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Immunostaining of DA neurons for GLU in vitro. To evaluate GLU staining of DA neurons, mass cultures of V TA, cerebellum, and hippocampus were immunostained for GLU and GABA. A, In vitro the majority of DA neurons in V M cultures (A 1 ) were GLUergic (A 2 ); occasional DA neurons (A 2 , arrow) were non-GLUergic. In the color merge (A 3 ), in which colocalization appears yellow, neuronal nuclei appear red, reflecting selective GLU staining. A neuron that is neither TH nor GLU is seen (A 3 , arrow). B, In a cerebellar culture in which granule cells, which are small and GLUergic, can be distinguished from Purkinje cells, which are large and GABAergic, only the putative large Purkinje cell stains for GABA (B 1 ), whereas the two granule cells do not stain (arrows). However, both the Purkinje cell as well as the granule cells appear GLUergic (B 2 ). This is seen more clearly in the color merge (B 3 ). In this experiment, all large neurons were GABA and GLU (n 16), whereas all small neurons were only GLU (n 40). This indicates that in vitro GABA neurons contain appreciable GLU, which is likely to be present as a precursor to GABA. C, Hippocampal neurons are either GLUergic (majority) or GABAergic (minority). In this culture, occasional cells stained for GABA (C 1 , arrow), whereas most stained for GLU
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Interactions between dopamine and glutamate play prominent roles in memory, addiction, and schizophrenia. Several lines of evidence have suggested that the ventral midbrain dopamine neurons that give rise to the major CNS dopaminergic projections may also be glutamatergic. To examine this possibility, we double immunostained ventral midbrain sectio...
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... and the others are almost entirely GABAergic (L. Lin and S. Rayport, unpublished observations), to ask whether the GLU immunoreactivity reflects neurotrans- mitter GLU. As in brain sections, we found by double immuno- staining that in vitro 84 5% of VTA DA neurons were GLU (n 1503 neurons in 12 cultures prepared on five separate culture days) (Fig. 2 A). We obtained similar levels of colocalization in SN cultures. We corroborated these results using a polyclonal GLU antiserum (Arnel, New York, NY) ( Hepler et al., 1988); more- over, a recent EM study using this antibody ( Smith et al., 1996) revealed significant GLU staining of DA neuron dendrites in the intact ...
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... cultures from brain regions with well character- ized cell types to verify further the specificity of the Glu2 GLU antiserum. In cultures from cerebellum, in which small cells are GLUergic granule cells and large cells are GABAergic Purkinje cells, we found that granule cells were GLU and GABA . Purkinje cells were GABA , but they were also GLU (Fig. 2 B). Similarly, in cultures from hippocampus (Fig. 2C) and nucleus accumbens (data not shown), most GLU cells were GABA , consistent with their being bona fide GLUergic neurons, whereas GABA cells were almost always GLU . This indicates that in vitro, Glu2 recognizes both GLUergic neurons and GABAergic neurons, in which GLU is likely to be ...
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... cell types to verify further the specificity of the Glu2 GLU antiserum. In cultures from cerebellum, in which small cells are GLUergic granule cells and large cells are GABAergic Purkinje cells, we found that granule cells were GLU and GABA . Purkinje cells were GABA , but they were also GLU (Fig. 2 B). Similarly, in cultures from hippocampus (Fig. 2C) and nucleus accumbens (data not shown), most GLU cells were GABA , consistent with their being bona fide GLUergic neurons, whereas GABA cells were almost always GLU . This indicates that in vitro, Glu2 recognizes both GLUergic neurons and GABAergic neurons, in which GLU is likely to be present as a GABA precursor. This differs from ...
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... to the situation in the intact VTA and SN, some DA neurons appear to be GABAergic. In a previous study, 2% of SN and 0.6% of VTA DA neurons in high-purity postnatal cultures were GABA ( Masuko et al., 1992). In our cultures (Fig. 2 D), 11 1.6% of DA neurons were GABA (n 299 cells in eight cultures). These TH /GABA VM neurons may derive from a minority population of SN reticulata neurons that send collateral projections to both the tectum and the striatum and contain both DA and GABA (Campbell et al., 1991). In contrast, hypothalamic DA neurons are extensively ...
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... (some are indicated by C 2 , arrows). D, In monkey V M, the majority of DA neurons (D 1 ) were GLUergic (D 2 ). Occasional DA neurons (D 2 , arrow) were non-GLUergic. This is shown as a color merge in D3. experiments). GLU staining of thin processes and varicosities (which we have shown previously to be axonal) was largely elim- inated by L-DON (Fig. 4C 2 ...
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... morphological observations in single-cell microcultures, in which we can be assured that all the processes arise from a single neuron, indicate that DA neurons indeed have two types of chemical synapses with distinct synaptic morphologies (Fig. 12). The synapses are segregated to different postsynaptic domains, with GLUergic terminals localized to proximal dendrites and the TH-GLU varicosities more peripherally distributed and appar- ently not contacting major dendritic branches. Taken together with the synaptic physiology, our morphological observations indicate that DA neurons ...
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... Our findings suggest that the E326K mutation in the GBA1 gene may lead to a reduction in the sodium currents and a decrease in the synaptic activity in DA neurons and these are also observed in sPD-derived neurons. Studies have shown that many DA neurons are also glutamatergic 69 and exert rapid synaptic currents by their glutamatergic synapses and slower modulatory actions via their dopaminergic synapses. Therefore, EPSCs are expected to be present when recording synaptic currents in our DA neuronal cultures, and we have measured these to be different in these PD models. ...
- Idan Rosh, Utkarsh Tripathi, Yara Hussein, Wote Amelo Rike, Jose Djamus, Boris Shklyar, Andreea Manole, Henry Houlden, Jurgen Winkler, Fred H. Gage & Shani Stern
Parkinson’s disease (PD) is a neurodegenerative disease with both genetic and sporadic origins. In this study, we investigated the electrophysiological properties, synaptic activity, and gene expression differences in dopaminergic (DA) neurons derived from induced pluripotent stem cells (iPSCs) of healthy controls, sporadic PD (sPD) patients, and PD patients with E326K- GBA1 mutations. Our results demonstrate reduced sodium currents and synaptic activity in DA neurons derived from PD patients with E326K- GBA1 mutations, suggesting a potential contribution to PD pathophysiology. We also observed distinct electrophysiological alterations in sPD DA neurons, which included a decrease in synaptic currents. RNA sequencing analysis revealed unique dysregulated pathways in sPD neurons and E326K- GBA1 neurons, further supporting the notion that molecular mechanisms driving PD may differ between PD patients. In agreement with our previous reports, Extracellular matrix and Focal adhesion pathways were among the top dysregulated pathways in DA neurons from sPD patients and from patients with E326K- GBA1 mutations. Overall, our study further confirms that impaired synaptic activity is a convergent functional phenotype in DA neurons derived from PD patients across multiple genetic mutations as well as sPD. At the transcriptome level, we find that the brain extracellular matrix is highly involved in PD pathology across multiple PD-associated mutations as well as sPD.
... However, it is possible that alternative mechanisms of self-excitation exist in vivo that could support our model and give rise to traveling waves. For example, DA fibers can co-release glutamate 54,[75][76][77][78] . Activation of nAChRs on DA might lead to glutamate release, which in turn would both excite CINs on a fast time scale through ionotropic glutamate receptors that would effectively provide self-excitation, and inhibit them more slowly by activation of metabotropic D2Rs 54 . ...
Striatal dopamine encodes reward, with recent work showing that dopamine release occurs in spatiotemporal waves. However, the mechanism of dopamine waves is unknown. Here we report that acetylcholine release in mouse striatum also exhibits wave activity, and that the spatial scale of striatal dopamine release is extended by nicotinic acetylcholine receptors. Based on these findings, and on our demonstration that single cholinergic interneurons can induce dopamine release, we hypothesized that the local reciprocal interaction between cholinergic interneurons and dopamine axons suffices to drive endogenous traveling waves. We show that the morphological and physiological properties of cholinergic interneuron – dopamine axon interactions can be modeled as a reaction-diffusion system that gives rise to traveling waves. Analytically-tractable versions of the model show that the structure and the nature of propagation of acetylcholine and dopamine traveling waves depend on their coupling, and that traveling waves can give rise to empirically observed correlations between these signals. Thus, our study provides evidence for striatal acetylcholine waves in vivo, and proposes a testable theoretical framework that predicts that the observed dopamine and acetylcholine waves are strongly coupled phenomena.
... The connectivity of the DA system is predominantly non-synaptic (Descarries et al., 2008;Ducrot et al., 2021;Wildenberg et al., 2021), with a majority of DA-releasing terminals not located in close apposition to a postsynaptic domain (Caillé et al., 1996;Descarries et al., 2008;Descarries et al., 1996;Descarries and Mechawar, 2000;Ducrot et al., 2021). A smaller synaptic subset of DA neuron terminals has the ability to co-release glutamate or GABA (Dal Bo et al., 2004;Mendez et al., 2008;Stuber et al., 2010;Sulzer et al., 1998;Tritsch et al., 2016;Tritsch et al., 2012). ...
Midbrain dopamine (DA) neurons are key regulators of basal ganglia functions. The axonal domain of these neurons is highly complex, with a large subset of non-synaptic release sites and a smaller subset of synaptic terminals from which in addition to DA, glutamate or GABA are also released. The molecular mechanisms regulating the connectivity of DA neurons and their neurochemical identity are unknown. An emerging literature suggests that neuroligins, trans-synaptic cell adhesion molecules, regulate both DA neuron connectivity and neurotransmission. However, the contribution of their major interaction partners, neurexins (Nrxns), is unexplored. Here, we tested the hypothesis that Nrxns regulate DA neuron neurotransmission. Mice with conditional deletion of all Nrxns in DA neurons (DAT::NrxnsKO) exhibited normal basic motor functions. However, they showed an impaired locomotor response to the psychostimulant amphetamine. In line with an alteration in DA neurotransmission, decreased levels of the membrane DA transporter (DAT) and increased levels of the vesicular monoamine transporter (VMAT2) were detected in the striatum of DAT::NrxnsKO mice, along with reduced activity-dependent DA release. Strikingly, electrophysiological recordings revealed an increase of GABA co-release from DA neuron axons in the striatum of these mice. Together, these findings suggest that Nrxns act as regulators of the functional connectivity of DA neurons.
... DA neuron synaptic transmission was first recognized as glutamate cotransmission in postnatal cell cultures. 20 DA neurons in the medial VTA projecting to the medial shell of the NAc 6,21,22 invariably elicit excitatory synaptic responses via glutamate cotransmission, 23,24 dependent on the expression of vesicular glutamate transporter VGlut2. 25,26 Glutamate cotransmission also generates slower but discrete synaptic currents in the lateral CPu via metabotropic glutamate receptors (mGluRs). ...
... DA neurons in the SN elicit D2R-mediated subsecond inhibitory synaptic responses in the CPu. 31 DA1/5R-mediated excitatory synaptic responses were first identified in the olfactory tubercle, 33 then in the lateral CPu. 27 These three neurotransmitters-identified as mediators of DA neuron synaptic transmission -engage five receptors: ionotropic glutamate receptors (iGluRs), mGluR1, DA D2R, DA D1/5 (D1-like) R, and GABA A R. 20,23,24,[27][28][29]31,[33][34][35] The corresponding synaptic actions can be categorized as either canonical synaptic transmission mediated by ion-channel-type receptors (iGluR and GABA A R) or as subsecond synaptic transmission mediated by G protein-coupled receptors (DAR and mGluR) activating effector channels to generate discrete synaptic currents, in contrast to volume transmission, which does not elicit discrete postsynaptic currents. ...
Dopamine neurons project to the striatum to control movement, cognition, and motivation via slower volume transmission as well as faster dopamine, glutamate, and GABA synaptic actions capable of conveying the temporal information in dopamine neuron firing. To define the scope of these synaptic actions, recordings of dopamine-neuron-evoked synaptic currents were made in four major striatal neuron types, spanning the entire striatum. This revealed that inhibitory postsynaptic currents are widespread, while excitatory postsynaptic currents are localized to the medial nucleus accumbens and the anterolateral-dorsal striatum, and that all synaptic actions are weak in the posterior striatum. Synaptic actions in cholinergic interneurons are the strongest, variably mediating inhibition throughout the striatum and excitation in the medial accumbens, capable of controlling their activity. This mapping shows that dopamine neuron synaptic actions extend throughout the striatum, preferentially target cholinergic interneurons, and define distinct striatal subregions.
... In contrast, activation of DA neurons within the NAc promotes reward seeking behavior (178)(179)(180)(181). In addition, A 10 DA neurons have been shown to express vesicular glutamate transporters 2 (VGlut2), which reportedly enhances DA storage, neuronal growth and survival, the density of DA innervation of the NAc, as well as promotes the corelease of DA and glutamate, the latter of which induces a fast excitatory postsynaptic current (182)(183)(184)(185). The knockout of VGlut2 gene, in contrast, reverses all the effects by abolishing glutamate release from A 10 DA neurons and reducing the excitatory signal to the NAc (184,185). ...
Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP), a pleiotropic neuropeptide, is widely distributed throughout the body. The abundance of PACAP expression in the central and peripheral nervous systems, and years of accompanying experimental evidence, indicates that PACAP plays crucial roles in diverse biological processes ranging from autonomic regulation to neuroprotection. In addition, PACAP is also abundantly expressed in the hypothalamic areas like the ventromedial and arcuate nuclei (VMN and ARC, respectively), as well as other brain regions such as the nucleus accumbens (NAc), bed nucleus of stria terminalis (BNST), and ventral tegmental area (VTA) – suggesting that PACAP is capable of regulating energy homeostasis via both the homeostatic and hedonic energy balance circuitries. The evidence gathered over the years has increased our appreciation for its function in controlling energy balance. Therefore, this review aims to further probe how the pleiotropic actions of PACAP in regulating energy homeostasis is influenced by sex and dynamic changes in energy status. We start with a general overview of energy homeostasis, and then introduce the integral components of the homeostatic and hedonic energy balance circuitries. Next, we discuss sex differences inherent to the regulation of energy homeostasis via these two circuitries, as well as the activational effects of sex steroid hormones that bring about these intrinsic disparities between males and females. Finally, we explore the multifaceted role of PACAP in regulating homeostatic and hedonic feeding through its actions in regions like the NAc, BNST, and in particular the ARC, VMN and VTA that occur in sex- and energy status-dependent ways.
... It is now recognized that DA neurons' role in diverse behaviors -from reward learning, to social motivation, to executive functions and motor output-results from a complex neuroanatomic and circuit organization through the midbrain, with differential gene expression profiles, electrophysiologic properties, and co-transmitter content (Watabe-Uchida et al., 2012;Beier et al., 2015;Lerner et al., 2015;Morales and Margolis, 2017;Zhang et al., 2017;Farassat et al., 2019;Poulin et al., 2020). For example, recent evidence, mainly from rodent models, indicates various DA neurons can co-contain glutamate (Sulzer et al., 1998;Root et al., 2016), and various neuropeptides (Seroogy et al., 1988;Bouarab et al., 2019;Pristera`et al., 2019;Nagaeva et al., 2020). ...
The ventral midbrain is the primary source of dopamine- (DA) expressing neurons in most species. GABA-ergic and glutamatergic cell populations are intermixed among DA-expressing cells and purported to regulate both local and long-range dopamine neuron activity. Most work has been conducted in rodent models, however due to evolutionary expansion of the ventral midbrain in primates, the increased size and complexity of DA subpopulations warrants further investigation. Here, we quantified the number of DA neurons, and their GABA-ergic complement in classic DA cell groups A10 (midline ventral tegmental area nuclei [VTA] and parabrachial pigmented nucleus [PBP]), A9 (substantia nigra, pars compacta [SNc]) and A8 (retrorubral field [RRF]) in the macaque. Because the PBP is a disproportionately expanded feature of the A10 group, and has unique connectional features in monkeys, we analyzed A10 data by dividing it into ‘classic’ midline nuclei and the PBP. Unbiased stereology revealed total putative DA neuron counts to be 210,238 +/- 17,127 (A10 = 110,319 +/- 9,649, A9= 87,399 +/-7,751 and A8=12,520 +/- 827). Putative GABAergic neurons were fewer overall, and evenly dispersed across the DA subpopulations (GAD67= 71,215 +/- 5,663; A10=16,836 +/- 2,743; A9=24,855 +/- 3,144 and A8=12,633 +/- 3,557). Calculating the GAD67/TH ratio for each subregion revealed differential balances of these two cell types across the DA subregions. The A8 subregion had the highest complement of GAD67-positive neurons compared to TH-positive neurons (1:1), suggesting a potentially high capacity for GABAergic inhibition of DA output in this region.
... Our results call for an expansion of this list, as SNc DA neurons rely exclusively on Gat1-mediated uptake as their source of GABA. This mechanism explains why GABA co-release is not detected in primary DA neuron cultures not supplemented with extracellular GABA (Sulzer et al., 1998) and raises the interesting possibility that GABA co-release may vary at individual synapses with extracellular GABA levels and Gat1 transporter activity. More importantly, our findings suggest that the function of GABA transporters extends beyond terminating GABA's actions in the synaptic cleft to include sustaining vesicular filling. ...
Dopamine (DA)-releasing neurons in the substantia nigra pars compacta (SNcDA) inhibit target cells in the striatum through postsynaptic activation of γ-aminobutyric acid (GABA) receptors. However, the molecular mechanisms responsible for GABAergic signaling remain unclear, as SNcDA neurons lack enzymes typically required to produce GABA or package it into synaptic vesicles. Here, we show that aldehyde dehydrogenase 1a1 (Aldh1a1), an enzyme proposed to function as a GABA synthetic enzyme in SNcDA neurons, does not produce GABA for synaptic transmission. Instead, we demonstrate that SNcDA axons obtain GABA exclusively through presynaptic uptake using the membrane GABA transporter Gat1 (encoded by Slc6a1). GABA is then packaged for vesicular release using the vesicular monoamine transporter Vmat2. Our data therefore show that presynaptic transmitter recycling can substitute for de novo GABA synthesis and that Vmat2 contributes to vesicular GABA transport, expanding the range of molecular mechanisms available to neurons to support inhibitory synaptic communication.
... Many recent studies have focused on co-transmission in mammalian reward systems, especially in the DAN of the VTA. The co-transmission of dopamine and glutamate in these neurons in developing and adult mammals (studies were performed in rats, mice, primates, including humans, in vitro and in vivo; dopaminergic characteristics are usually detected by the presence of tyrosine hydroxylase (TH), glutamate according to VGluT2 transporter) is sufficiently documented (Lapish et al., 2006;Vaaga et al., 2014;Sulzer et al., 1998;Stuber et al., (Vogt et al., 2014). A, B -Visualization of two main sets of dopaminergic cells projecting to the mushroom body (PAM and PPL1 clusters), dashed line -the position of the mushroom body, scale bars -50 µm, C -scheme of basic bidirectional modulation of reward / punishment value by different groups of dopaminergic neurons in the mushroom body and formation of appetitive /aversive memories in associative learning. ...
In classical neuroscience, Dale´s principle postulates that neuronal identity is conferred by the specific neurotransmitter that it releases. However, the brain might be more tractable to specific situations regardless of specific specialisation which may contradict this principle. Hence, this constrained approach of how we perceive and study the nervous system must be revisited and revised, specifically by studying the dopaminergic system. We presume a relatively flexible change in the dopaminergic system due to neuronal activity or environmental changes. While the parallel between the reward system of mammals and insects is generally well accepted, herein, we extend the idea that the insect nervous system might also possess incredible plasticity, similar to the mammalian system. In this review, we critically evaluate the available information about the reward system in vertebrates and invertebrates, emphasising the dopaminergic neuronal plasticity, a challenge to the classical Dale’s principle. Thus, neurotransmitter switching significantly disrupts the static idea of neural network organisation and suggests greater possibilities for a dynamic response to the current life context of organisms.
... It is now recognized that DA neurons' role in diverse behaviors--from reward learning, to social motivation, to executive functions and motor output--results from a complex neuroanatomic and circuit organization through the midbrain, with differential gene expression profiles, electrophysiologic properties, and co-transmitter content (Watabe-Uchida et al., 2012;Beier et al., 2015;Lerner et al., 2015;Morales and Margolis, 2017;Zhang et al., 2017;Farassat et al., 2019;Poulin et al., 2020). For example, recent evidence, mainly from rodent models, indicates various DA neurons can co-contain glutamate (Sulzer et al., 1998;Root et al., 2016), and various neuropeptides (Seroogy et al., 1988;Bouarab et al., 2019;Pristera et al., 2019;Nagaeva et al., 2020). ...
The ventral midbrain is the primary source of dopamine- (DA) expressing neurons in most species. GABA-ergic and glutamatergic cell populations are intermixed among DA-expressing cells and purported to regulate both local and long-range dopamine neuron activity. Most work has been conducted in rodent models, however due to evolutionary expansion of the ventral midbrain in primates, the increased size and complexity of DA subpopulations warrants further investigation. Here, we quantified the number of DA neurons, and their GABA-ergic complement in classic DA cell groups A10 (midline ventral tegmental area nuclei [VTA] and parabrachial pigmented nucleus [PBP]), A9 (substantia nigra, pars compacta [SNc]) and A8 (retrorubral field [RRF]) in the macaque. Because the PBP is a disproportionately expanded feature of the A10 group, and has unique connectional features in monkeys, we analyzed A10 data by dividing it into ‘classic’ midline nuclei and the PBP. Unbiased stereology revealed total putative DA neuron counts to be 210,238 +/− 17,127 (A10 = 110,319 +/− 9,649, A9= 87,399 +/−7,751 and A8=12,520 +/− 827). Putative GABAergic neurons were fewer overall, and evenly dispersed across the DA subpopulations (GAD67= 71,215 +/− 5,663; A10=16,836 +/− 2,743; A9=24,855 +/− 3,144 and A8=12,633 +/− 3,557). Calculating the GAD67/TH ratio for each subpopulation revealed differential balances of these two cell types across the DA subpopulations. The A8 subpopulation had the highest complement of GAD67-positive neurons compared to TH-positive neurons (1:1), suggesting a potentially high capacity for GABAergic inhibition of DA output in this region.
HIGHLIGHTS
The A10 subpopulation expands laterally and caudally in nonhuman primates
The A10, A9, and A8 subpopulations comprise 52%, 42% and 6% of DA neurons
GABAergic neurons are more evenly dispersed
The A8 subpopulation has the highest ratio of GABA: DA neurons
... 9,10 Previous studies have demonstrated that dopamine neurons possess the machinery for co-release of other neurotransmitters, including glutamate and GABA, but to what extent, if any, co-release events spatially overlap remains insufficiently understood. [11][12][13][14][15] Electrochemical and microdialysis assays in midbrain regions have shown that somatodendritic release of dopamine constitutes an important component of dopamine signaling, with notable implications in disease and behavior. [16][17][18][19][20][21] However, the spatial and temporal dynamics of dendritic release events and their regulatory mechanisms remain poorly characterized. ...
Chemical neurotransmission constitutes one of the fundamental modalities of communication between neurons. Monitoring release of these chemicals has traditionally been difficult to carry out at spatial and temporal scales relevant to neuron function. To understand chemical neurotransmission more fully, we need to improve the spatial and temporal resolutions of measurements for neurotransmitter release. To address this, we engineered a chemi-sensitive, two-dimensional nanofilm that facilitates subcellular visualization of the release and diffusion of the neurochemical dopamine with synaptic resolution, quantal sensitivity, and simultaneously from hundreds of release sites. Using this technology, we were able to monitor the spatiotemporal dynamics of dopamine release in dendritic processes, a poorly understood phenomenon. We found that dopamine release is broadcast from a subset of dendritic processes as hotspots that have a mean spatial spread of ~3.2 μm (full width at half maximum) and are observed with a mean spatial frequency of 1 hotspot per ~7.5 μm of dendritic length. Major dendrites of dopamine neurons and fine dendritic processes, as well as dendritic arbors and dendrites with no apparent varicose morphology participated in dopamine release. Remarkably, these release hotspots colocalized with Bassoon, suggesting that Bassoon may contribute to organizing active zones in dendrites, similar to its role in axon terminals.
