Substance P induces the reversible formation of varicosities in the dendrites of rat brainstem neurons

Abstract

This study investigated the ability of substance P (Sub P) to induce dendritic varicosities (DVs) or beads in neurons of the rostral ventromedial medulla (RVM) of the rat. Microinjection of 5-200 pmol Sub P in the RVM produced a concentration-dependent increase in the number of DVs in distal dendrites of RVM neurons that were immunoreactive for the neurokinin-1 receptor, but not serotonin. The effect was reversible, as DVs were essentially absent 2 and 4h after microinjection. Fluoro-Jade B labeled neurons were not evident in the RVM 4 days after microinjection of Sub P, although such neurons were present 4 days after microinjection of a neurotoxic dose of kainate. Bath application of Sub P to brainstem slices for a period as brief as 30s also produced DVs in neurokinin-1 immunoreactive RVM neurons. Prior exposure to L-703606 prevented the formation of DVs by Sub P, implicating the neurokinin-1 receptor, a Gq type of G protein coupled receptor, in the formation of DVs by Sub P. Finally, stabilization of microtubules by prior exposure to taxol also prevented the formation of DVs, consistent with the idea that increases in intracellular Ca(2+) lead to the formation of DVs secondary to a disruption of the linear arrays of microtubules in dendrites. These data establish a mechanistic basis for the formation of DVs by Sub P and support further studies to test the hypothesis that the formation of DVs is a morphological mechanism by which neurons can regulate their responses to inhibitory or excitatory inputs.

Figure 1

(A) Representative photomicrograph of dendritic varicosities (DVs) in neurokinin-1 receptor expressing neurons of the rostral ventromedial medulla (RVM) demonstrating the clarity with which DVs could be viewed with a 60× N.A. 1.4 objective and 10× eyepiece objective. The vertical line drawn perpendicular to the dendrite’s trajectory illustrates how the diameter of a DV was determined. Note that the segments between the DVs are constricted. (B) Representative photomicrographs of DVs in neurokinin-1 receptor expressing neurons of the rostral ventromedial medulla 1 hr after microinjection of 100 pmol substance P. (C) Few DVs were observed in neurokinin-1 receptor expressing neurons in the RVM after microinjection of saline. (D) Substance P did not produce DVs in serotonergic neurons in the RVM. Scale bar is 10 μm in panel A and 100 μm in panels B–D. Arrowheads indicate DVs. Where not visible in panels B–D, the microinjection site was within 250 – 500 microns.

Figure 2

(A) Microinjection of Substance P (Sub P) in the rostral ventromedial medulla (RVM) of adult rats produced dendritic varicosities (DVs) in a dose-dependent manner. Values are the mean ± S.E.M. of the total number of DVs counted in serial sections through the RVM of 3–9 rats. (B) Time course of DV formation in the RVM after microinjection of 100 pmol Sub P. Values are the mean and S.E.M. of the total number of DVs counted in serial sections through the RVM of 3–6 rats.

Figure 3

The effects of substance P (Sub P) are concentration- and time-dependent and mediated by neurokinin-1 receptors (NK-1R) as determined in brainstem slices from juvenile rats. (A) Negligible numbers of dendritic varicosities (DVs) are present in brainstem slices incubated for as long as 6 hrs before transfer to the recording chamber. The induction of DVs in NK-1R immunoreactive neurons in the RVM is highly dependent on (B) the concentration (illustrated for a 30 s duration of exposure) and (C) duration of exposure to Sub P. (D) Pretreatment with 0.1 (open bars) or 1.0 μM (hatched bars) L703,606 prevents the formation of DVs by 30 sec exposure to 1 μM Sub P, but does not decrease the number of DVs by itself. The stippled bar is ACSF only (control). * P< 0.05, ** P < 0.01 compared to control. † P < 0.05, ‡ P < 0.01 compared to Sub P. Data are mean and S.E.M. of determinations in 4–9 slices obtained from 3–6 rats.

Figure 4

The production of dendritic varicosities (DVs) involves microtubules, and does not alter passive membrane properties of neurokinin-1 receptor (NK-1R) expressing neurons in the RVM. (A) Pretreatment with the microtubule stabilizer taxol (10 μM) prevents the formation of DVs by 30 sec exposure to 1 μM substance P. Data are the mean ± S.E.M of determinations in 3–4 slices. (B) Current-voltage relationship for three NK-1R expressing RVM neurons before (circles) and 15 min after a 30 sec exposure to 1 μM Sub P (squares). (C) Brief exposure to Sub P did not alter passive membrane properties of NK-1R-expressing RVM neurons. Data are mean ± S.E.M. for three neurons.

Figure 5

Representative micrographs of Fluoro-jade B staining of (A) hippocampal neurons 4 days after i.p. injection of 10 mg/kg kainate or (B) RVM neurons four days after microinjection of 1.2 nmol kainate into the RVM. Insert in panel A is a high magnification image of the boxed area. Fluoro-jade B staining was not observed in the RVM four days after microinjection of (C) 100 pmol Sub P, a dose known to produce DVs in RVM neurons, or (D) saline. Arrows identify the microinjection site. Scale bars are 100 μm.

Figure 6

Schematic illustrating how dendritic varicosities (DVs) and the intervening constrictions can alter the electrotonic spread of potentials from dendrites to the soma, effectively isolating the soma from excitatory or inhibitory drive. This figure does not represent actual data, but is a graphic illustration of the known passive electrical properties of dendrites. V_o: initial membrane potential. V_d: voltage at distance d from the source. d: distance from the source on the dendrite. r_m: membrane resistance. r_i: internal resistance. The space constant, also known as λ, is the square root of the ratio of r_m and r_i.