Sympathetic Hyperactivity and Age Affect Segregation and Expression of Neurotransmitters

Abstract

Sympathetic neurons of the rat superior cervical ganglion (SCG) can segregate their neurotransmitters and co-transmitters to separate varicosities of single axons. We have shown that transmitter segregation is a plastic phenomenon and that it is correlated with the strength of synaptic transmission. Here, we determined whether sympathetic dysfunction occurring in stress and hypertension was correlated with plastic changes of neurotransmitter segregation. We characterized the expression of the markers, L-glutamic acid decarboxylase of 67 kDa (GAD67) and vesicular acetylcholine (ACh) transporter (VAChT) in the SCG of cold stressed and spontaneously hypertensive rats (SHR). Considering that the SCG comprises a heterogeneous neuronal population, we explored whether the expression and segregation of neurotransmitters would also have an intraganglionic heterogeneous distribution in ganglia of stressed and hypertensive rats. Furthermore, since hypertension in SHR is detected around 8-10 weeks, we evaluated expression and segregation of ACh and GABA in adult hypertensive (12-week old (wo)) and young pre-hypertensive (6-wo) SHR. We found an increase in segregation of ACh and GABA with no change in transmitter expression in ganglia of stressed animals. In contrast, in SHR, there was an increase in GABA expression, although segregation did not vary. Segregation showed a caudo-rostral gradient in controls but not in the ganglia of stressed animals. GABA expression showed a rostro-caudal gradient in adult SHR, which was not present in young 6-wo rats. In young SHR, ACh increased and, unexpectedly, segregation of ACh and GABA was higher than in adults. Data suggest that ACh and GABA segregation increases in acute sympathetic hyperactivity like stress, but does not vary in chronic hyperactivity such as in hypertension. Changes in segregation are age-dependent and might be involved in the mechanisms underlying stress and hypertension.

Keywords: GABA; SHR; acetylcholine; co-transmission; hypertension; stress; sympathetic ganglia.

Figure 1

Cold-stress increased expression of tyrosine hydroxylase (TH) mRNA and protein in adrenal medulla (AM). (A) Conventional RT-PCR analysis performed on total RNA obtained from AM showing an increase (17.5%) in TH mRNA after 5 days of cold-stress. RT-PCR assay was performed for the amplification of TH mRNA fragment of 646 bp. (B) Western blot (WB) analysis of TH protein (56 kDa) expression showing an increase (1.6-fold) after cold-stress. Graphs show quantification of TH relative to actin, mean ± SEM and the significance level. Actin was used as the housekeeping gene for RT-PCR and as the loading control for WB assays. ^*P < 0.05.

Figure 2

All sympathetic preganglionic neuronal cell bodies positive for L-glutamic acid decarboxylase of 67 kDa (GAD67), co-expressed ChAT. Double immunostaining in sections of spinal cord for GAD67 (red) and ChAT (green). (A) Low magnification of transverse section of spinal cord depicting the whole gray region, the sympathetic nuclei are shown with boxes. (B) Micrograph of transverse section of spinal cord in cold-stressed rats (n = 4). (C) Micrograph of transverse section of spinal cord in spontaneously hypertensive rats (SHR) adult 12-week old (wo) rats (n = 4). (D) Micrograph of longitudinal section of spinal cord in SHR young 6-wo rats (n = 3). GAD67-IR cell bodies co-expressing ChAT are shown with arrows. Scale bar = 100 μm (A), 10 μm (B–D).

Figure 3

Cold-stress increased segregation of acetylcholine (ACh) and GAD67. (A) Merged images showing the immunolabeling of GAD67 (red), vesicular ACh transporter (VAChT; green) and co-occurrence of both labels (yellow) in superior cervical ganglion (SCG) from control and cold-stressed rats. (B) Graphs showing percent segregation of VAChT/GAD67 in SCG of stressed rats with respect to control. Stress increased the percentage of segregation in the whole ganglion; 67.8 ± 4.4% compared to 50.1 ± 3.6% in control rats (P < 0.05). The caudo-rostral ganglionic regionalization of VAChT/GAD67 segregation found in control animals (rostral 37.1 ± 3.4%, caudal 55.4 ± 5.1%; P < 0.05) was no longer present in stressed rats (rostral 63.7 ± 5.7%, caudal 72.4 ± 2.0%; P > 0.05). Scale bar = 10 μm; n = 5. ^*P < 0.05.

Figure 4

Expression of GAD67 and VAChT in SCG was not modified by cold-stress. (A) GAD67 showed similar presence in control and stressed rats (0.053 ± 0.003% in control and 0.064 ± 0.005% in stress; P > 0.05). GAD67 showed a rostro-caudal gradient in control rats (0.07 ± 0.01% rostral vs. 0.04 ± 0.01% caudal; P < 0.05) that was not present in stressed rats (0.07 ± 0.01% rostral vs. 0.06 ± 0.01% caudal; P > 0.05). (B) VAChT in SCG from control and stressed rats showed no differences in whole ganglia (1.09 ± 0.05% in control and 1.23 ± 0.03% in stress; P > 0.05), or in ganglionic regions in control rats (1.20 ± 0.07% rostral vs. 0.93 ± 0.08% caudal; P < 0.05) and in stress (1.20 ± 0.07% rostral vs. 1.28 ± 0.05% caudal; P > 0.05). Scale bar = 25 μm; n = 5. ^*P < 0.05.

Figure 5

Hypertension did not modify segregation of GAD67/VAChT in SCG from SHR of adult or young rats. However, segregation was larger in young than in adult SHR rats. (A) Merged images showing the co-occurrence (yellow) of two labels: GAD67 (red) and VAChT (green) in 12- and 6- wo SHR and WKy rats. (B) Regional distribution of the level of segregation of GAD67/VAChT in 12- and 6-wo SHR and Wistar Kyoto (WKy) rats. Percent of ACh and GABA segregation in the whole ganglion was larger in young than in adult SHR and WKy rats (68.3 ± 1.1% at 6 wo and 43.5 ± 5.4% at 12 wo; P < 0.001) for SHR and (68.9 ± 1.4% at 6 wo and 40.8 ± 3.1% at 12 wo; P < 0.001) for WKy. This larger segregation in young than in adult rats was also found at intraganglionic regional level, in SHR rostral (68.6 ± 1.6% at 6 wo and 40.0 ± 6.0% at 12 wo; P < 0.001) caudal (69.0 ± 1.5% at 6 wo and 47.2 ± 8.5% at 12 wo; P < 0.01); while in WKy rostral (67.8 ± 2.1% at 6 wo and 40.0 ± 6.9% at 12 wo; P < 0.001) caudal (67.6 ± 3.2% at 6 wo and 50.3 ± 4.9% at 12 wo; P < 0.001). Scale bar = 10 μm; n = 5 for all groups. ^*P < 0.01.

Figure 6

Hypertension increased GAD67 expression in 12- and 6-wo rats and VAChT expression in 6-wo animals. (A) Immunostaining of SCG for GAD67 at 12- and 6-wo SHR and WKy rats. (B) GAD67-containing varicose fibers increased in adult and young SHR rats in the whole ganglion (0.12 ± 0.03% in SHR and 0.03 ± 0.01% in WKy at 12 wo; P < 0.01; in young animals: 0.37 ± 0.01% in SHR and 0.140 ± 0.003% in WKy; P < 0.001). At 12 wo in SHR rostral expression of GAD67 was larger than in caudal region (rostral 0.10 ± 0.02%; caudal 0. 020 ± 0.007%; P < 0.05). (C) Immunostaining of SCG for VAChT in 12 and 6 wo in SHR and WKy rats. (D) Although there were no changes in VAChT expression between SHR and WKY at 12 wo (0.79 ± 0.19%, in SHR; 0.52 ± 0.13%, in WKy, P > 0.05), VAChT expression was greater in SHR at 6 wo (1.13 ± 0.05% in SHR and 0.75 ± 0.05%, in WKy; P < 0.01). At 12 wo, there was a rostro-caudal gradient in VAChT expression in both strands SHR (rostral 0.70 ± 0.02%; caudal 0.29 ± 0.02%; P < 0.001); WKy (rostral 0.60 ± 0.01%; caudal 0.39 ± 0.01%; P < 0.001). At 6 wo, there was no regional distribution in VAChT expression SHR (1.11 ± 0.08% rostral and 1.16 ± 0.06% caudal, P > 0.05); WKy (0.78 ± 0.06% rostral and 0.74 ± 0.04% caudal, P > 0.05). Scale bar = 25 μm; n = 5 for all groups. ^*P < 0.05.