus lineae lateralis posterior (LLLp) and in the medullary electric organ pacemaker nucleus (pm). FMRFi fibers and nerve endings were found in the bulbus olfactorius, in medial areas of the telencephalon, and rather densely in the rostral diencephalon. Ventrocaudally to most of the hypothalamic nuclei the occurrence of immunoreactive fibres increased; many coursed to the pituitary through the pituitary stalk. FMRFi fibres also appeared in the deep layers of the tectum opticum, in the torus semicircularis, in the medial and lateral medulla and below the pacemaker nucleus. Wherever FMRFamide-immunoreactivity occurred fibres and nerve endings could be found in close contact with blood vessels.