Glutamate signaling proteins and tyrosine hydroxylase in the locus coeruleus of alcoholics

Abstract

It has been postulated that alcoholism is associated with abnormalities in glutamatergic neurotransmission. This study examined the density of glutamate NMDA receptor subunits and its associated proteins in the noradrenergic locus coeruleus (LC) in deceased alcoholic subjects. Our previous research indicated that the NMDA receptor in the human LC is composed of obligatory NR1 and regulatory NR2C subunits. At synapses, NMDA receptors are stabilized through interactions with postsynaptic density protein (PSD-95). PSD-95 provides structural and functional coupling of the NMDA receptor with neuronal nitric oxide synthase (nNOS), an intracellular mediator of NMDA receptor activation. LC tissue was obtained from 10 alcohol-dependent subjects and eight psychiatrically healthy controls. Concentrations of NR1 and NR2C subunits, as well as PSD-95 and nNOS, were measured using Western blotting. In addition, we have examined tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of norepinephrine. The amount of NR1 was lower in the rostral (-30%) and middle (-41%) portions of the LC of alcoholics as compared to control subjects. No differences in the amounts of NR2C, PSD-95, nNOS and TH were detected comparing alcoholic to control subjects. Lower levels of NR1 subunit of the NMDA receptor in the LC implicates altered glutamate-norepinephrine interactions in alcoholism.

Figure 1

Relationship between the optical density values of NR1, NR2C, PSD-95, nNOS, TH, actin and total protein concentration in the human LC. Wells were loaded with 10, 20, and 40 µg of total human LC protein.

Figure 2

Immunoblots of NR1, NR2C, PSD-95, nNOS, TH and actin from a representative pair of control/alcoholic subjects used in the analysis. Shown are blots from three separate anatomical levels of the LC: rostral (R), middle (M), and caudal (C). Each well was loaded with 20 µg of total protein. Additionally, each gel was loaded with three concentrations of LC standard (dissected from a healthy control subject) consisting of 10–40 µg of total protein.

Figure 3

Amounts of NR1, NR2C, PSD-95, and nNOS immunoreactivities in the LC from control subjects (open bars; n=8) and alcohol dependent subjects (filled bars; n=10). Immunoreactivities were measured at the rostral, middle and caudal portions of the LC. Asterisks indicate statistical significance (p<0.01) Comparing amounts of immunoreactivity in alcoholic subjects to control subjects at the same anatomical level.

Figure 4

Amount of TH immunoreactivity and neuromelanin containing cell numbers from control subjects (open bars; n=8) and alcohol dependent subjects (filled bars; n=10). Immunoreactivities and LC cell numbers were estimated at the rostral, middle and caudal portions of the LC.

Figure 5

Body mass index (BMI) in psychiatrically normal control subjects (open bars, n=8) and in subjects diagnosed with alcohol dependence (filled bars; n=10). Each bar represents the mean ± SEM. There was no significant difference in BMI comparing alcoholics to control subjects.