The present study reveals disruption of key components of the PSD and coupling to the endocytic zone in the amygdala of human heroin, cocaine and polysubstance heroin-cocaine users that strongly imply disturbances in the regulation of synaptic plasticity.
Regardless of the nature of the illicit drug abused, there was a strong positive correlation between GluA1 and PSD-95 mRNA expression levels that was not observed in control subjects. Similarly, correlation structure analysis of the network of proteins related to glutamatergic neurotransmission revealed that the GluA1—PSD-95 relationship specifically distinguished heroin abusers from controls. These findings are intriguing given that PSD-95 induces GluA1 delivery into synapses, which is coupled to the strengthening of excitatory synapses during experience-driven learning (6
). In addition, trafficking of GluA1 into the active synaptic site is consistently observed in relation to drug-seeking behavior in animal models (33
Most of the information garnered to date about amygdala dysfunction of synaptic plasticity derive from studies of fear conditioning that is widely used to examine associative emotional memory formation (37
), which is of critical importance in the etiology of addiction. As such, similar neurobiological mechanisms are likely to play a significant role in both fear conditioning and the development of addiction disorders (38
). It is well documented that fear conditioning induces strengthening of excitatory synapses within the lateral and basal amygdala nuclei and requires trafficking of GluA1 into synapses (40
). Increased GluA1 in the plasma membrane has been reported following fear conditioning although the total amount of GluA1 mRNA and protein levels are unchanged (43
). The GluA1 and PSD-95 correlation observed in our study was also not accompanied by alteration in the total GluA1 or PSD-95 mRNA or protein levels. It is therefore possible that functional rearrangement of GluA1 subunits is masked when measuring total levels of GluA1 similar to that observed in morphine-exposed rats (44
). It would therefore be tempting to speculate that the strong coupling between GluA1 and PSD-95 in drug abusers represent an induction of synaptic GluA1 that leads to strengthening of synaptic connectivity and increased responsiveness of the amygdala during, for example, relapse.
In addition to the strong association evident between GluA1 and PSD-95, the relationship detected between Homer and dynamin-3 in heroin abusers would also strongly suggest enhanced availability of AMPA receptors at glutamatergic synapses and thus potentiated synaptic transmission. Homer is concentrated to the PSD and together with dynamin-3 is localized to the lateral spine membrane with a distribution that spans the PSD and endocytic zone (32
). In vitro
studies have demonstrated that the physical link between dynamin-3 and Homer positions the endocytic zone near the PSD to maintain cycling AMPA receptors at the synapse (31
). Those investigations provide clear evidence that synapses lacking a PSD directly linked to the endocytic zone results in depletion of AMPA receptors at the synapse thereby leading to a reduction of excitatory synaptic transmission. Our data demonstrating that the physical coupling between Homer 1b/c and dynamin-3 was positively correlated to blood morphine levels would be in line with an upregulation of AMPA transmission upon drug intake.
While the recent use of heroin was associated with enhanced Homer 1b/c—dynamin-3 interaction, both PSD proteins were significantly increased irrespective of blood morphine levels suggesting persistent disturbance of the excitatory synapse than only a rapid dynamic modulation due to the immediate pharmacological action of the drug. Enhanced Homer 1 b/c protein in the lateral amygdala was also evident in cocaine users emphasizing the important upregulation of the scaffolding protein in association with both psychostimulant and opiate drugs. Despite the critical role of Homer 1b/c and dynamin-3 in synaptic plasticity, no data are currently available in regard to dynamin-3's potential role in addiction and only limited investigations have directly evaluated amygdala Homer regulation in relation to behavior. To date, studies have examined either transgenic animals with a global developmental knockout of Homer 1 or have focused on manipulating Homer specifically in the nucleus accumbens (21
). Such studies, based on locomotor sensitization as the behavioral indicator of addiction sensitivity, have led to the speculation that overexpression of Homer reduces addiction vulnerability (21
). However, no animal investigation has evaluated Homer regulation specifically in the amygdala, thus brain region-specific disturbances in the long Homer isoforms might underlie different components of the addiction phenotype. This is particularly relevant since it has been recently documented, for example, that Homer 1b/c is differentially altered in the nucleus accumbens (decreased) and prefrontal cortex (increased) after early drug withdrawal in animals that self-administered cocaine (48
). Moreover, the prefrontal cortical increase of Homer 1b/c was only evident in animals that experienced daily, extended access to cocaine self-administration (48
), an animal model mimicking loss of control over drug intake, compared to animals with only short access to the drug. Such findings implicate an important contribution of compulsive drug use to the cortical Homer 1b/c alterations. It remains to be studied whether allocortical amygdala Homer 1 b/c alterations are more comparable to those evident in the prefrontal cortex in contrast to that seen in the nucleus accumbens. The current results obtained by the direct investigation of human abusers provide a significant foundation to guide future animal studies in evaluating novel amygdala molecular targets as potential contributors to the short-term and long-term regulation of behaviors more reflective of the human addiction condition.
Another interesting observation of the present study was the association of glutamatergic markers with aging. In addition to demonstrating a decline of GluA1 with age that has been observed in the hippocampus in rats (49
), the current findings document for the first time an age-related decline in regard to the interaction between dynamin-3 and Homer 1b/c. Such impairment of the PSD and the coupling to the endocytic zone would be consistent with the known reduction of synaptic plasticity and concomitant deficit in learning and memory as seen during normal aging (50
There are inherent limitations with post-mortem human brain studies with confounds such as drug use history, co-morbidity with psychiatric disorders, and lifestyle characteristics that are difficult to validate. In addition, studies of the post-mortem human brain only allow evaluation at a fixed time point; thus following the trajectory of neurobiological measures with behaviors over time is not feasible and it is impossible to know the state of these systems prior to drug use. Nevertheless it is critical to expand our understanding of what is a quintessential human condition by the direct study of the human brain and that can provide a foundation for future experimental animal models in which causal relationship to specific addiction-related behaviors can be determined.
In conclusion, our study reveals dynamic synchronization of PSD-95 and GluA1 in the amygdaloid complex of human drug abusers. The fact that enhanced GluA1-PSD-95 coupling, well established to reflect strengthening of excitatory synapses, was evident in heroin, cocaine, and polysubstance users is consistent with the hypothesis that potentiated glutamatergic long-term plasticity is a common feature of drug abuse. Upregulation of amygdala dynamin-3 and Homer 1b/c levels together with potentiation of their physical interaction suggests abnormality of the PSD and endocytic zone structural network in the amygdala of drug abusers. Such disturbances might be a fundamental component of the pathophysiology underlying addiction disorder.