Dopamine, acting at the D1 family receptors (D1R) is critical for the functioning of the amygdala, including fear conditioning and cue-induced reinstatement of drug self administration. However, little is known about the different contributions of the two D1R subtypes, D1 and D5. W identified D1-immunoreactive patches in the primate that appear similar to the intercalated cell masses reported in the rodent; however, both receptors were present across the subdivisions of the primate amygdala including the basolateral amygdala (BLA). Using immunoelectron microscopy, we established that both receptors have widespread distributions in BLA. The D1R subtypes colocalize in dendritic spines and terminals, with D1 predominant in spines and D5 in terminals. Single cell PCR confirmed that individual BLA projection neurons express both D1 and D5 mRNA. The responses of primate BLA neurons to dopamine and D1R drugs were studied using in vitro slices. We found that responses were similar to those previously reported in rat BLA neurons and included a mixture of postsynaptic and presynaptic actions. Given this we investigated the distribution of D1R in the rat BLA and found that there were similarities between the species, such as more prominent D5 localization to presynaptic structures. The higher affinity of D5 for dopamine suggests that presynaptic actions may predominate in the BLA at low levels of dopamine, while postsynaptic effects increase and dominate as dopaminergic drive increases. The results presented here suggest a complex action of dopamine on BLA circuitry that may evolve with different degrees of dopaminergic stimulation.
Stress is a growing public health concern and can lead to significant disabilities. The neural response to stressors is thought to be dependent on the extended amygdala. The basolateral amygdala (BLA) is responsible for associations of sensory stimuli with emotional valence and is thought to be involved in stress-induced responses. Previous behavioral and electrophysiological experiments demonstrate that, in response to stress, changes occur in glutamatergic neurotransmission within the BLA and, in particular in transmission at AMPA receptors. Given the established role of AMPA receptors in memory and synaptic plasticity, we tested the hypothesis that stress produces alterations in the distribution of these receptors in a way that might account for stress-induced alterations in amygdala circuitry function. We examined the subcellular localization of GluR1 subunits of the AMPA receptor and the electrophysiological characteristics of BLA principal neurons in an animal model of unpredictable stress. Compared to controls, we demonstrated an increase in the ratio of labeled spines to labeled dendritic shafts in the BLA of rats 6 and 14 days post-stress, but not 1 day post-stress. Furthermore, the frequency of mini-EPSCs was increased in stressed animals without a change in general membrane properties, mini-EPSC amplitude, or in paired pulse modulation of glutamate release. Taken together, these data suggest that the shift of GluR1-containing AMPA receptors from dendritic stores into spines may be in part responsible for the persistent behavioral alterations observed following severe stressors.
The actions of dopamine D1 family receptors (D1R) depend upon a signal transduction cascade that modulates the phosphorylation state of important effector proteins, such as glutamate receptors and ion channels. This is accomplished both through activation of protein kinase A (PKA) and the inhibition of protein phosphatase-1 (PP1). Inhibition of PP1 occurs through PKA-mediated phosphorylation of DARPP-32 or the related protein inhibitor-1 (I-1), and the availability of DARPP-32 is essential to the functional outcome of D1R activation in the basal ganglia. While D1R activation is critical for prefrontal cortex (PFC) function, especially working memory, the functional role played by DARPP-32 or I-1 is less clear. In order to examine this more thoroughly, we have utilized immunoelectron microscopy to quantitatively determine the localization of DARPP-32 and I-1 in the neuropil of the rhesus monkey PFC. Both were distributed widely in the different components of the neuropil, but were enriched in dendritic shafts. I-1 label was more frequently identified in axon terminals than was DARPP-32, and DARPP-32 label was more frequently identified in glia than was I-1. We also quantified the extent to which these proteins were found in dendritic spines. DARPP-32 and I-1 were present in small subpopulations of dendritic spines, (4.4 and 7.7% and respectively), which were substantially smaller than observed for D1R in our previous studies (20%). Double-label experiments did not find evidence for colocalization of D1R and DARPP-32 or I-1 in spines or terminals. Thus, at the least, not all prefrontal spines which contain D1R also contain I-1 or DARPP-32, suggesting important differences in D1R signaling in the PFC compared to the striatum.
The anterolateral cell group of the bed nucleus of the stria terminalis (BNSTALG) serves as an important relay station in stress circuitry. Limbic inputs to the BNSTALG are primarily glutamatergic and activity-dependent changes in this input have been implicated in abnormal behaviors associated with chronic stress and addiction. Significantly, local infusion of acetylcholine (ACh) receptor agonists into the BNST trigger stress-like cardiovascular responses, however, little is known about the effects of these agents on glutamatergic transmission in the BNSTALG. Here, we show that glutamate- and ACh-containing fibers are found in close association in the BNSTALG. Moreover, in the presence of the acetylcholinesterase inhibitor, eserine, endogenous ACh release evoked a long-lasting reduction of the amplitude of stimulus-evoked EPSCs. This effect was mimicked by exogenous application of the ACh analogue, carbachol, which caused a reversible, dose-dependent, reduction of the evoked EPSC amplitude, and an increase in both the paired pulse ratio and coefficient of variation, suggesting a presynaptic site of action. Uncoupling of postsynaptic G-proteins with intracellular GDP-β-S, or application of the nicotinic receptor antagonist, tubocurarine, failed to block the carbachol effect. In contrast, the carbachol effect was blocked by prior application of atropine or M2 receptor-preferring antagonists, and was absent in M2/M4 receptor knockout mice, suggesting that presynaptic M2 receptors mediate the effect of ACh. Immuno-electron microscopy studies further revealed the presence of M2 receptors on axon terminals that formed asymmetric synapses with BNST neurons. Our findings suggest that presynaptic M2 receptors might be an important modulator of the stress circuit and hence a novel target for drug development.
D1 family receptors (D1R) in prefrontal cortex (PFC) are critical for normal cognition and are implicated in pathological states such as schizophrenia. The two D1R subtypes, D1 and D5, cannot be pharmacologically distinguished but have important functional differences. To understand their contributions to cortical function, we quantified their localization in the neuropil of primate PFC. We identified different patterns of distribution for the two receptors that showed variation across cortical laminae. Although D1 was enriched in spines and D5 in dendrites, there was considerable overlap in their distribution within neuronal compartments. To determine whether the D1 and D5 receptors are localized to separate populations of synapses, we employed double-labeling methods. We found the two receptors colocalized and quantified the overlap of their distribution in spines and axon terminals of prefrontal cortical area 9 in the Macaca mulatta monkey. The two receptors are found in partially overlapping populations, such that the D5 receptor is found in a subpopulation of those spines and terminals that contain D1. These results indicate that dopamine activation of the two D1R subtypes does not modulate disparate populations of synapses onto dendritic spines in prefrontal cortical area 9; rather, dopamine can activate D1 and D5 receptors on the same spines, plus an additional group of spines that contains only D1. The implications of these results for the dose-dependent relationship between D1R activation and PFC function are discussed.
Working memory (WM) is a core cognitive process that depends upon activation of D1 family receptors (D1R) and inhibitory interneurons in the prefrontal cortex (PFC). D1R are comprised of the D1 and D5 subtypes, and D5 has a 10-fold higher affinity for dopamine. Parvalbumin (PV) and calretinin (CR) are 2 interneuron populations that are differentially affected by D1R stimulation and have discrete postsynaptic targets, such that PV interneurons provide strong inhibition to pyramidal cells, whereas CR interneurons inhibit other interneurons. The distinct properties of both the D1R and interneuron subtypes may contribute to the “inverted-U” relationship of D1R stimulation and WM ability. To determine the prevalence of D1 and D5 in PV and CR interneurons, we performed quantitative double-label immunoelectron microscopy in layer III of macaque area 9. We found that D1 was the predominant D1R subtype in PV interneurons and was found mainly in dendrites. In contrast, D5 was the predominant D1R subtype in CR interneurons and was found mainly in dendrites. Integrating these findings with previously published electrophysiological data, we propose a circuitry model as a framework for understanding the inverted-U relationship between dopamine stimulation of D1R and WM performance.
As part of a study of antipsychotic drug treatment in monkeys, we developed a technique to provide chronic, constant-rate, gastric drug infusion in nontethered rhesus macaques. This method allowed us to mimic the osmotic release oral delivery system currently used in humans for continuous enteral drug delivery. Rhesus macaques (n = 5) underwent gastric catheter placement by laparotomy. After the catheters were secured to the stomach, the remaining catheter length was exited through the lateral abdomen, tunneled subcutaneously along the back, and connected to a 2-mL osmotic pump enclosed in a subcutaneous pocket. Osmotic pumps were changed every 2 to 4 wk for 1 y and remained patent for the duration of the study. Four complications (including cutting of the catheter, incisional dehiscence at the pump site, and loss of 1 catheter into the abdominal cavity requiring catheter replacement) occurred among the 80 pump changes performed during the yearlong study. At necropsy, histopathologic examination of the catheter implant sites revealed mild changes consistent with a foreign-body reaction. Our results indicate that the gastric catheter and osmotic pump system was well tolerated in rhesus macaques for as long as 12 mo after placement and suggest that this system will be an attractive option for use in studies that require chronic, constant-rate, gastric drug infusion in nontethered monkeys.
Mutations in DJ-1 cause autosomal recessive, early-onset Parkinson's disease (PD). The precise function and distribution of DJ-1 in the central nervous system remain unclear. In this study, we performed a comprehensive analysis of DJ-1 expression in human, monkey, and rat brains using antibodies that recognize distinct, evolutionarily conserved epitopes of DJ-1. We found that DJ-1 displays region-specific neuronal and glial labeling in human and non-human primate brain, sharply contrasting the primarily neuronal expression pattern observed throughout rat brain. Further immunohistochemical analysis of DJ-1 expression in human and non-human primate brains showed that DJ-1 protein is expressed in neurons within the substantia nigra pars compacta and striatum, two regions critically involved in PD pathogenesis. Moreover, immunoelectron microscopic analysis revealed a selective enrichment of DJ-1 within primate striatal axons, presynaptic terminals, and dendritic spines with respect to the DJ-1 expression in prefrontal cortex. Together, these findings indicate neuronal and synaptic expression of DJ-1 in primate subcortical brain regions and suggest a physiological role for DJ-1 in the survival and/or function of nigral-striatal neurons.
Blockade of D2 family dopamine receptors (D2Rs) is a fundamental property of antipsychotics, and the degree of striatal D2R occupancy has been related to antipsychotic and motor effects of these drugs. Recent studies suggest the D2R occupancy of antipsychotics may differ in extrastriatal regions compared with the dorsal striatum. We studied this issue in macaque monkeys by using a within-subjects design. [18F]fallypride positron emission tomography scans were obtained on four different doses of risperidone and paliperidone (the 9-OH metabolite of risperidone) and compared with multiple off-drug scans in each animal. The half-life of the two drugs in these monkeys was determined to be between 3 and 4 h, and drug was administered by a constant infusion through an intragastric catheter. The D2R occupancy of antipsychotic was determined in the caudate, putamen, ventral striatum, and four prefrontal and temporal cortical regions and was related to serum and cerebrospinal fluid drug levels. Repeated 2-week treatment with risperidone or paliperidone did not produce lasting changes in D2R binding potential in any region examined. As expected, D2R binding potential was highest in the caudate and putamen and was approximately one-third that level in the ventral striatum and 2% of that level in the cortical regions. We found dose-dependent D2R occupancy for both risperidone and paliperidone in both basal ganglia and cortical regions of interest. We could not find evidence of regional variation in D2R occupancy of either drug. Comparison of D2R occupancy and serum drug levels supports a target of 40 to 80 ng/ml active drug for these two atypical antipsychotics.