Micrographs of VRIs retrogradely labeled with biocytin injected into the middle portion of the motor layer and visualized with DAB. A: Biocytin was injected into middle portion of motor layer (arrowhead, ML). Labeled axons (arrow) run across the fiber layer (FL) and neuronal somata are labeled retrogradely in sensory layer (asterisk, SL). B: Higher magnification picture indicated with an asterisk in A. Retrogradely labeled somata are distributed throughout the sensory layer. Most VRIs have round or spindle shape soma and extend dendrites radially inward and outward. Few cells have tangentially-oriented processes. C–F: Higher magnification images of retrogradely labeled VRIs. Primary afferent fibers terminate in layers IV, VI and IX (Morita and Finger, 1985). C: Unipolar neurons in Layer III (arrowheads) extend their basal dendrites to layer IX (arrows). D: Bipolar neuron in layer V (arrowhead) extends its dendrites both inward and outward. Dorsal dendrite branches in layer IV and ventral one in layers VI and IX (arrows). E: Unipolar neuron in layer IX (arrowhead) directs its dendrite outward and branch in the layers IV and VI (arrow). F: Rare neurons in layer IX showed multipolar somata and possess a dendrite oriented tangentially (white arrows). Scale Bar = 200 µm (A), 50 µm (B), 20µm (C–F).

A: Micrographs showing triple labeling with micro-ruby labeled vagal motoneurons (red), biocytin labeled VRI axon terminals (green), and calretinin immunohistochemistry (blue) in the motor layer. Calretinin marks two distinct cell populations (Ikenaga et al., 2006). Small calretinin-positive, micro-ruby-negative neurons (c) are GABAergic interneurons of the superficial part of the motor layer. Calretinin-positive motoneurons (a) are magenta and represent a subset of the motoneuronal population. This picture is stack of 10 confocal images spaced by 1 µm. B: Higher magnification single confocal image plane showing neurons a and b from panel A. Green-labeled puncta from VRIs appear to contact the somata of both outer (calretinin-positive) and inner (calretinin-negative) motoneurons. C: Apparent synaptic contact between calretinin positive smaller interneuron (c) and VRIs terminals (arrow) is observed. Scale bar = 50 µm (A), 20 µm (B, C).

Topographic organization of the goldfish vagal reflex system. A, B: Micrographs showing the results of small biocytin injections into the motor layer. Restricted injection into dorsal motor layer (arrow, A1) labeled VRIs only in the dorsal sensory layer (A2), not in the ventral one (A3). In the case of ventral injection (arrow, B1), labeled VRIs were distributed in the ventral sensory layer (B3), not in the dorsal (B2). These in vitro findings confirm our previous in vivo tracer study (Goehler & Finger, 1992). C, D: Micrographs (left) and graphs (right) showing calcium imaging experiment with topographic stimulation. Micrographs were captured under visible light after the recording to indicate the site of the stimulating electrode. Each black and red trace represents the signals obtained from the regions indicated as white and red squares, respectively, in the micrographs. When the stimulating electrode was placed in the dorsal sensory layer (white arrowhead, C1), calcium responses were elicited following the electrical stimulation in the dorsal motor layer, not in the ventral one (C2). Ventral sensory layer stimulation induced calcium responses in the ventral but not dorsal motor layer (D2). The same vagal lobe slice was used in C and D. Bar scale = 500 µm (A1, B1), 100 µm (A2, 3, B2, 3).

Effects of Mg²⁺-free conditions and application of 10 µM AP-5, competitive NMDA receptor antagonist for the vagal motoneurons activity. A: Traces illustrating the evoked calcium responses of the vagal motoneurons by electrical stimulation under control conditions in standard aCSF (upper) ; in Mg²⁺-free aCSF (second) -- note that the enhance of the responses; in the presence of 10 µM AP-5 dissolved in Mg²⁺-free aCSF for 9 minutes (third) -- note the elimination of enhanced response; and during washout of AP-5 following 30 minutes in Mg²⁺-free aCSF (lower). B: Histogram showing average of the vagal motoneuron calcium responses after the incubation into Mg²⁺-free aCSF, AP-5 application, and washout. Responses were normalized to control response prior to Mg²⁺-free aCSF incubation. n = 13 cells from 3 slices.

Schematic diagram showing the essential circuitry of the vagal reflex system involved in intraoral food-sorting. Primary Gustatory Inputs from the palatal organ (roof of the mouth) and gill arches (floor of the mouth) terminate within different sublaminae of the sensory layer of the vagal lobe. The primary afferents synapse with dendrites of interneurons (not shown) and Vagal Reflex Interneurons which send axons across the fiber layer to terminate on both interneurons and Vagal Lobe Motoneurons. Both the primary afferents and the Vagal Reflex Interneurons release glutamate which acts on post-synaptic AMPA/kainate (AMPA+) and NMDA receptors (R’s) on the postsynaptic cells.

Anatomy and circuitry of the vagal lobe. A: Dorsal view of the exposed goldfish brain; anterior to the left. The solid vertical line indicates the level of the section shown in B (data from Huesa et al., 2008). Cblm, cerebellum; Tel, telencephalon; TeO, optic tectum; SpC, spinal cord. B: Nissl stained transverse goldfish vagal lobe. The lobe is divisible into three main layers: superficial sensory layer, central fiber layer, and deeper motor layer. C: Hypothesized vagal reflex circuit for the goldfish food sorting behavior. Gustatory information from taste buds of oral cavity (palatal organ and gill arches) is conveyed by the vagus nerve through the fiber layer and terminates in the sensory layer (1). Vagal primary afferent fibers activate vagal reflex interneurons (VRIs) which project through the fiber layer (2) and terminate on the vagal motoneurons (3). These motoneurons innervate muscles of the palatal organ and activate them to manipulate food particles in the oral cavity. D: Micrograph showing retrogradely labeled motor neurons and their dendritic ramifications in the superficial part of the motor layer following labeling of the nerve root with rhodamine dextran. The motor layer in the same orientation as panel B. E: Confocal micrograph of the sensory layers following filling with rhodamine dextran of a few primary afferent fibers from the palatal organ. Each fiber terminates in a restricted domain of either layer vi with some collateral endings in layer ix. No primary afferent fibers terminate in the motor layer.

A Micrograph of a VRIs in the sensory layers of the vagal lobe retrogradely labeled (green) by a biocytin injection into the motor layer. As shown in the previous figure, retrogradely labeled neurons are present in superficial, middle and deep layers of the sensory layers but dendrites ramify mostly within layers ix and vi, two layers of termination of primary afferent fibers. Box D shows the area enlarged in panels D and D’. Scale bar = 50 µm. B. Confocal image through the neuropil of layer vi in a different section than that shown in A. The arrows indicate several points of apparent contact between the primary afferent fibers (magenta) and the dendrites (green) of the retrogradely labeled VRI neurons. C. Z-stack confocal image (14 @ 0.25 µm = 3.25µm thickness) showing a VRI cell body in layer vi apparently being contacted by a labeled primary afferent fiber (magenta). D. Small z-stack confocal image (14 @ 0.25 µm = 3.25µm thickness) showing close apposition between labeled primary afferent fiber (magenta) and two retrogradely labeled VRIs (green). The area indicated by the arrow is shown enlarged in a single plane confocal image in D’.

Calcium imaging with vagal motoneurons shows the existence of a reflex pathway from sensory to motor layers. Compare this image with Fig. 1B, C. A: Injections of the calcium sensitive dye, calcium green dextran, into the vagus nerve retrogradely label vagal motoneurons visible as bright spots of the motor layer (ML) in this in vitro vagal lobe slice preparation. The stimulating electrode was placed into the sensory layer (SL, white arrowhead). Bright fluorescence also is visible in fascicles of fibers within the fiber layer (FL) between the black rectangle and the white arrowhead. B: Higher magnification image indicated as a rectangle in panel A. Individual labeled motoneurons can be distinguished clearly. C1–3: Graphs of intracellular calcium responses of the vagal motoneurons. Each black and red trace represents the signals obtained from the regions of interest indicated as black and red squares in B respectively. In normal aCSF (C1), intracellular calcium levels increased following the electrical stimulation (arrowhead, left). These responses were suppressed in Ca²⁺-free aCSF (C2) and recovered in normal aCSF (C3). D, E: Effect of cutting the fiber layer (FL) between the sensory layer where the stimulating electrode is placed (white arrowhead) and motor layer where calcium responses are recorded. The red color indicates regions with significant elevation in the Ca⁺⁺ signal. In the intact condition, stimulation of the sensory layer evokes a Ca⁺⁺ response in the motoneurons (D2). After the fiber layer is cut (dotted line inE1), the motor layer responses were eliminated (E2). In this case, Ca⁺⁺ responses around the stimulating electrode remained, indicating that the electrical stimulation of the sensory layer was still effective.

Effects of the antagonist for AMPA/kainate receptors, DNQX, on the responses of vagal motoneurons. A: Micrograph of living vagal lobe slice for the pharmacological experiment captured by CCD camera. In pharmacological experiments, the sensory layer was cut off and the stimulating electrode (white arrowhead) was placed in fiber layer (FL) to directly stimulate axons of VRIs. B: Graph showing one example of the evoked vagal motoneuron calcium responses by electrical stimulation from single slice under control conditions with aCSF (upper), in the presence of 10 µM DNQX for 9 minutes (middle), and during recovery from DNQX following 45 minutes in normal aCSF (lower). Note that evoked calcium responses were suppressed by application of 10 µM DNQX. C: Histogram showing average of the vagal motoneuron calcium responses after DNQX application and washout. Responses were normalized to control response prior to DNQX application. n = 17 cells from 3 slices.

A: As in Fig 8A, the first three traces show calcium responses of the vagal motoneurons under various conditions. Application of 10µM AP-5 eliminated the enhanced calcium responses but small responses remained (center panel). The remaining responses were removed by the further addition of 10µM DNQX (9minutes). B: Histogram showing average of the vagal motoneuron calcium responses after the incubation into Mg²⁺-free aCSF, AP-5 and DNQX application, and washout. Responses were normalized to the control response prior to Mg²⁺-free aCSF incubation. n = 14 cells from 3 slices.