Descending 5-hydroxytryptamine raphe inputs repress the expression of serotonergic neurons and slow the maturation of inhibitory systems in mouse embryonic spinal cord

Abstract

Spontaneous synchronous rhythmic activities are a common feature of immature neuronal networks. Although the mechanisms underlying such activities have been studied extensively, whether they might be controlled by modulatory information remains questionable. Here, we investigated the role of descending serotonergic (5-HT) inputs from the medulla to the spinal cord in the maturation of rhythmic activity. We found that in spinal cords maintained, as a whole, in organotypic culture without the medulla, the maturation of spontaneous activity is similar to that found in spinal cords developed in utero. Interestingly, in organotypic cultures without the medulla (i.e., devoid of descending inputs), numerous intraspinal neurons expressed 5-HT, unlike in spinal cords cultivated in the presence of the medulla or matured in utero. We demonstrated that this 5-HT expression was specifically dependent on the absence of 5-HT fibers and was repressed by 5-HT itself via activation of 5-HT(1A) receptors. Finally, to verify whether the expression of 5-HT intraspinal neurons could compensate for the lack of descending 5-HT fibers and play a role in the development of spontaneous activity, we blocked the 5-HT synthesis using p-chlorophenylalanine methyl ester in cultures devoid of the medulla. Surprisingly, we found that this pharmacological treatment did not prevent the development of spontaneous activity but accelerated the maturation of intraspinal inhibition at the studied stages. Together, our data indicate that descending 5-HT raphe inputs (1) repress the expression of spinal serotonergic neurons and (2) slow the maturation of inhibitory systems in mouse spinal cord.

Fig. 1.

Spinal spontaneous rhythmic activities in acute (in vitro) and organotypic culture preparations.A, Schematic of the spinal cord organotypic culture, illustrating the opening of the dorsal side at E12.5 (top), and a photomicrograph of a 24 hr cultured spinal cord preparation (lumbar part). d, Dorsal;m, midline (dotted line);v, ventral. B, Spontaneous activities can be recorded extracellularly in acute in vitropreparations from ventral roots. C, Spontaneous activities are also recorded in vitro using pipettes closely apposed to the ventral gray matter. D, In an organotypic culture devoid of ventral roots, ventral gray matter recordings were used to monitor spontaneous rhythmic activities.Gray traces are raw data of activity; black traces are integrated activity. In B–D, thetwo bottom traces show one burst (delineated by dotted lines) on an expanded time scale.

Fig. 2.

Spontaneous rhythmic bursts of activity recorded from lumbar spinal cords in vitro or in organotypic cultures, with or without the medulla, exhibited a dramatic reduction of the interburst period accompanied by a large increase of variability. Note that these changes occurred between E14.5 and E18.5in vitro and between E12.5 plus 2 DIC and E12.5 plus 6 DIC in organotypic culture. A, Spontaneous bursts of activity recorded in vitro recurred with a regular period of spontaneous activity of ∼3.2 min at E12.5 and E14.5 but became very erratic at E18.5. B, In spinal cords maintained in organotypic cultures at E12.5 with the medulla (E12.5; start of culture, 0 DIC), bursts of activity also recurred with a regular spontaneous activity period of ∼2 min after 2 DIC and became very irregular after 6 DIC. C, Cultures without the medulla also exhibited an identical evolution in their spontaneous bursts of action potentials. D, E, Quantitative analysis of the interburst periods in each experimental condition and index of variability of these periods given by the CV (SD/mean of periods). Values represent the mean ± SEM of three to six experiments (number in parentheses). *p < 0.05 (one-way ANOVA followed by Tukey test).

Fig. 3.

Similar maturation of the excitatory glutamatergic synaptic transmission in utero and in organotypic cultures. A, At E12.5, 4 mm kynurenate did not alter the ongoing spontaneous activity. B,C, Same treatment at E18.5 and E12.5 plus 6 DIC resulted in complete abolition of the spontaneous rhythmic activities.D, Quantitative analysis of kynurenate effects; the number of preparations is in parentheses.

Fig. 4.

Similar maturation of the inhibitory synaptic transmission in utero and in organotypic cultures.A, GABA_A–glycinergic receptor blockage [30 μm bicuculline and 5 μm strychnine (Bicu-Stry)] reduced the duration of spontaneous bursts at E12.5 in vitro (see vertical dotted line). B, C, Adding the same GABA_A–glycinergic synaptic transmission antagonists 2 d later either at E14.5 in vitro or after 2 DIC induced an opposite effect leading to an increase of the duration of spontaneous bursts; this increase became larger at E18.5 in vitro and after 6 DIC. Gray areas, Extension of burst duration during drug application. The end of the burst was considered to occur when the integrated signal fell below zero.D, Quantitative analysis of bicuculline–strychnine effects measured as the percentage of decrease or increase of the burst duration. **p < 0.01 (t test between E14.5 and E18.5); ***p < 0.001 (t test between E12.5 plus 2 DIC and E12.5 plus 6 DIC).

Fig. 5.

Caudal raphe neurons sent descending fibers into the spinal cord in utero or in organotypic culture.A, In an acute E18.5 preparation, 5-HT labeled neurons were detected at the medulla level (top), and their 5-HT descending axons reached the lumbar level of the spinal cord (bottom). B, In organotypic culture preparation, a similar nucleus containing 5-HT-immunostained neurons was found at the medulla level; these neurons sent fibers into the caudal levels of the cultured spinal cord. The white dashed line delimits the fourth ventricle (IV);white arrowheads point to the terminal end (growth cone) of a serotonergic axon. The schematic on theright represents the descending 5-HT inputs (lines) from raphe somata (blacks dots).cerv, Cervical; med, medulla;lumb, lumbar; th, thoracic;L, lateral; R, rostral. As in Figures 6and 7, the vertical dotted lines on the rightschematic drawings represent the location of the central canal.

Fig. 6.

Expression of 5-HT intraspinal neurons in organotypic cultures without the medulla. A, After 2 DIC, the immunostaining procedure allowed detection of 5-HT-labeled somata (black arrowheads). B, After 6 DIC, the same protocol revealed a stronger staining of 5-HT neurons extending into their neuritic processes (white arrowheads). L, Lateral; R, rostral. On the right schematic drawings, black dots indicate the presence of intraspinal 5-HT-immunoreactive somata.

Fig. 7.

Experimental evidence for a repressive role of 5-HT descending inputs on the expression of 5-HT intraspinal neurons.A, Serotonin (5 μm) added to the culture medium resulted in the absence of 5-HT staining in the preparation without medulla. B, Blockade of 5-HT_1A by 10 μm spiroxatrine revealed 5-HT intraspinal labeled somata (black arrowheads) and long 5-HT fibers (white arrowheads). antag, Antagonist.L, lateral; R, rostral. The dashed line in the top panel in B delimits the fourth ventricle. On the right schematic drawings,vertical solid lines represent the descending 5-HT axons andblack dots represent the 5-HT raphe and intraspinal somata.

Fig. 8.

The blockade of GABA_A–glycine receptors revealed an increase in the duration of the spontaneous rhythmic bursts in pCPA-treated organotypic cultures after 2 DIC.A, As shown in Figure 4A, at E12.5, an application of 30 μm bicuculline and 5 μm strychnine (Bicu-Stry) induced a decrease of spontaneous burst duration. B, After 2 d of culture in control medium (E12.5 plus 2 DIC, untreated cultures), the same application of bicuculline–strychnine induced an increase in burst duration (gray area). C, pCPA-treated E12.5 plus 2 DIC cultures (10 μm pCPA) exhibited a larger increase of burst duration (gray areas) after GABA_A–glycine receptor blockage. D, Quantitative analysis revealed a significant difference between E12.5 plus 2 DIC controls and pCPA-treated preparations. **p < 0.01 (t test).