Reduced vesicular monoamine transport disrupts serotonin signaling but does not cause serotonergic degeneration

Abstract

We previously demonstrated that mice with reduced expression of the vesicular monoamine transporter 2 (VMAT2 LO) undergo age-related degeneration of the catecholamine-producing neurons of the substantia nigra pars compacta and locus ceruleus and exhibit motor disturbances and depressive-like behavior. In this work, we investigated the effects of reduced vesicular transport on the function and viability of serotonin neurons in these mice. Adult (4-6 months of age), VMAT2 LO mice exhibit dramatically reduced (90%) serotonin release capacity, as measured by fast scan cyclic voltammetry. We observed changes in serotonin receptor responsivity in in vivo pharmacological assays. Aged (months) VMAT2 LO mice exhibited abolished 5-HT1A autoreceptor sensitivity, as determined by 8-OH-DPAT (0.1 mg/kg) induction of hypothermia. When challenged with the 5HT2 agonist, 2,5-dimethoxy-4-iodoamphetamine (1 mg/kg), VMAT2 LO mice exhibited a marked increase (50%) in head twitch responses. We observed sparing of serotonergic terminals in aged mice (18-24 months) throughout the forebrain by SERT immunohistochemistry and [(3)H]-paroxetine binding in striatal homogenates of aged VMAT2 LO mice. In contrast to their loss of catecholamine neurons of the substantia nigra and locus ceruleus, aged VMAT2 LO mice do not exhibit a change in the number of serotonergic (TPH2+) neurons within the dorsal raphe, as measured by unbiased stereology at 26-30 months. Collectively, these data indicate that reduced vesicular monoamine transport significantly disrupts serotonergic signaling, but does not drive degeneration of serotonin neurons.

Keywords: 5-HT1A; 5-HT2; Dorsal raphe; Parkinson's disease; Serotonin; Synaptic vesicle; VMAT2.

Fig. 1

Genetic VMAT2 suppression causes reduced synaptic release of 5-HT in the SNr. Synaptic release of 5-HT in the SNr was measured with fast scan cyclic voltammetry A. Top color plot, adult (4–6 months) VMAT2 WT animals exhibited a peak release of 1 µM 5-HT in the SNr in response to electric stimulation (20 pulses, 100 Hz, 350 µA, 4 ms, monophasic). Bottom color plot. VMAT2 LO littermates had drastically reduced peak release. Representative color plots for single recordings are shown. B. VMAT2 LO mice exhibited a 90% reduction in mean peak release (*p = 0.02). C. There was no significant difference in the synaptic clearance rate of 5-HT, as reflected by tau. Four sample recordings were taken at 3 sites within the lateral SNr of each mouse; n = 3 mice per genotype.

Fig. 2

VMAT2 LO mice have an ablated hypothermic response to the 5-HT1A agonist 8-OH-DPAT. Aged (18–24 months) wildtype and VMAT2 LO mice were injected with saline or 8-OHDPAT (0.1 mg/kg; i.p.) to assess thermic response to 5-HT1A activation. Graph represents the mean of differences between core body temperature taken at baseline and 20 min following injection for each animal. Wildtype animals exhibited a −4° change in core body temperature, while VMAT2 LO mice had an ablated response of −1° (****p < 0.0001, n = 7–8 mice per group).

Fig. 3

VMAT2 LO mice have increased sensitivity to the 5-HT2 agonist DOI. Wildtype and LO mice (4–6 months of age) were injected with saline or DOI (1 mg/kg) and placed in a cage for 10 min video-recorded observation of head twitches. When challenged with DOI, VMAT2 LO mice exhibited a 79% increase in the number of head twitches over wildtype mice (****p < 0.0001, n = 8–9 mice per group,).

Fig. 4

Serotonergic innervation is preserved in aged VMAT2 LO mice. Immunohistochemical staining against SERT reveals no substantial change in serotonergic innervation between aged (24 months) wildtype and VMAT2 LO mice. Regions depicted are the somatomotor cortex (A, 200× magnification), SNr (B, 400× magnification, inset at 25×), and dorsal striatum(C, 400× magnification, inset at 25×) D. [3 H]-paroxetine binding in striatal homogenates reveals no change in binding sites between aged (22–24 months) wildtype and VMAT2 LO mice (n = 6, p = 0.9).

Fig. 5

Preservation of the dorsal raphe nucleus in aged VMAT2 LO mice. A. Immunohistochemical staining against TPH2 in the dorsal raphe shows normal cytoarchitecture in VMAT2 LO mice at 24 months of age (images taken at 50× magnification). B. Unbiased stereological sampling revealed no significant change in the quantity of TPH2 + neurons in the dorsal raphe between aged WT and VMAT2 LO mice; the quantity of Nissl + neurons (inclusive of TPH2 + neurons) was also unchanged. Analysis was performed at section intervals of 3, with a total of 6–7 dorsal raphe sections per animal. The Gundersen coefficient of error was <0.1 for all animals (n = 5 mice, p = 0.73).