Voltage-sensitive dye recording of glossopharyngeal nerve-related synaptic networks in the embryonic mouse brainstem

Abstract

The glossopharyngeal nerve (N.IX) transfers motor and sensory information related to visceral and somatic functions, such as salivary secretion, gustation and the control of blood pressure. N.IX-related neural circuits are indispensable for these essential functions. Compared with the strenuous analysis of morphogenesis, we are only just starting to elucidate the functiogenesis of these neural circuits during ontogenesis. In the present study, we applied voltage-sensitive dye recording to the embryonic mouse brainstem, and examined the functional development of the N.IX-related neural circuits. First, we optically identified the motor nucleus (the inferior salivatory nucleus (ISN)) and the first-order sensory nucleus (the nucleus of the tractus solitarius (NTS)). We also succeeded in recording optical responses in the second/higher-order sensory nuclei via the NTS, including the parabrachial nucleus. Second, we pursued neuronal excitability and the onset of synaptic function in the N.IX-related nuclei. The neurons in the ISN were excitable at least at E11, and functional synaptic transmission in the NTS was first expressed at E12. In the second/higher-order sensory nuclei, synaptic function emerged at around E12-13. Third, by mapping optical responses to N.IX and vagus nerve (N.X) stimulation, we showed that the distribution patterns of neural activity in the NTS were different between the N.IX and the N.X from the early stage of ontogenesis. We discuss N.IX-related neural circuit formation in the brainstem, in comparison with our previous results obtained from chick and rat embryos.

Keywords: APV, dl-2-amino-5-phosphonovaleric acid; CNQX, 6-cyano-7- nitroquinoxaline-2,3-dione; CNS, central nervous system; Development; EPSP, excitatory postsynaptic potential; Glossopharyngeal nerve; ISN, inferior salivatory nucleus; N.IX, glossopharyngeal nerve; N.X, vagus nerve; NTS, nucleus of the tractus solitarius; Neural circuit formation; Optical recording; PBN, parabrachial nucleus; Synaptogenesis; VSD, voltage-sensitive dye; Voltage-sensitive dye.

Fig. 1

(A) Optical recording of neural responses to N.IX stimulation in an E12 mouse en bloc preparation. The recording was made with the ventral side up and with a magnification of x25. The cut end of the N.IX was electrically stimulated with a depolarizing pulse (8 μA/5 msec) using a suction electrode. The relative position of the image of the preparation is drawn on the recording, and signals detected on the stimulated side are shown. The direction of the arrow in the lower right of the figure indicates an increase in transmitted light intensity (a decrease in dye absorption), and the length of the arrow represents the stated value of the fractional change. (B) Enlarged traces of optical signals detected in positions 1–4 in A. The signals detected in the rostro-medial region (gray shadow: positions 1 and 2) consisted of a fast spike-like signal (indicated by arrowheads), whereas those detected in the lateral region (positions 3 and 4) exhibited two components: a fast spike-like signal (indicated by arrowheads) and a long-lasting slow signal (indicated by asterisks). In this and other recordings, signal averaging of two trials was performed offline. N.IX, glossopharyngeal nerve; N.X, vagus nerve.

Fig. 2

(A) Optical recording of neural responses to N.IX stimulation in an E12 mouse medulla slice preparation. The recording was made with the caudal side up. The fast signals observed near the root of the N.IX were reversed in polarity with hyperpolarizing stimulation (data not shown), suggesting that they were not active responses but electrotonic potentials. (B) Enlarged traces of optical signals detected in the dorso-lateral region (a position indicated by an asterisk in A). The upper trace is a signal detected in a physiological solution, and the lower trace is a signal in a solution containing both APV (200 μM) and CNQX (5 μM). The slow signal was eliminated in the presence of these glutamate receptor antagonists.

Fig. 3

1020-site optical recordings of neural responses to N.IX (A) and N.X (B) stimulation in an E14 mouse brainstem en bloc preparation. The recordings were made with a magnification of x10 to detect optical responses from a wide region of the brainstem. Either N.IX or N.X stimulation elicited neural responses in three areas (Areas 1–3) on the stimulated side. Area 1 (pink shadow) was located at the caudal level of the N.IX/N.X root, and corresponded to the NTS. Areas 2 and 3 (orange and green shadows, respectively) were discerned in the rostral medulla and pons, respectively. On the contralateral side, small slow signals were detected in several positions as typified by arrowheads. The colored-response areas were determined by eye, whereas the precise maps are shown in Fig. 5B. G.VIII, vestibulo-cochlear ganglion.

Fig. 4

Enlarged traces of the N.IX-related optical signals recorded from Areas 1–3 and the contralateral side of an E14 en bloc brainstem. The vertical line indicates the onset timing of the signal in Area 1. Significant delays of the onsets existed between the signal in Area 1 and the signals in other areas. This suggests that the latter signals correspond to polysynaptic responses in the second/higher-order nuclei.

Fig. 5

Contour line maps of the amplitude of the slow signal in response to N.IX (left panels) and N.X (right panels) stimulation in an E13 (A) and E14 (B) en bloc brainstem preparation. The numerals on the contour lines indicate the fractional change multiplied by 10⁴. Maps in B were obtained from the preparation shown in Fig. 3.

Fig. 6

Typical examples of the slow signal area and the peak locations of the slow signal amplitude. Cases 1 and 2 were obtained from E13 preparations, and case 3 was obtained from an E14 preparation. The slow signal areas (ΔI/I ≥ 1.0 × 10⁻⁴) are illustrated with lines, and locations of the peak are shown with filled circles. Blue lines and circles correspond to N.IX responses, and red ones to N.X responses.