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Which brain regions are D1 dopamine receptors expressed, and which brain regions are D2 dopamine receptors expressed?

Which brain regions are D1 dopamine receptors expressed, and which brain regions are D2 dopamine receptors expressed?


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This is a follow-up question to If D1 receptors stimulate adenylate cyclase (through GPCRs) and D2 receptors inhibit it, then why do mutations in both have similar effects?.

As a further question - I'd like to ask: do D1 dopamine receptors have the same (excitatory) effect everywhere in the brain? And do D2 dopamine receptors have the same (inhibitory) effect everywhere in the brain?


As to your main question, I imagine the Paul Allan Brain Atlas has what you're looking for. In 3D even.

Offhand, I cannot be of assistance for the second question.


D1 Dopamine Receptor

Introduction

The D1 dopamine receptor is activated by the catecholamine dopamine and is a member of the rhodopsin-like dopamine receptor family of the 7-transmembrane superfamily of receptors . The D1 receptor is one of two mammalian D1-like receptor subtypes, D1 and D5 . The D1 dopamine receptor is a postsynaptic or heterosynaptic (i.e., located on the terminals of non-dopaminergic neurons) receptor that couples to the heterotrimeric G proteins Gs and Golf to stimulate adenylate cyclase activity and cyclic AMP accumulation. The D1 receptor is the most abundant of the D1-like receptors and appears to be the subtype responsible for most of the effects attributed to stimulation of this receptor type Jose et al (1998) , Holmes et al (2001) .


Background

Dopamine plays a key role in the regulation of various physiological functions of normal brain including reward, locomotion, behavior, learning, and emotion. It is not then surprising that the dysregulation of the dopaminergic system has been linked to pathophysiology of many diseases, such as Alzheimer's disease, schizophrenia, Parkinson's disease, attention deficit hyperactivity disorder, depression and drug addiction [1–3], leading to the clinical use of drugs that target dopamine neurotransmission in the treatment of these disorders.

Five subtypes of dopamine receptors (D1R-D5R), belonging to the G-protein-coupled receptor (GPCR) superfamily have been cloned, through which dopamine transduces its various effects. Dopamine receptors are subdivided into D1-like (D1, D5) and D2-like (D2, D3, D4) receptor subclasses [1–3], with the D1 and D2 receptors being the major subtypes. The most studied dopamine signaling pathway is the modulation of cyclic AMP production, with D1-like receptors activating cyclic AMP production through Gs/olf, and D2-like receptors inhibiting adenylyl cyclase (AC) activity through Gi/o proteins [2]. This results in a bidirectional modulation of this pathway and related proteins, such as protein kinase A (PKA) and DARPP-32 (dopamine and cAMP regulated protein) [4]. Other important dopamine signaling pathways have also been reported, including the modulation of the Akt-GSK3 pathway [5] and the activation of the PAR4 signaling pathway [6].

For some actions of dopamine, such as the control of motor behavior [7] or dopamine-mediated reward processes in nucleus accumbens [8], a concomitant stimulation of D1 and D2 receptors is required, a phenomenon known as the "requisite" D1/D2 synergism [9]. In this type of synergism, D1 and D2 receptor-specific drugs potentiate the effect exerted by each other when delivered together, but are ineffective when administered separately [9]. The combined, but not separate, administration of a selective D1 and a selective D2 agonist was shown to be necessary for the dopamine-stimulated expression of immediate-early gene c-fos in striatal neurons [10] and in electro-physiological studies where both receptors were indeed responsible for GABA release in striatum [11]. The participation of both D1 and D2 receptors was also required for evoking neural and behavioral sensitization to cocaine [12] and for evoking the changes in behavior and basal ganglia output [13, 14]. All these observations are other evidence for the presence of not only a synergism between dopamine D1 and D2 receptors, but an obligatory participation of both receptors to generate this synergism.

One explanation for how the well documented synergistic effects seen between D1 and D2 receptors [15, 16] may be achieved is through the formation of heterooligomers between the two receptors, as it has been shown for many GPCRs [17–19]. Dopamine receptors, all subtypes included, in addition to their ability to exist as homomers, were shown to form different heteromeric complexes with other receptors (reviewed in 20). The presence of D1-D2 receptor heteromers with unique functional properties was first shown in transfected cells using different methods [21–24] as described below. Initially, the notion of heteromerization observed for many GPCRs and its functional relevance was not completely clear in physiological conditions and was in some cases regarded with a degree of skepticism, but at least for the D1-D2 receptor heteromer we have shown evidence of occurrence under physiological conditions in native tissues with emerging important functional relevance.

For D1 and D2 receptors, the presence of two anatomically segregated sets of neurons, forming the striatonigral D1-enriched direct pathway and the striatopallidal D2-enriched indirect pathway is commonly recognized, with D1R localizing to the dynorphin (DYN)-expressing neurons, and D2R localizing to the enkephalin (ENK)-expressing neurons [25, 26]. Recent studies emanating from fluorophore-tagged promoter elements of D1R and D2R in bacterial artificial chromosome (BAC) transgenic mice [27] allowed an evaluation of the proportions of striatal neurons expressing D1R, D2R, or both [28–32]. There were, however, variations in the levels of expression of EGFP between one line and another [32], resulting in incomplete labeling of a significant proportion of striatal medium spiny neurons (MSNs) [28]. While this method supported the segregation between the D1-enriched direct pathway and the striatopallidal D2-enriched indirect pathway, a certain fraction of MSNs (

17%) expressing both receptors was predicted in the NAc shell, whereas only

5-6% of MSNs were calculated to co-express both receptors in the dorsal striatum [30–32]. These BAC-calculated colocalization data are consistent with our data and the numerous other reports indicating a colocalization of D1R and D2R in neurons in culture or in situ with higher D1R and D2R co-localization observed in cultured striatal neurons (60 to 100%) than in the adult striatum [33–40].

Presence of dopamine D1-D2 receptor heteromers in brain

Several reports indicated the presence of a D1-like receptor activating IP3 production and/or increasing intracellular calcium in neurons in culture or slices from different brain regions, including striatum, hippocampus, and cortex [41–44]. However, the cloned D1R was devoid of such effects when expressed in different host cells (reviewed in 17 and 20) and persisted in a D1 receptor null mouse model [45]. We then demonstrated that dopamine D1 and D2 receptors form functional heterooligomeric complexes in cells and in vivo [21–23, 40, 46] and that the mobilization of intracellular calcium was in fact a unique signaling pathway resulting from the activation of this D1-D2 heteromeric receptor complex [21, 23, 40].

The presence of the D1-D2 receptor heteromer was demonstrated by different techniques including coimmunoprecipitating both receptors from rat striatum, as well as from cells coexpressing D1R and D2R [21, 40], and by different methodologies using the fluorescence resonance energy transfer (FRET) technique in cells [22, 24], in striatal neurons [40, 47] and different brain regions [40, 46].

Interestingly, in adult rat brain, coexpressed dopamine D1 and D2 receptors were present in a unique subset of neurons coexpressing both DYN and ENK neuropeptides in different brain regions, including nucleus accumbens (NAc), caudate-putamen (CP), ventral pallidum, globus pallidus (GP), and entopeduncular nucleus [46], with some inter-regional variation. The lowest proportion (

6-7%) of D1R-expressing neurons that coexpress D2R was shown in the CP [40, 46], whereas the highest proportion (

59%) of D1R-expressing neurons that coexpress D2R was observed in GP [46]. A substantial number (

20-30%) of D1R neurons that coexpress D2R was also observed in NAc [40, 46], consistent with the anatomical findings resulting from BAC transgenic mice [30–32].

The direct interaction of D1R and D2R to form heteromers in brain was shown by confocal FRET technique using two methodologies [40, 46, 47]. The confocal FRET technique demonstrated clearly and directly the presence of the D1-D2 receptor heteromer in striatal neurons [40, 47] and in brain in situ[40, 46]. In NAc, acceptor photobleaching-based FRET showed a high FRET efficiency of

20%) as with a second quantitative confocal FRET, that further quantified the parameters of the interaction between D1R and D2R to calculate the FRET efficiency and the assessment of the distance separating both fluophore-tagged receptors [40, 46]. In NAc, interactions between colocalized D1R and D2R (Figure 1) displayed high FRET efficiency (

20%) and a relative distance of 5-7 nm (50-70 Å) (Table 1), synonymous with a close proximity between D1 and D2 receptors and indicative of D1-D2 heteromer formation. In contrast, although an indication of D1-D2 heteromer formation in CP was observed, the parameters, FRET efficiency (

5%) and the relative distance of 8-9 nm (80-90 Å) between the receptors suggested that in CP either D1R-D2R interaction was weaker, or fewer D1-D2 receptor heteromers were formed, and/or lower order of D1-D2 oligomers than in the NAc was present [40, 46].

Example of Confocal FRET analysis of D1 and D2 receptor interaction in a medium spiney neuron from the core region of rat nucleus accumbens. Anti-D2-Alexa 350 (green) and anti-D1-Alexa 488 (red) were used as donor and acceptor dipoles. The FRET signal was detected and measured in microdomains [regions of interest (ROIs)] within the neuron coexpressing D1 and D2 receptors. Analysis shows the FRET efficiency and the distance separating the dipoles.

D1-D2 receptor heteromer-induced signaling pathway and its physiologic relevance

The specific activation of the D1-D2 receptor heteromer in postnatal striatal neurons [40], and from cells co-expressing D1R and D2R [21, 23] resulted in the intracellular release of calcium from stores sensitive to activation of inositol triphosphate receptors (IP3-R). This rise in intracellular calcium was rapid, transient, independent of extracellular calcium influx, and involved the activation of Gq protein, and phospholipase C (PLC) [21, 23, 40]. This calcium signal resulted in an increase in the phosphorylated-activated form of CaMKIIα in postnatal striatal neurons [40] and rat striatum [23]. The use of dopamine D1 -/- , D2 -/- and D5 -/- receptor null mice indicated clearly that the calcium-CaMKIIα signaling pathway exclusively involved both D1R and D2R within a functional complex [23, 40], and was different from the calcium signal generated by the activation of D5R or the D2-D5 receptor heteromer [48, 49].

Intracellular calcium plays key roles in many neuronal functions including the regulation of synaptic transmission [50]. The intracellular calcium signaling pathway activated through the dopamine D1-D2 receptor heteromer resulted in CaMKIIα activation and BDNF production in striatal neurons in culture as well as in the nucleus accumbens of adult rats, leading ultimately in cultured postnatal striatal neurons to enhanced dendritic branching [40]. Both CaMKIIα and BDNF have been shown to be involved in synaptic plasticity. While evidence has indicated that CaMKIIα is a critical regulator of synaptic plasticity in neurons [51–54] with 50% of CaMKIIα-deficient mice presenting changes in behavior and learning [55], BDNF has been shown to modulate the branching and growth of axons, dendrites and spines (reviewed in 56). For example, BDNF was shown to be released from cell bodies and dendrites of cortical neurons and regulated the branching of dendrites in adjacent neurons [57]. The BDNF effect on the dendritic morphology and also on spine morphology (reviewed in 56) would be of great importance in the modulation of neuronal and synaptic function and plasticity [58]. The neurotrophin signaling transduced through BDNF receptor TrkB has been recently reported to be involved in the control of the size of the striatum by modulating the number of medium spiny neurons (MSNs), with deletion of the gene for the TrkB receptor in striatal progenitors leading to the loss of almost 50% of MSNs without affecting striatal interneurons [59]. Also, the BDNF signaling through TrkB was shown to be involved in the induction and the maintenance of synaptic plasticity, through its long-term potentiation (LTP) component [60]. The other component, long-term depression (LTD) was shown to involve BDNF signaling through the receptor p75 in hippocampal slices from p75-deficient mice [61]. BDNF plays also an important role in the modulation of neurotransmitter release, a key step in synaptic plasticity [56]. The release of glutamate for example involves PLC and BDNF through a mechanism involving a rise in intracellular calcium via a release from IP3 receptor-sensitive stores [62, 63]. It is very interesting to draw the parallel between these mechanisms by which CaMKII and BDNF modulate synaptic plasticity and the signaling pathway revealed with the activation of dopamine D1-D2 receptor heteromer in the striatum [40], which also involves PLC, the intracellular calcium release from IP3 receptor-sensitive stores, CaMKII activation and BDNF production. This suggests that the D1-D2 receptor heteromer-mediated signaling pathway may play an essential role in synaptic plasticity, notably in its LTP component [20, 40, 49], the dysregulation of which may lead to alterations in cognition, learning, and memory that contribute to the pathophysiology of dopamine-related disorders such as schizophrenia or drug addiction [20, 40, 46, 49].

Further, we showed that in rat striatum amphetamine administration significantly increased the affinity of SKF 83959, a specific D1-D2 receptor heteromer agonist [64], by 10-fold for the D1-D2 receptor heteromer and increased the proportion of the D1-D2 heteromer in the agonist-detected high affinity state [46]. GTPγS binding studies indicated that the D1-D2 heteromer was functionally supersensitive in response to repeated increases in dopamine transmission following amphetamine administration [46]. In addition to increasing the activity and sensitivity of D1-D2 receptor heteromers, amphetamine also increased the D1-D2 receptor heteromer density in the NAc as assessed by FRET technique [46].

Interestingly, the increase in the proportion of D1-D2 heteromers in the high affinity state was also detected in schizophrenia globus pallidus (GP) [46]. Amphetamine treatment leading to increased dopamine transmission and behavioral sensitization has been used as an animal model for schizophrenia [65], since schizophrenia has been linked to increased dopamine transmission [66]. Moreover, the different components of calcium signaling, including Gq proteins, PLC, and CaMKII were shown to be affected in the brains of schizophrenia patients [67]. Given these facts, the findings showing an increase in the proportion of D1-D2 heteromers in high affinity state in both schizophrenia and chronic amphetamine treatment may indicate a preponderant role of the D1-D2 receptor heteromer-mediated calcium-CaMKII-BDNF signaling pathway in both drug addiction and schizophrenia.

This D1-D2 receptor heteromer-calcium signal may represent a first common biochemical bridge between the dopaminergic system-CaMKII-BDNF, synaptic plasticity and the occurrence of drug addiction and schizophrenia. The finding that the activation of CaMKIIα was necessary for the induction of behavioral sensitization to drugs [68], a physiological phenomenon that also requires the coactivation of D1 and D2 dopamine receptors [14], provides additional evidence of the important role of dopamine D1-D2 receptor heteromer-calcium signal in drug addiction.

After years of some skepticism surrounding the physiological presence and relevance of GPCR homo- and hetero-oligomers, there is ample evidence for the presence in the brain of a unique entity, the D1-D2 receptor heteromer, with a unique signaling pathway different from the signals generated by each receptor homomer, with a physiological relevance and high importance in at least two major pathologies, schizophrenia and drug addiction, making the D1-D2 receptor an interesting therapeutic target for these disorders.


Cellular colocalization of dopamine D1 mRNA and D2 receptor in rat brain using a D2 dopamine receptor specific polyclonal antibody

1. The main objective of this work was to investigate the extent of cellular colocalization of dopamine D1 and D2 receptors in the rat brain. A double labeling technique, that combined immunocytochemical labeling of the D2 receptor using polyclonal antibodies raised against the third intracellular loop of the short isoform of the human D2 receptor in combination with in situ hybridization detecting D1 mRNA expression, was designed to accomplish this goal. 2. The specificity of the antisera obtained was confirmed by immunoprecipitation assay, Western blot analysis, and immunocytochemistry on D2R transfected cells and murine brain tissue. Western blot using the D2 receptor antibody revealed a specific broad band centered at 67 kDa in transfected cells and a major protein of 88 kDa corresponding to D2R expressed in the caudate-putamen, to a lesser extent in the cortex, and not at all detected in the hypothalamic region. 3. The content of neurons double-labeled for D1/D2 receptors was observed at in differing intensities in the dorsal endopiroform nucleus, the intercalated nucleus of amygdala, the anterior part of the cortical nucleus amygdala, the nucleus of the lateral olfactory tract, the piriform cortex, the parabrachial nucleus, the supraoptic nucleus and the parabigeminal nucleus. All other regions of the brain revealed neurons expressing either D1 or D2 dopamine receptors but not both at that same time. 4. These results clearly demonstrated that specific neurons expressed both receptors D1 and D2, and that this colocalization was restricted to particular regions of the rat brain.


Localization of D1 dopamine receptor mRNA in brain supports a role in cognitive, affective, and neuroendocrine aspects of dopaminergic neurotransmission.

Expression of a D1 dopamine receptor was examined in the rat brain by using a combination of in situ hybridization and in vitro receptor autoradiography. Cells expressing D1 receptor mRNA were localized to many, but not all, brain regions receiving dopaminergic innervation. The highest levels of hybridization were detected in the caudate-putamen, nucleus accumbens, and olfactory tubercle. Cells expressing D1 receptor mRNA were also detected throughout the cerebral cortex, limbic system, hypothalamus, and thalamus. D1 receptor mRNA was differentially expressed in distinct regions of the hippocampal formation. Dentate granule cells were labeled in dorsal but not ventral regions, whereas the subicular complex was prominently labeled in ventral but not dorsal regions. Intermediate to high levels of D1 binding sites, but no hybridizing D1 receptor mRNA, were detected in the substantia nigra pars reticulata, globus pallidus, entopeduncular nucleus, and subthalamic nucleus. In these brain regions, which are involved in the efferent flow of information from the basal ganglia, D1 receptors may be localized on afferent nerve terminals originating in other brain regions. These results indicate that in addition to a role in control of motor function, the D1 receptor may also participate in the cognitive, affective, and neuroendocrine effects of dopaminergic neurotransmission.


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Molecular characterization of individual D3 dopamine receptor-expressing cells isolated from multiple brain regions of a novel mouse model

Among dopamine receptors, the expression and function of the D3 receptor subtype is not well understood. The receptor has the highest affinity for dopamine and many drugs that target dopamine receptors.In this paper, we examined, at the single cell level, the characteristics of D3 receptor-expressing cells isolated from different brain regions of male and female mice that were either 35 or 70 days old. The brain regions included nucleus accumbens, Islands of Calleja, olfactory tubercle,retrosplenial cortex, dorsal subiculum, mammillary body,amygdala and septum. The expression analysis was done in the drd3-enhanced green fluorescent protein transgenic mice that report the endogenous expression of D3 receptor mRNA. Using single cell reverse transcriptase PCR, we determined if the D3 receptor-expressing fluorescent cells in these mice were neurons or glia and if they were glutamatergic, GABAergic or catecholaminergic. Next, we determined if the fluorescent cells co-expressed the four other dopamine receptor subtypes, adenylate cyclase V(ACV) isoform, and three different isoforms of G protein coupled inward rectifier potassium (GIRK) channels. The results suggest that D3 receptor is expressed in neurons,with region-specific expression in glutamatergic and GABAergic neurons. The D3 receptor primarily coexpressed with D1 and D2 dopamine receptors with regional, sex and age-dependent differences in the coexpression pattern. The percentage of cells co-expressing D3 receptor and ACV or GIRK channels varied significantly by brain region, sex and age. The molecular characterization of D3 receptor-expressing cells in mouse brain reported here will facilitate the characterization of D(3) receptor function in physiology and pathophysiology.

Figures

Representative image of a sagittal…

Representative image of a sagittal section of brain obtained from an adult drd3…

Representative images of coronal brain…

Representative images of coronal brain sections obtained from an adult drd3 -EGFP mouse.…

A flowchart depicting the experimental…

A flowchart depicting the experimental plan for the single cell RT-PCR analysis followed…

Sensitivity, linearity and reproducibility of…

Sensitivity, linearity and reproducibility of the RT-PCR used in this study. a Raw…

Co-expression of D 3 dopamine…

Co-expression of D 3 dopamine receptor with other dopamine receptor subtypes, ACV and…

Co-expression of D 3 dopamine…

Co-expression of D 3 dopamine receptor with other dopamine receptor subtypes, ACV and…

Co-expression of D 3 dopamine…

Co-expression of D 3 dopamine receptor with other dopamine receptor subtypes, ACV and…

D 3 receptor-expressing fluorescent cells…

D 3 receptor-expressing fluorescent cells are found in different molecular layers in cingulate…

Co-expression of D 3 dopamine…

Co-expression of D 3 dopamine receptor with other dopamine receptor subtypes, ACV and…

Co-expression of D 3 dopamine…

Co-expression of D 3 dopamine receptor with other dopamine receptor subtypes, ACV and…

Co-expression of D 3 dopamine…

Co-expression of D 3 dopamine receptor with other dopa-mine receptor subtypes, ACV and…

Co-expression of D 3 dopamine…

Co-expression of D 3 dopamine receptor with other dopamine receptor subtypes, ACV and…

Co-expression of D 3 dopamine…

Co-expression of D 3 dopamine receptor with GAD65 and VGLUT1 in the amygdala.…

Co-expression of D 3 dopamine…

Co-expression of D 3 dopamine receptor with other dopamine receptor subtypes, ACV and…

D 3 receptor-expressing fluorescent cells…

D 3 receptor-expressing fluorescent cells also express D 3 receptor protein. Representative images…


Dopamine D1 or D2 receptor-expressing neurons in the central nervous system

X. Y. W. and T. M. contributed equally to this work.Present address: Department of Neurobiology and Collaborative Innvation Center for Brain Science, School of Basic Medicine, The Fouth Military Medical University, Xi'an, China.Search for more papers by this author

Department of Neuroscience and Experimental Therapeutics, College of Medicine, Texas A&M University Health Science Center, Bryan, TX, USA

X. Y. W. and T. M. contributed equally to this work.Search for more papers by this author

Department of Neuroscience and Experimental Therapeutics, College of Medicine, Texas A&M University Health Science Center, Bryan, TX, USA

Department of Neuroscience and Experimental Therapeutics, College of Medicine, Texas A&M University Health Science Center, Bryan, TX, USA

Department of Neuroscience and Experimental Therapeutics, College of Medicine, Texas A&M University Health Science Center, Bryan, TX, USA

Department of Neuroscience and Experimental Therapeutics, College of Medicine, Texas A&M University Health Science Center, Bryan, TX, USA

Department of Neuroscience and Experimental Therapeutics, College of Medicine, Texas A&M University Health Science Center, Bryan, TX, USA

Correspondence to: Jun Wang, Department of Neuroscience and Experimental Therapeutics, College of Medicine, Texas A&M University Health Science Center, 2106 Medical Research and Education Building, 8447 Riverside Pkwy, Bryan, TX 77807-3206, USA. E-mail: [email protected] Search for more papers by this author

Department of Neuroscience and Experimental Therapeutics, College of Medicine, Texas A&M University Health Science Center, Bryan, TX, USA

X. Y. W. and T. M. contributed equally to this work.Present address: Department of Neurobiology and Collaborative Innvation Center for Brain Science, School of Basic Medicine, The Fouth Military Medical University, Xi'an, China.Search for more papers by this author

Department of Neuroscience and Experimental Therapeutics, College of Medicine, Texas A&M University Health Science Center, Bryan, TX, USA

X. Y. W. and T. M. contributed equally to this work.Search for more papers by this author

Department of Neuroscience and Experimental Therapeutics, College of Medicine, Texas A&M University Health Science Center, Bryan, TX, USA

Department of Neuroscience and Experimental Therapeutics, College of Medicine, Texas A&M University Health Science Center, Bryan, TX, USA

Department of Neuroscience and Experimental Therapeutics, College of Medicine, Texas A&M University Health Science Center, Bryan, TX, USA

Department of Neuroscience and Experimental Therapeutics, College of Medicine, Texas A&M University Health Science Center, Bryan, TX, USA

Department of Neuroscience and Experimental Therapeutics, College of Medicine, Texas A&M University Health Science Center, Bryan, TX, USA

Correspondence to: Jun Wang, Department of Neuroscience and Experimental Therapeutics, College of Medicine, Texas A&M University Health Science Center, 2106 Medical Research and Education Building, 8447 Riverside Pkwy, Bryan, TX 77807-3206, USA. E-mail: [email protected] Search for more papers by this author

Abstract

Dopamine signals mainly through D1 receptors (D1Rs) and D2 receptors (D2Rs) D1R-expressing or D2R-expressing neurons contribute to distinct reward and addictive behaviors. Traditionally, transgenic mice expressing green fluorescent protein (GFP) under D1R or D2R promoters are used for fluorescent verification in electrophysiology studies, whereas Cre mice are employed for behavioral research. However, it is unknown whether the same neuronal populations are targeted in GFP and Cre mice. Additionally, while D1Rs and D2Rs are known to be expressed in different striatal neurons, their expression patterns outside the striatum remain unclear. The present study addressed these two questions by using several transgenic mouse lines expressing fluorescent proteins (GFP or tdTomato) or Cre under the control of D1R or D2R promoters. We found a high degree of overlap between GFP-positive and Cre-positive neurons in the striatum and hippocampus. Additionally, we discovered that D1Rs and D2Rs were highly segregated in the orbitofrontal cortex, prefrontal cortex, dorsal and ventral hippocampus, and amygdala:

4–34 percent of neurons co-expressed these receptors. Importantly, slice electrophysiological studies demonstrated that D1R-positive and D1R-negative hippocampal neurons were functionally distinct in a mouse line generated by crossing Drd1a-Cre mice with a Cre reporter Ai14 line. Lastly, we discovered that chronic alcohol intake differentially altered D1R-positive and D2R-positive neuron excitability in the ventral CA1. These data suggest that GFP and Cre mice target the same populations of striatal neurons, D1R-expressing or D2R-expressing neurons are highly segregated outside the striatum, and these neurons in the ventral hippocampal may exert distinct roles in alcohol addiction.

Figure S1. Expression of D1Rs and D2Rs in the ventral medial prefrontal cortex (vmPFC). Two male D1-tdTomatoD2-CreSnap25 mice were used to examine co-localization of D1Rs and D2Rs in the vmPFC. Representative images show the D1R + neurons (A, red), the D2R + neurons (B, green) and some co-localization (C, yellow), as indicated by the arrows. Note that the percentage of overlapped D1R + and D2R + neurons did not differ (D). n.s., not significant, P > 0.05, paired t-test. n = 15 sections. D, dorsal L, lateral. Scale: 20 μm

Table S1. Summary of overlapping rates of D1R + and D2R + neurons in key brain regions of male and female mice. *indicates numbers of sections and mice. Data were expressed as mean ± SE. dmPFC, dorsomedial prefrontal cortex dDG, dorsal dentate gyrus vCA1, ventral hippocampal CA1 BLA, basolateral amygdala. t-test was used to determine the significance

Table S2. Comparison of the overlapping rates of D1R + and D2R + neurons in the layers II and V/VI of the dmPFC. Data were expressed as mean ± SE. n = 11 sections from two male mice per group. t-test was used to determine the significance

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MATERIALS & METHODS

Animals

Experiments used either heterozygous D1-tdTomato mice (Ade KK et al. 2011), or heterozygous D1-tdTomato mice crossed with either homozygous GAD-Cre (Taniguchi H et al. 2011), PV-Cre (Hippenmeyer S et al. 2005), SOM-Cre (Taniguchi H et al. 2011), VIP-Cre (Taniguchi H et al. 2011) or heterozygous 5HT3a-Cre mice (Gerfen CR et al. 2013). Mice were bred on a C57BL/6J background with the exception of D1-tdTomato x VIP-Cre mice, which were mixed background. All mice were purchased from Jackson laboratories. Mice of both sexes were used, and no differences were found. All experimental procedures were approved by the University Animal Welfare Committee of New York University.

Stereotaxic injections

Mice aged 4-7 weeks were deeply anesthetized with a mixture of ketamine (10 mg/mL) and xylazine (0.1 mg/mL) and head fixed in a stereotax (Kopf Instruments). A small craniotomy was made over the injection site, using coordinates relative to Bregma (dorsoventral, mediolateral, and rostrocaudal, respectively): prefrontal cortex (PFC) = −2.1, ±0.4, +2.2 mm claustrum (CLA) = −3.6, −3.2, +1.6 mm (injected at 5 degrees to the upright) mediodorsal thalamus (MD) = −3.6, −0.3, −0.5 mm ventromedial thalamus (VM) = −3.4, −2.7, −0.4 mm (injected at 30 degrees to the upright) ventral tegmental area (VTA) = −4.5, −0.5, −2.95 mm basolateral amygdala (BLA) = −4.9, −3.2, −1.2 mm, dorsomedial striatum (STR) = −3.2, −1.1, +1.1 mm, pontine nucleus (pons) = −4.7, +0.5, - 4.0 mm. For retrograde labeling, pipettes were filled with Cholera Toxin Subunit B (CTB) conjugated to either Alexa-488 or −647 (Life Technologies). Virus varied between experiment: Cre-dependent labeling of interneurons = AAV9-CAG-FLEX-EGFP-WPRE (UPenn) labeling putative pyramidal neurons = AAV1-CaMKII-EGFP-WPRE (UPenn) axon anatomy = AAV-DJ-hSyn1-mCherry-IRES-eGFP-Syb2 (SynaptoTag, Stanford) retrograde-Cre and associated axon anatomy = AAVrg-EF1a-mCherry-IRES-Cre (Addgene) and AAV1-EF1a-DIO-eYFP-WPRE (UPenn). Borosilicate pipettes with 5-10 μm tip diameters were backfilled, and between 130-550 nL of solution was pressure injected using a Nanoject III (Drummond), with 30 s spacing between injections. The pipette was subsequently left in place for an additional 5 min, allowing time to diffuse away from the pipette tip, before being slowly retracted from the brain. Animals were returned to their cages for between 1-3 weeks before being used for recording or anatomy, or for 4-6 weeks in the case of the SynaptoTag virus injections.

Slice preparation

Mice aged 6-8 weeks were anesthetized with a lethal dose of ketamine (25 mg/mL) and xylazine (0.25 mg/mL) and perfused intracardially with ice-cold external solution containing the following (in mM): 65 sucrose, 76 NaCl, 25 NaHCO3, 1.4 NaH2PO4, 25 glucose, 2.5 KCl, 7 MgCl2, 0.4 Na-ascorbate, and 2 Na-pyruvate (295-305 mOsm), and bubbled with 95% O2/5% CO2. Coronal slices (300 μm thick) were cut on a VS1200 vibratome (Leica) in ice-cold external solution, before being transferred to ACSF containing the following (in mM): 120 NaCl, 25 NaHCO3, 1.4 NaH2PO4, 21 glucose, 2.5 KCl, 2 CaCl2, 1 MgCl2, 0.4 Na-ascorbate, and 2 Na-pyruvate (295-305 mOsm), bubbled with 95% O2/5% CO2. Slices were kept for 30 min at 35°C, before being allowed to recover for 30 min at room temperature. Intrinsic properties were recorded at 30−32°C. To facilitate stable recordings of cells with very high input resistance, modulation of VIP+ interneurons was performed at room temperature. Modulation of pyramidal cells was performed at both 30−32°C and room temperature, with no differences observed across these conditions, so results were pooled for analysis.

Electrophysiology

Whole-cell recordings were obtained from neurons across all layers of the prelimbic subdivision of PFC. Neurons were identified by infrared-differential interference contrast, as previously described (Chalifoux JR and AG Carter 2010). Neuronal identity was established by the presence or absence of tdTomato, EGFP and Alexa-conjugated CTB under fluorescent illumination. Borosilicate pipettes (2-6 MΩ) were filled with internal solution comprising (in mM): 135 K-gluconate, 7 KCl, 10 HEPES, 10 Na-phosphocreatine, 4 Mg2-ATP, 0.4 Na-GTP and 0.5 EGTA, 290–295 mOsm, pH 7.3, with KOH. For a subset of experiments, 30 μM Alexa Fluor 594 was included for 2-photon imaging, in which case dye was allowed to diffuse throughout the dendrites and axons for at least 20 min before imaging.

Electrophysiology recordings were made with a Multiclamp 700B amplifier (Axon Instruments), filtered at 4 kHz, and sampled at 10 kHz. Series resistance was typically <20 MΩ for pyramidal neurons and <30 MΩ for VIP+ interneurons. Current-clamp recordings were performed in the presence of the synaptic blockers CPP (10 μM), NBQX (10 μM) and Gabazine (10 μM). Dopamine receptor pharmacology was performed using wash-in of the selective D1-type dopamine receptor agonist SKF-81297 (10 μM) and the selective antagonist SCH-23390 (10 μM). Modulation experiments involved 5 min of baseline firing, either in the presence or absence of SCH-23390, followed by bath application of SKF-81297. Firing modulation was calculated by comparing the average number of action potentials evoked per stimulus in this baseline epoch with a 5-minute window starting 10 minutes after initial SKF-81297 application. All chemicals were purchased from Sigma or Tocris Bioscience.

Two-photon microscopy

Two-photon imaging was performed on a custom microscope, as previously described (Chalifoux JR and AG Carter 2010). Briefly, a Ti:Sapphire laser (Coherent) tuned to 810 nm was used to excite Alexa Fluor 594 to image morphology with a 60x 1.0 NA objective (Olympus). Three-dimensional reconstructions of dendritic morphologies were performed using NeuronStudio (Wearne et al., 2005), while two-dimensional tracing of dendrites and axons for figures was performed using Neurolucida (MBF Bioscience). Dendrite analysis was performed by summing the total, apical or basal dendrite length contained within 10 μm concentric rings emanating from the soma and plotting these as a function of distance from the soma.

Histology and fluorescence microscopy

Mice were anesthetized with a lethal dose of ketamine (25 mg/mL) and xylazine (0.25 mg/mL) and perfused intracardially with 0.01 M phosphate buffered saline (PBS) followed by 4 % paraformaldehyde (PFA) in 0.01 M PBS. Brains were fixed in 4 % PFA in 0.01 M PBS for 4-12 hours at 4°C. Slices were prepared at a thickness of 40-60 μm (Leica VT 1000S vibratome). For enhanced detection of tdTomato signal in D1-tdTomato mice slices were stained with antibodies against RFP. For antibody labeling, slices were washed once in PBS (0.01 M), once in PBS-T (0.2 % Triton-X100), then blocked in PBS-T with 1 % w/v bovine serum albumin (BSA) for one hour, all at room temperature. Primary antibody incubation (Rabbit anti-red fluorescent protein, 600-401-379, Rockland, 1:1000 Mouse anti-calretinin, MAB1568, Millipore, 1:1000 Mouse anti-parvalbumin, MAB1572, Millipore, 1:2000 Rat anti-somatostatin, MAB354, Millipore, 1:400) was performed at 4 ° C overnight. Slices were then washed 4x in PBS at RT before incubating with secondary antibody (Goat anti-rabbit Alexa 594, ab150080, AbCam, 1:400 Goat anti-rat Alexa 647, 21247, Fisher-Invitrogen, 1:200 Goat anti-mouse Alexa 647, ab150119, Abcam, 1:200) in PBS-T + BSA for 1 hour at room temperature. Slices were washed a further 3x in PBS before being mounted under glass coverslips on gelatin-coated slides using ProLong Gold antifade reagent with DAPI (Invitrogen). Whole-brain images were acquired using a slide-scanning microscope (Olympus VS120) with a 10x 0.25 NA or 20x 0.75 NA objective. Excitation wavelengths were 387, 485, 560 and 650 nm for DAPI, FITC, TRITC and Cy5, respectively. PFC images were acquired using a confocal microscope (Leica SP8) with 10x 0.4 NA or 20x 0.75 NA objective. Excitation wavelengths were 405, 488, 552 and 638 nm for DAPI, FITC, TRITC and Cy5, respectively. Image processing involved adjusting brightness and contrast using ImageJ (NIH). Cell counting was performed in a 400 x 1000 μm region of interest across the depth of the prelimbic prefrontal cortex.

In situ hybridization

Mice were anesthetized with a lethal dose of ketamine (25 mg/mL) and xylazine (0.25 mg/mL) and perfused intracardially with chilled 0.01 M PBS. The brain was extracted and immediately submerged in isopentane cooled on dry ice. Tissue was coated in O.C.T. media (Tissue Tek) and stored in an airtight container at −80°C until sectioning. 10 μm sections were taken on a cryostat at −20°C and mounted on Superfrost Plus microscope slides (Fisher) and stored at −80°C. In situ hybridization of Mm-Drd1a-C2 and tdTomato-C3 probes was performed using a standard RNAscope protocol for flash frozen tissue from ACD bio. Slides were mounted under glass coverslips using ProLong Gold antifade reagent with DAPI (Invitrogen). Images were acquired using a confocal microscope (Leica SP8) with 20x 0.75 NA or 40x 1.3 NA oil immersion objective.

Data analysis

Electrophysiology and imaging data were acquired using National Instruments boards and custom software written in MATLAB (MathWorks). Off-line analysis was performed using custom software written in Igor Pro (WaveMetrics). Input resistance was measured using the steady-state response to a −50 pA current injection for pyramidal neurons and −10 or −20 pA for interneurons. The membrane time constant (tau) was measured using exponential fits to these same hyperpolarizations. Voltage sag due to h-current was calculated by taking the minimum voltage in the first 200 ms, subtracting the average voltage over the final 100 ms, and dividing by the steady-state value. Spike frequency adaptation was calculated by calculating the ratio of the initial inter spike interval (ISI) and final ISI in response to a 500 ms depolarizing current pulse which evoked >5 action potentials. For cell counting as a function of layer, individual cells were assigned a distance from the midline (top of layer 1), binned in 25 μm increments across the depth of PFC, and then assigned into individual layers. Layers were defined based on peaks in neuron density (Table 2), which gave defined ranges for each layer (Table 4). Data was collected from at least 3 slices per animal, with a minimum of 3 mice per projection class / interneuron subtype. Cell-by cell mRNA puncta analysis was performed using a circular region of interest (17.5 μm in diameter) placed over the soma of individual neurons and manual counting of puncta for Drd1a and tdTomato.

Experimental design and statistical analysis

Summary data are reported in the text and shown in figures as arithmetic mean ± SEM, unless otherwise stated. Statistical comparisons were performed in GraphPad Prism (version 7.0c) using a two-tailed non-parametric Mann-Whitney U test. Significance was defined as p < 0.05.


Neurobiology of Attention Deficit Hyperactivity Disorder (ADHD) – A Primer

ADHD is a neurodevelopmental disorder characterised by symptoms of inattention, impulsivity and locomotor hyperactivity.

The prevalence of ADHD in children and adolescents is estimated to be 5.3% (worldwide) [Polanczyk, 2007] and between 4.4% -5.2% in adults between 18-44 years of age. [Young and Goodman, 2016]

Traditionally thought to be a disorder of childhood and adolescents, there is increasing evidence that the condition is prevalent in adulthood and can lead to significant disability.

ADHD frequently persists into adulthood with up to 60% of children continuing to meet diagnostic criteria during adulthood. [Faraone and Biederman, 2005]

As the child grows, the clinical presentation of ADHD is likely to change with inattention more likely to persist compared to hyperactivity, which tends to diminish with age. [Faraone et al., 2006]

However, some adult patients will only meet symptom criteria for adult ADHD without ever meeting the criteria during childhood. This is possibly indicative of a late-onset variant of ADHD. [Faraone and Biederman, 2016]

In this article, we focus on the neurobiology of ADHD and the different models hypothesised in the genesis of the condition.

CLINICAL PRESENTATION OF ADHD

The main symptom domains in ADHD are inattention and hyperactivity-impulsivity. Below are the DSM – 5 criteria for ADHD. ([American Psychiatric Association, 2013]

ANATOMICAL BRAIN CHANGES IN ADHD

1. The most consistent finding in ADHD is an overall reduction in total brain size with specific changes in the caudate nucleus, prefrontal cortex white matter, corpus callosum and cerebellar vermis. [Tripp and Wickens, 2009]

2. Swanson in 2007 noted that the caudate nucleus and globus pallidus, parts of the basal ganglia which both contain a high density of dopamine receptors, are smaller in ADHD. [Swanson et al., 2007]

3. Ventral striatum, which is part of the reward pathway, tends to be reduced in ADHD and there is a negative correlation between ventral striatum and childhood hyperactivity and impulsivity. [Tripp and Wickens, 2009]

4. There is a reduction in cortical thickness which is associated with the DRD4 7-repeat allele. This regional thinning resolves in adolescence and is associated with a better clinical outcome. [Shaw et al., 2007]

5. Diffusion transfer imaging shows alterations in frontal and cerebellar white matter in children and adolescents.

6. The frontostriatal circuit has been convincingly implicated in ADHD. Both DTI and functional MRI (fMRI) show abnormalities in the frontostriatal connectivity and function. [Faraone et al., 2015]

7. fMRI shows reduced activation of prefrontal cortex and striatal regions.

ROLE OF THE PREFRONTAL CORTEX IN ADHD

The prefrontal cortex is essential for executive functioning, allowing us to:

The dorsal and lateral prefrontal cortex regulates attention and motor responses while the ventral and medial portion regulates emotion.

It is the last part of the brain to mature, and maturation only occurs in late adolescence.

The two key receptors that are situated in the prefrontal cortex are dopamine D1 receptor and alpha-2A adrenoreceptors.

The prefrontal cortex exhibits a Goldilocks phenomenon being highly dependent on a balanced neurochemical environment for proper functioning.

ADHD is associated with genetic changes that weaken catecholamine signalling and slow prefrontal cortex (PFC) maturation in some cases.

The following is from Arnsten’s excellent article. [Arnsten, 2009]

Functions of the PFC:

1.Regulation of Top-down attention

  • Concentrate and sustain attention especially under boring conditions.
  • Focus on material that is important but not salient by suppressing processing of irrelevant stimuli and enhancing processing of relevant stimuli.
  • Inhibits internal and external distractions.
  • Divides and shift attention in multi-tasking.
  • Responsible for attention regulation through its effect on the sensory cortices.

2. Regulation of Behaviour

  • Prefrontal cortex is responsible for inhibition of inappropriate behaviour
  • Can guide behavioural output by projections to the motor and the premotor cortices along with the basal ganglia and cerebellum.

3. Regulation of emotions

  • The ventromedial prefrontal cortex (VMPFC) has projections to amygdala, hypothalamus and nucleus accumbens and weakens reactions to disinhibited aggressive impulses and emotional dysregulation.
  • Abnormalities of the VMPFC can lead to conduct disorder like symptoms

Functions of parietal and temporal lobes in the regulation of attention (Bottom-up processing of attention):

  • The parietal and temporal lobes provide bottom-up processing, i.e., process stimuli according to inherent salience.
  • The ventral and the dorsal temporal stream are responsible for visual features location of things
  • The parietal lobe is responsible for orienting attention to parts of space and of time.

OTHER BRAIN PATHWAYS INVOLVED

1.Executive Network in ADHD: [Faraone et al., 2015]

2. Alerting network

3. Default mode network (DMN)

The DMN is a network of brain regions that are active when in a ‘resting state’ and tend to be negative correlated with attention networks.

Negative correlations between the DMN and the frontoparietal control network are weaker in patients with ADHD than in people who do not have the disorder.

A metanalysis of 55 MRI studies found that : [Cortese et al., 2012]

Children with ADHD have:

  • hypoactivation in frontoparietal and ventral attention networks
  • hyperactivation in default mode networks, ventral attention and somatomotor areas

Adults with ADHD have:

  • hypoactivation in the frontoparietal area.
  • hyperactivation in the visual, dorsal attention, and default networks.
ROLE OF NEUROTRANSMITTERS IN ADHD - FOCUS ON DOPAMINE AND NORADRENALINE

The neurotransmitter dopamine is implicated as the main mediator of the brain’s reinforcement signal.

Dopamine cell bodies lie in the pars compacta of the substantia nigra (SN) and the ventral tegmental area (VTA).

We cover the dopamine pathways in more detail here.

The substantia nigra projects to the dorsolateral striatum and is responsible for motor control.

The ventromedial projections from the ventral tegmental area (VTA) are responsible for cognitive and affective function.

The Cellular Actions of Dopamine:
The physiological effects of dopamine transmission in the brain are mediated by G-protein coupled receptors.

There are five key receptors: D1 to D5. Each of these is situated in different parts of the brain and perform different functions.

Dopamine receptors and the role of the D1 receptor:

D1 and D2 are uniformly expressed throughout the striatum and play an important part in the reward pathway. [Arnsten, 2009]

  • D4 and D5 receptors are found at lower levels in the striatum and moderate levels in the prefrontal cortex.
  • Low to moderate levels of D1 receptor stimulation can improve prefrontal cortex functioning.
  • Stimulation of D1 receptors weakens the neuronal signal and is responsible for decreasing ‘noise’ by pruning inappropriate connections.
  • Excessive D1 receptor stimulation (such as occurs during stress) impairs PFC function by weakening too many network connections.

The dopamine cells tend to have two firing modes:

  • clock-like rhythmic firing
  • burst-like firing in response to events connected with reward.

Normally, cells tend to respond approximately 200 ms after the delivery of an unexpected reward.

Individuals with ADHD tend to have altered dopamine signalling which leads to altered reinforcement sensitivity. [Luman et al., 2010]

The DAT1 gene variable number tandem repeat (VNTR) is known to be associated with ADHD and may explain the delayed, altered reinforcement sensitivity. [Tripp and Wickens, 2009] (See Dopamine transfer deficit theory later)

The Cellular Actions of Noradrenaline (NA): [Arnsten, 2009]

  • At moderate levels, noradrenaline can improve prefrontal cortex functioning by stimulating postsynaptic α2A receptors
  • NA activation of the α2A receptor strengthens the neuronal signal and hence strengthens network connectivity
  • Higher levels of NA can impair prefrontal cortex function by stimulating α1 receptors

Thus, DA and NE have complementary beneficial actions with NA increasing the signal and DA reducing noise, and an optimal balance of both neurotransmitters is required for proper PFC functioning.

The Connection to Pyramidal Glutamate Neurons:

The prefrontal cortex has pyramidal glutamate neurons. These neurons can “keep in mind” information to help guide attention and behaviour in a thoughtful manner. [Arnsten, 2009]

The neurons in these networks interact with other pyramidal cells through synapses on dendritic spines which contain NA alpha-2A receptors or D1 receptors.

These pyramidal glutamate neurons are inhibited by GABA, which in turn is suppressed by the D4 receptor.

Thus, activation of the D4 receptors suppresses GABA, which in turn activates the pyramidal glutamate neurons.

Not much research has been carried out on the D4 receptor however the D4 receptor can be stimulated by NA and DA and deficient stimulation of the D4 receptor can impair PFC functioning by weakening glutamate release.

ADHD PATHOPHYSIOLOGY - NEUROCHEMICAL DEFICIT THEORIES

The reward network underpins two key dopamine related neurochemical deficit theories.

1. Dynamic Developmental Theory:
The dynamic developmental theory hypothesises that there is a dysfunction of dopamine transmission in the frontal-limbic circuits, which is responsible for a steeper delay-of-reinforcement gradient and slower effects of extinction. [Sagvolden et al., 2005]

The model proposes that due to the steep delay of reinforcement there is a critical window during which reinforcement of behaviour can occur in individuals with ADHD. The steep and shorter delay of reinforcement is caused due to lower levels of tonic dopamine.

Thus, a reinforcer loses its value relatively quickly, which makes it difficult to change behaviour. Only short sequences of responses can be reinforced due to the short critical window in which behaviour can be reinforced.

Children therefore, tend to respond better to immediate rewards over delayed rewards and only show learning when rewards are received immediately and frequently.

Furthermore, due to the lower tonic dopamine levels, there is only a blunted dip in the phasic dopamine after the omission of the reward. Thus, there is a slower extinction of behaviour.

In normal children, the gradient is not as steep and is gradual, thus allowing a greater window of opportunity where children can obtain adequate reinforcement from delayed rewards.

2. Dopamine Transfer Deficit Theory:

The dopamine system uses previous instances of reinforcement to produce anticipatory dopamine release. [Tripp and Wickens, 2008]

According to the dopamine transfer deficit, this assumes that there is a normal tonic level of dopamine but the phasic dopamine response to reinforcement is altered.

The model proposes that the anticipatory dopamine cell firing is disturbed whereby the dopamine response does not transfer to earlier and earlier predictors of response requiring actual instances of reinforcement for control of behaviour rather than predicted behaviour.

In children with ADHD the phasic dopamine cell response to cues that predict reinforcement is reduced in amplitude to the point of being ineffective and similarly when the reward is taken away there is a blunting of the phasic dopamine decrease response leading to slower extinction of behaviour.

The dopamine transfer deficit explains the symptoms of inattention as the child fails to give close attention to details and makes careless mistakes and cannot maintain on-task behaviour as there is an absence of the continuous reinforcement of attending by anticipation of dopamine release.

Similarly, it also explains hyperactivity and impulsivity, where the child leaves the seat in the classroom where remaining seated is expected due to lack of effective reinforcement, i.e., lowered phasic dopamine response to rewards.

Impulsivity may also be explained as there is a delay between the target behaviour and actual reinforcement.

The fidgetiness may be due to activating effects of dopamine due to excitability of striatal neurons.

Hence, specific abnormalities in reward sensitivity include: [Luman et al., 2010]

  1. Greater emphasis on immediate rewards than delayed rewards.
  2. Poorer performance under partial or discontinuous reinforcement schedules.
  3. Impaired reinforcement learning and acquisition of behaviour.
  4. Impaired integration of earlier reinforcers.
  5. Impaired ability to change behaviour in response to changes in reinforcement contingencies.
  6. Impaired response to conditioned than to actual reinforcement.
  7. Problems with adding new contingency information in the working memory.
  8. Behavioural inhibition less under the influence of cues of aversive stimuli.
  9. Lower level of tonic dopamine in frontal-limbic circuitry in the brain and hypodopaminergic state.
  10. Smaller phasic dopamine response to actual rewards.
  11. Slower shift in dopamine from actual reward to reward cues.
  12. Reduced phasic anticipatory dopamine release in striatum to reward cues.
  13. Slower rate of extinction.
ALTERNATE NEUROBIOLOGICAL MODELS

The observed clinical variability of ADHD may indicate the possibility of multiple developmental pathways. Since Barkley theorised in 1997 [Barkley, 1997] that normal behavioural inhibition was necessary for attention and executive function, there have been a number of subsequent neurobiological models proposed.

Most of these models have been attributed to delayed development within the later maturing areas of the brain that affect attention, decision-making, and reinforcement learning.

1.Behavioural neuroenergetics model: [Killeen, 2013]

This model involves the inadequate production of lactate by astrocytes in the brain.

Astrocytes take up glucose from blood vessels and convert it to glycogen and lactate, with the latter being released for the neurons to metabolise into energy (ATP). Therefore, the insufficient provision of neuronal energy creates a state of hypo-energy.

There is an approximate loss of 15-25% of neurocognitive energy that can be applied to any one task, which results in attention drifting and mental fatigue.

2. The state regulation model: [Hegerl and Hensch, 2014]

This model postulates that there is a dysregulation in the regulation of vigilance (brain arousal), which underlies the attention deficits in ADHD.

This model has pathogenetic relevance to both ADHD and mania whereby unstable or low vigilance can induce an excessive autoregulatory attempt to stabilise vigilance. This is how periods of hyperactivity are proposed to occur.

3. Executive dysfunction theory: [Baroni and Castellanos, 2014]

Advances in MRI techniques have shown researchers that the observed phenotypic variations in ADHD are a result of impairments to top-down cognitive processes that are important for organising behaviour. The executive control network is implicated here.

The implications of disrupted executive processes and functions affect reward-related processing, inhibition, vigilance, reaction time variability, and emotional lability.

4.Delay Aversion Theory: [Sonuga-Barke et al., 1992]

This model proposes that escape from delay is a key reinforcer for children with ADHD, as the delay appears to have a negative association.

When delay cannot be reduced, children will engage in behaviours that reduce the perception of the length of delay or engage in behaviours that act as immediate reinforcers such as fidgeting and attending to alternative stimuli.

In terms of neurobiological mechanisms, the delay aversion is due to a reduced efficiency of dopamine in reward circuits signalling future rewards and a steeper and shorter delay-of-reinforcement gradient in children with ADHD.

5. Dual Pathway model: [Sonuga-Barke, 2003]

Sonuga-Barke incorporated the delay aversion model within the dual pathway model.

According to the dual pathway model, it proposes there are two independent neurocircuitries linked to ADHD, namely the ventrolateral and dorsolateral corticostriatal circuitry subserving executive and inhibitory processes and mesolimbic-ventrostriatal circuitry subserving motivational and reward processes, so there are abnormalities in executive processes and motivational processes.

6. Tripartite Pathway Model: [Sonuga-Barke et al., 2010]

This model is a refinement of the dual pathway theory and explains the neuropsychological heterogeneity of ADHD as a combination of one or more deficits in inhibitory control, motivational control, and temporal processing.

Three pathways are involved indicating ADHD subtypes:

1. Dysfunction of the prefrontal cortex is likely to result in a
reduced ability to exert control.

2. Dysfunction in dorsal striatum might lead to differences in the ability to predict what events are going to occur, whereas dysfunction in ventral striatum is more likely to lead to deficits in motivation and reward processing.

3. Dysfunction of the cerebellum is likely associated with problems in the ability to predict when events are going to occur and other problems with timing. The fronto-cerebellar circuit may be involved in temporal processing. The cerebellum has outputs to both the prefrontal cortex and the basal ganglia. [Durston et al., 2011]

THE FUTURE

The neurobiology of ADHD is complex and involves multiple brain pathways. Two key neurotransmitters highlighted in the pathogenesis of ADHD are dopamine and noradrenaline.

As neuroimaging advances, different subtypes of ADHD may emerge involving distinct pathways giving rise to a specific set of symptoms. By combining this with an understanding of the neurotransmitters we may be able to develop and target treatments for better outcomes.



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