Transcellular induction of neuropeptide Y expression by NT4 and BDNF

Abstract

The transcellular signaling of neurotrophins is postulated, but evidence is scarce. We now show that a small number of NT4- and BDNF-overexpressing neurons in the cortical explant of thalamocortical cocultures rapidly evoked a Trk receptor-dependent upregulation of neuropeptide Y (NPY) mRNA in interneurons. In contrast to BDNF, the action of NT4 was independent of calcium influx through NMDA receptors and L-type calcium channels. NPY neurons vastly outnumbered the neurotrophin-overexpressing neurons (mostly pyramidal cells), arguing for a spread of the neurotrophin signal via axonally connected neuronal populations. Furthermore, NT4 transfection of one explant of axonally connected corticocortical cocultures evoked significantly larger numbers of NPY neurons in both explants. Delivery of the signal was not by diffusion of neurotrophins via the medium. Moreover, cortical NPY neuron numbers increased after NT4 and BDNF transfection of a cocultured tectal explant innervated selectively by cortical layer V pyramidal neurons. The transcellular induction of NPY suggests a source-to-sink model for axonal transport and a local cortical redistribution of TrkB ligands to interneurons competent for NPY expression.

Fig. 1.

NPY-mRNA-expressing neurons in cortical explants of 70-DIV thalamocortical cocultures. (A) NPY neuron numbers remained low after 60- to 70-DIV EGFP expression. Transfection of EGFP+BDNF (B) and EGFP+NT4 (C) (expression from 60 to 70 DIV) strongly increased NPY neuron numbers. The Insets show that EGFP-fluorescent BDNF and NT4 transfectants (Right Inset)do not express NPY mRNA (Left Inset). (D) NPY neuron numbers had increased already 16 h after NT4 transfection. (E) Mean percentage with SEM of NPY-mRNA-expressing neurons in the experimental conditions of A–D. Letters on the x axis match the labeling of the pictures in this and the following figures. White matter is oriented downwards. (Scale bar: 300 μm.) **, P < 0.01; ***, P < 0.001.

Fig. 2.

Dependence on Trk receptors and calcium influx. (A) Low density of NPY neurons in BDNF-transfected cultures treated with K252a. (B) Low density of NPY neurons in NT4-transfected cultures treated with K252a. (C) Blocking L-type calcium channels with nifedipine and NMDA receptors with APV efficiently prevented NPY up-regulation after BDNF transfection but not after NT4 transfection (D). (E) Representative plot of the distribution of EGFP/NT-4-expressing neurons (triangles) and nonneuronal cells (mostly glia; circles) and NPY neurons (small dots) in a thalamocortical coculture at 70 DIV treated with nifedipine and APV concurrent to transfection. (F) NT4 overexpression failed to induce NPY in cultures activity-deprived with 10 mM Mg²⁺ (chr. Mg). (G) Mean percentage (with SEM) of NPY neurons in the experimental conditions of A–F. White matter is oriented downward (A–E). (Scale bars: A–D, 300μm; E, 500 μm.) ***, P < 0.001.

Fig. 3.

Circumstantial evidence for axonal delivery of NT4 in corticocortical cocultures. (A) Conditioned medium from NT4-transfected cocultures fails to promote NPY in untransfected age-matched cocultures. This result argues against a diffusible action of the neurotrophin. (B) Transfection of NT4 (expression from 60 to 70 DIV) strongly up-regulated the NPY expression in the transfected explant of corticocortical OTC and also in the nontransfected explant (C). (D) Mean percentage with SEM of NPY neurons in the experimental conditions of A–C in comparison with untransfected visual corticocortical OTC (VC-VC control). White matter is oriented downward. (Scale bar: 300 μm.) **, P < 0.01.

Fig. 4.

Circumstantial evidence for axonal delivery of NT4 in corticotectal cocultures. (A) NPY expression remained low in activity-deprived VC cultures (VC deprived). Even resumption of activity failed to increase the NPY expression (20 DIV under 10 mM Mg²⁺ and subsequently 10 DIV recovery in 2 mM Mg²⁺; VC recovery). (B) The cortical explant of corticotectal cocultures displays low numbers of NPY neurons after 15-DIV activity deprivation and transfection with EGFP from 20 to 25 DIV. (C) After NT4 transfection of the tectal explant, NPY neuron numbers increased in the cortical explant. (D) After BDNF transfection of the tectal explant, NPY neuron numbers increased in the cortical explant, and BDNF was more effective than NT4 (see A). (E) DiI injection into the tectal explant of a corticotectal coculture activity-deprived for 20 DIV. Despite the absence of activity, layer V pyramidal cells with apical dendrites reaching layer I were retrogradely labeled, suggesting that their morphological differentiation was not impaired (19). White lines, borders of the cortical explant; black line, border of the tectal explant. Roman numerals mark cortical layers. White matter is oriented downward. (F and G) The cortical expression of NT4 and BDNF mRNA was analyzed by RT-PCR. (F) The NT4 mRNA expression was not altered after tectal transfection with BDNF or NT4 plasmids. Transfection with EGFP plasmids in 25-DIV spontaneously active cultures and in recovery cultures (15 DIV deprived, then 10 DIV active; level was set to 1 for normalization) were used as controls, and the initial activity deprivation did not alter cortical NT4 expression. (G) The same was observed for cortical BDNF mRNA expression in the control conditions, and cortical BDNF mRNA was not changed after tectal NT4 transfection. However, cortical BDNF mRNA expression increased after tectal BDNF transfection. EGFP, transfection with EGFP plasmid alone; BDNF, transfection with EGFP+BDNF plasmid; NT4, transfection with EGFP+NT4 plasmid. (Scale bars: 300 μm.) **, P < 0.01; ***, P < 0.001.

Fig. 5.

Effects of neutralization of BDNF and NT4. (A) Neutralizing NT4 after tectal NT4 transfection prevented the induction of NPY. (B) Neutralizing BDNF after tectal BDNF transfection prevented the induction of NPY. (C) Neutralization of BDNF after tectal transfection of NT4 did not prevent the induction of NPY. (D) It seems that BDNF needs endogenous NT4 signaling as a cofactor. In contrast, neutralization of BDNF in corticotectal OTC does not prevent the up-regulation of NPY by tectal NT4 transfection. (E) Mean percentage (with SEM) of NPY neurons in the experimental conditions of A–D. White matter is oriented downward. (Scale bar: 300 μm.) ***, P < 0.001, tested against VC-SC-deprived (Fig. 4B).

Fig. 6.

The source-to-sink model. NT4 overexpressers in the tectal explant () become a source of NT4. Nonoverexpressing pyramidal neurons in layer V of the cortical explant () represent a first sink of NT4 and retrogradely import the factor (a). Now, layer V pyramidal neurons () accumulate NT4 and represent a source of NT4 for cortical interneurons (). They, in turn, represent the second sink of NT4 and either retrieve somatic or dendritic released imported NT4 (b) or receive imported NT4 by anterograde delivery via pyramidal cell axon collaterals (c). The model also explains the results obtained in corticocortical cocultures; in addition, transfected projection neurons may directly deliver NT4 anterogradely to neurons in nontransfected explants.