Pharmacological target sites for restoration of age-associated deficits in NMDA receptor-mediated norepinephrine release in brain

Abstract

Aging affects virtually all organs of the body, but perhaps it has the most profound effects on the brain and its neurotransmitter systems, which influence a wide range of crucial functions, such as attention, focus, mood, neuroendocrine and autonomic functions, and sleep cycles. All of these essential functions, as well as fundamental cognitive processes such as memory, recall, and processing speed, utilize neuronal circuits that depend on neurotransmitter signaling between neurons. Glutamate (Glu), the main excitatory neurotransmitter in the CNS, is involved in most neuronal excitatory functions, including release of the neurotransmitter norepinephrine (NE). Previous studies from our lab demonstrated that the age-associated decline in Glu-stimulated NE release in rat cerebral cortex and hippocampus mediated by NMDA glutamate receptors, as well as deficits in dendritic spines, and cognitive functions are fully rescued by the CNS stimulant amphetamine. Here we further investigated Glu-stimulated NE release in the cerebral cortex to identify additional novel target sites for restoration of Glu-stimulated NE release. We found that blockade of alpha-2 adrenergic receptors fully restores Glu-stimulated NE release to the levels of young controls. In addition, we investigated the density and responsiveness of NMDA receptors as a potential underlying neuronal mechanism that could account for the observed age-associated decline in Glu-stimulated NE release. In the basal state of the receptor (no added glutamate and glycine) the density of NMDA receptors in the cortex from young and aged rats was similar. However, in contrast, in the presence of 10 μM added glutamate, which opens the receptor channel and increases the number of available [³H]-MK-801 binding sites within the channel, the density of [³H]-MK-801 binding sites was significantly less in the cortex from aged rats.

Keywords: NMDA receptors; aging; alpha‐2‐adrenergic receptors; norepinephrine release.

FIGURE 1

The effect of aging on [³H]‐NE release from young (2–3 months old) and aged (18–24 months old) Fischer 344 rat cortical brain slices and its rescue. In (a), the descriptive experimental timeline indicates total fractional NE release from young rat cortical slices (n = 7) in response to a 1 mM Glu stimulation (at 24–26 min time points) and 30 mM K⁺ (at 32–34 min time points). In (b), the age‐associated decline in the Glu‐stimulated [³H]‐NE release was rescued using 3 μM MPH in the cerebral cortex tissue slices from male (solid symbol) and female (open symbol) F344 rats (young rats n = 12 and aged rats n = 10). (c) 30 mM K⁺‐ evoked [³H]‐NE release in young versus aged rats. Each bar is the mean ± SEM of net fractional release (%) in each group after subtracting the basal fractional release (~1.49% of the total tritium content). Data were analyzed using two‐way ANOVA with Tukey's multiple comparison tests: ***p = 0.001, ****p < 0.0001, whereas the effect of aging on K⁺‐evoked release was analyzed using an unpaired t‐test. NE, norepinephrine; Glu, glutamate; KCL, potassium chloride; MPH, methylphenidate.

FIGURE 2

Presynaptic α2‐adrenoceptors receptors modulate the glutamate‐stimulated release of [³H] ‐NE in aged Fischer 344 rat cortical brain slices. In (a), α2‐adrenoreceptors receptors agonist, Clonidine, decreases glutamate (Glu)‐stimulated [³H]‐NE release in a concentration‐dependent manner in young rat cortex (n = 3–5). In (b), [³H]‐NE release was potentiated by antagonizing α2‐adrenoceptors receptors in the cerebral cortex from young (n = 4–11) and aged (n = 4–8) with 10 μM phentolamine, idazoxan, and mirtazapine. Each data point represents a duplicate of one animal. Data were analyzed using two‐way ANOVA with Dunnett's multiple comparison test; *p ≤ 0.05, **p ≤ 0.01, ****p < 0.0001; ^## p < 0.01, ^### p < 0.001 (# for aged rats comparison using a mixed‐effect analysis followed by Dunnett's multiple comparison test).

FIGURE 3

Characterizing the cellular localization of the NMDA receptors regulating [³H] ‐NE release in young rat cortical brain slices. 1 mM Glu‐stimulated‐[³H]‐NE releases in the cerebral cortex tissue slices from young rats (n = 3–7) in the presence 1 & 3 μM of the TTX, voltage‐gated Na channel blocker, 10 μM MK‐801, and a combination of MK‐801 and TTX. Data are expressed as mean (±SEM) of net fractional release (stimulated—basal), with each data point representing a duplicate from one animal. Data were analyzed using a mixed‐effect analysis followed by Dunnett's multiple comparison test ****p < 0.0001. NE, norepinephrine; Glu, glutamate; TTX, tetrodotoxin.

FIGURE 4

The stimulatory effect of Glutamate Vs. NMDA on [³H] ‐NE release in young rat cortical brain slices. In (a), the concentrations‐response curves of glutamate and NMDA‐stimulated [³H] ‐NE releases in the cerebral cortex tissue slices from young rats (n = 4). In (b), the 1 mM glutamate and NMDA stimulated NE release in the presence and absence of 1.2 mM magnesium in the cerebral cortex tissue slices from young rats (n = 3). Data were analyzed using an unpaired t‐test. *p ≤ 0.05, **p ≤ 0.01 NE, norepinephrine; Glu, glutamate; NMDA, N‐methyl‐d‐aspartate; Mg²⁺, magnesium.

FIGURE 5

Age‐associated changes in the expression of NMDA receptors freely solubilize subunits in the cortical rat tissue homogenate. A representative Western blot for NMDA receptors subunits in the cerebral cortex in (a); and in (b) quantified results for GluN1, GluN2A, and GluN2B expressions in young and aged rats (n = 5). Data are expressed as mean (±SEM) normalized to young rats, with each data point representing a duplicate from one animal. Data were analyzed using an unpaired t‐test. *p ≤ 0.05. NMDA, N‐methyl‐d‐aspartate).

FIGURE 6

Effect of aging on [³H]‐MK‐801 binding to NMDA receptors in the young (2–3 months old) and aged (18–24 months old) rat cortical tissue membrane. In (a) saturation curve of [³H]‐MK‐801 binding to NMDA receptors in young rats in the presence of 10 μM Glu and Gly (n = 4). The non‐linear least‐squares fitting of the saturation isotherm yielded K _d and B _max values of 1.8 nM and 970 fmol/mg of protein, respectively. Both total and non‐specific binding of [³H]‐MK‐801 is shown in the curve, and the specific [³H]‐MK‐801 binding is presented with 95% CI in dotted lines. Inset: Saturation data graphed as Scatchard plots. Whereas in (b), the binding of 10 nM [³H]‐MK‐801 in +/− 10 μM Glu and Gly in young and aged rats (n = 7), each performed in triplicate and repeated twice. In (c), the % increases after subtracting baseline binding from the binding in the presence of 10 μM Glu and Gly. Baseline Binding values represent [³H]‐MK‐801 binding without exogenous addition of Glu and Gly. Data were analyzed using a Mixed‐effect analysis followed by Tukey's multiple comparison tests. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001; whereas the % of increase over the baseline was analyzed using an unpaired t‐test: **p ≤ 0.01. NMDA, N‐methyl‐d‐aspartate; Glu, Glutamate; Gly, Glycine; K _d, dissociation constant; B _max, Maximum Binding; n_H, Hill Coefficient).