Development of hippocampal mossy fiber synaptic outputs by new neurons in the adult brain

New neurons are continuously generated in restricted regions of the adult mammalian brain. Although these adult-born neurons have been shown to receive synaptic inputs, little is known about their synaptic outputs. Using retrovirus-mediated birth-dating and labeling in combination with serial section electron microscopic reconstruction, we report that mossy fiber en passant boutons of adult-born dentate granule cells form initial synaptic contacts with CA3 pyramidal cells within 2 weeks after their birth and reach morphologic maturity within 8 weeks in the adult hippocampus. Knockdown of Disrupted-in-Schizophrenia-1 (DISC1) in newborn granule cells leads to defects in axonal targeting and development of synaptic outputs in the adult brain. Together with previous reports of synaptic inputs, these results demonstrate that adult-born neurons are fully integrated into the existing neuronal circuitry. Our results also indicate a role for DISC1 in presynaptic development and may have implications for the etiology of schizophrenia and related mental disorders.     

Protein kinase LKB1 regulates polarized dendrite formation of adult hippocampal newborn neurons

Adult-born granule cells in the dentate gyrus of the rodent hippocampus are important for memory formation and mood regulation, but the cellular mechanism underlying their polarized development, a process critical for their incorporation into functional circuits, remains unknown. We found that deletion of the serine-threonine protein kinase LKB1 or overexpression of dominant-negative LKB1 reduced the polarized initiation of the primary dendrite from the soma and disrupted its oriented growth toward the molecular layer. This abnormality correlated with the dispersion of Golgi apparatus that normally accumulated at the base and within the initial segment of the primary dendrite, and was mimicked by disrupting Golgi organization via altering the expression of Golgi structural proteins GM130 or GRASP65. Thus, besides its known function in axon formation in embryonic pyramidal neurons, LKB1 plays an additional role in regulating polarized dendrite morphogenesis in adult-born granule cells in the hippocampus.Granule cells are continuously being generated in the dentate gyrus of the adult hippocampus (1). These adult-born neurons play an important role in memory formation (2–4) and mood regulation (5, 6). Typically, developing granule cells assume a bipolar morphology, with highly branched dendrites extending toward the molecular layer, and a long thin axon projecting into the hilar area (7). This asymmetric structure provides the anatomical basis for directional information flow within the neuron, receiving input from the entorhinal cortex at the dendrite and sending axonal output to the CA3 region. However, little is known about the mechanism responsible for the polarization of these newborn neurons within the adult tissue environment, including the specification of a proper number of axons and dendrites, as well as the extension of these processes with proper orientation relative to the layer structure of the dentate gyrus.The serine/threonine protein kinase liver kinase B1 (LKB1) is a well-known regulator of cell polarity; it was originally identified as one of the six master regulators of anterior–posterior axis of the Caenorhabditis elegans zygote (8). Growing evidence indicates that LKB1 also plays important roles in cellular polarization in epithelial and other nonneural tissues in Drosophila and vertebrates (9–11). In the rodent central nervous system, LKB1 was shown to regulate axon formation and cell migration in embryonic cortical pyramidal neurons (12–14). However, it is unclear whether LKB1 also regulates other asymmetrical aspects of neuronal development, such as the polarized dendrite formation. Moreover, because LKB1 is also expressed in the adult brain, whether it plays a role in the morphogenesis of newborn neurons within the adult tissue environment remains to be determined.In this study, we used a retrovirus-mediated gene-transfer approach (15) to delete the LKB1 allele in adult-born hippocampal granule cells, and found that LKB1 is essential for polarized initiation of a single primary dendrite from the soma and oriented growth of its arbor toward the molecular layer. We also obtained evidence that the effect of LKB1 on polarized dendrite development is mediated by regulating the distribution of the Golgi apparatus in the cytoplasm. Together, the data from this study demonstrate a unique function of LKB1 in dendrite morphogenesis and suggest a cellular mechanism underlying its action.     

Astrocyte-derived neurons provide excitatory input to the adult striatal circuitry

Astrocytes have emerged as a potential source for new neurons in the adult mammalian brain. In mice, adult striatal neurogenesis can be stimulated by local damage, which recruits striatal astrocytes into a neurogenic program by suppression of active Notch signaling (J. P. Magnusson et al., Science 346, 237–241 [2014]). Here, we induced adult striatal neurogenesis in the intact mouse brain by the inhibition of Notch signaling in astrocytes. We show that most striatal astrocyte-derived neurons are confined to the anterior medial striatum, do not express established striatal neuronal markers, and exhibit dendritic spines, which are atypical for striatal interneurons. In contrast to striatal neurons generated during development, which are GABAergic or cholinergic, most adult astrocyte-derived striatal neurons possess distinct electrophysiological properties, constituting the only glutamatergic striatal population. Astrocyte-derived neurons integrate into the adult striatal microcircuitry, both receiving and providing synaptic input. The glutamatergic nature of these neurons has the potential to provide excitatory input to the striatal circuitry and may represent an efficient strategy to compensate for reduced neuronal activity caused by aging or lesion-induced neuronal loss.

The striatum is a central brain structure important for the initiation and coordination of motor functions, sensory processing, and reward (1–3). The striatal neuronal composition is dominated by a vast majority (∼95%) of projection neurons, the so called medium spiny neurons (MSNs), and a small yet diverse population of interneurons. The vast majority of striatal neurons is GABAergic, apart from one population of cholinergic interneurons, which provide most of the cholinergic input to the network (4). GABAergic interneurons provide synaptic inhibition to MSNs with cell type–dependent characteristics such as the location of inhibitory synapses (MSN cell bodies or dendrites), synaptic dynamics (depression and facilitation), and connection probability (5). This intricate synaptic connectivity is essential for normal striatal function as it shapes the activity of MSNs, thus determining the downstream flow of information from the striatum to the output nuclei of the basal ganglia. There are several types of GABAergic interneurons, and their classification is an ongoing effort, facilitated by recently discovered, new types of neurons and neuronal markers (6–9).Most striatal neurons are generated during early development. There is evidence for adult striatal neurogenesis in the uninjured human brain (10) and possibly some nonhuman primates and to a much smaller degree in rats and rabbits (11). For their abundance throughout the brain and similarity to adult neural stem cells, parenchymal astrocytes have emerged as a potential source for new neurons in nonneurogenic brain regions (12). Indeed, in response to experimental stroke or an excitotoxic lesion, some astrocytes in the mouse striatum can generate neurons (13–16). In both lesions striatal astrocytes are recruited into a neurogenic program (15, 16), which is mediated through suppression of active Notch signaling (15). Striatal neurogenesis in mice can also be evoked by deletion of the Notch downstream effector RBPj-κ in astrocytes in the otherwise intact striatum (15) and offers a model system to study adult-born striatal neurons. Furthermore, deletion of RBPj-κ in striatal astrocytes in the context of stroke increases the efficacy of astrocyte recruitment and boosts stroke-induced striatal neurogenesis (17). Therapeutically interesting, adult neurogenesis by striatal astrocytes can be further amplified by epidermal growth factor infusion (18). However, it is currently unclear whether astrocyte-derived neurons become functional, which neuronal properties they possess, and whether they can integrate into the adult striatal circuitry.To better understand the function of adult striatal neurogenesis and harness its therapeutic potential, we capitalized on the ability to induce astrocyte-derived striatal neurogenesis in the adult mouse brain by cell-specific inhibition of the Notch signaling pathway. We show that most astrocyte-derived neurons constitute a previously unknown subset of striatal neurons with distinct electrophysiological properties and morphological features, which possess the capacity to synaptically integrate into the adult striatal circuitry and provide glutamatergic input to striatal projection neurons.     

Melatonin synergizes with citalopram to induce antidepressant‐like behavior and to promote hippocampal neurogenesis in adult mice

Adult hippocampal neurogenesis is affected in some neuropsychiatric disorders such as depression. Numerous evidence indicates that plasma levels of melatonin are decreased in depressed patients. Also, melatonin exerts positive effects on the hippocampal neurogenic process and on depressive‐like behavior. In addition, antidepressants revert alterations of hippocampal neurogenesis present in models of depression following a similar time course to the improvement of behavior. In this study, we analyzed the effects of both, citalopram, a widely used antidepressant, and melatonin in the Porsolt forced swim test. In addition, we investigated the potential antidepressant role of the combination of melatonin and citalopram (MLTCITAL), its type of pharmacological interaction on depressive behavior, and its effect on hippocampal neurogenesis. Here, we found decreased immobility behavior in mice treated with melatonin (<14–33%) and citalopram (<17–30%). Additionally, the MLTCITAL combination also decreased immobility (<22–35%) in comparison with control mice, reflecting an antidepressant‐like effect after 14 days of treatment. Moreover, MLTCITAL decreased plasma corticosterone levels (≤13%) and increased cell proliferation (>29%), survival (>39%), and the absolute number of ‐associated new neurons (>53%) in the dentate gyrus of the hippocampus. These results indicate that the MLTCITAL combination exerts synergism to induce an antidepressant‐like action that could be related to the modulation of adult hippocampal neurogenesis. This outcome opens the opportunity of using melatonin to promote behavioral benefits and hippocampal neurogenesis in depression and also supports the use of the MLTCITAL combination as an alternative to treat depression.     

A small molecule accelerates neuronal differentiation in the adult rat

Adult neurogenesis occurs in mammals and provides a mechanism for continuous neural plasticity in the brain. However, little is known about the molecular mechanisms regulating hippocampal neural progenitor cells (NPCs) and whether their fate can be pharmacologically modulated to improve neural plasticity and regeneration. Here, we report the characterization of a small molecule (KHS101) that selectively induces a neuronal differentiation phenotype. Mechanism of action studies revealed a link of KHS101 to cell cycle exit and specific binding to the TACC3 protein, whose knockdown in NPCs recapitulates the KHS101-induced phenotype. Upon systemic administration, KHS101 distributed to the brain and resulted in a significant increase in neuronal differentiation in vivo. Our findings indicate that KHS101 accelerates neuronal differentiation by interaction with TACC3 and may provide a basis for pharmacological intervention directed at endogenous NPCs.     

Tangential migration of neuronal precursors of glutamatergic neurons in the adult mammalian brain

In a classic model of mammalian brain formation, precursors of principal glutamatergic neurons migrate radially along radial glia fibers whereas GABAergic interneuron precursors migrate tangentially. These migration modes have significant implications for brain function. Here we used clonal lineage tracing of active radial glia-like neural stem cells in the adult mouse dentate gyrus and made the surprising discovery that proliferating neuronal precursors of glutamatergic granule neurons exhibit significant tangential migration along blood vessels, followed by limited radial migration. Genetic birthdating and morphological and molecular analyses pinpointed the neuroblast stage as the main developmental window when tangential migration occurs. We also developed a partial “whole-mount” dentate gyrus preparation and observed a dense plexus of capillaries, with which only neuroblasts, among the entire population of progenitors, are directly associated. Together, these results provide insight into neuronal migration in the adult mammalian nervous system.The nervous system is formed by migration of neuronal precursors and immature neurons to specific locations during development. The classic radial unit hypothesis of mammalian brain development postulates that in the developing neocortex, glutamatergic, excitatory, principal neurons migrate radially to form discrete information-processing columns of ontogenetic origin (1), whereas GABAergic, inhibitory, modulatory interneurons migrate tangentially across columns (2). Neurogenesis persists in the adult mammalian brain in two primary regions and is thought to follow the classic migration model (3, 4). In the subventricular zone (SVZ) of the lateral ventricles, new neurons generated from neural precursors migrate tangentially to the olfactory bulb to become GABAergic interneurons (5, 6). In contrast, in the subgranular zone (SGZ) of the dentate gyrus, new neurons generated from radial glia-like neural stem cells (RGLs) migrate radially into the granule cell layer to become principal glutamatergic granule cells (7). Due to technical challenges, migratory patterns have only been examined at the cell-population level, and thus we still lack detailed information about the spatial relationship between individual precursors and their progeny in vivo. Both adult neurogenic niches are highly vascularized, and this property is hypothesized to play a critical role in adult neurogenesis (3). In both adult SVZ (8–10) and SGZ (11), proliferating progenitor cells are in close association with the vasculature, yet the functional role of the vasculature in the niche remains to be fully explored.Contrary to the classic model, our recent clonal lineage tracing of individual quiescent RGLs showed tangential distribution of glutamatergic granule neurons with respect to their parental RGL in the adult dentate gyrus (12). We therefore systematically investigated the migration pattern and trajectory of these newborn cells. Using a clonal lineage-tracing approach that preferentially targets active RGLs in the adult mouse dentate gyrus, thereby birthdating their newborn progeny in vivo, we found significant tangential distribution of newborn neuroblasts from their parental RGL. Furthermore, neuroblasts directly contact the vascular network, suggesting an important function of blood vessels as a substrate for migration. Together, our results reveal a previously unidentified mode of glutamatergic neuronal migration under physiological conditions in the adult mammalian brain.     

Chronic treatment with melatonin stimulates dendrite maturation and complexity in adult hippocampal neurogenesis of mice

In the course of adult hippocampal neurogenesis, the postmitotic maturation and survival phase is associated with dendrite maturation. Melatonin modulates the survival of new neurons with relative specificity. During this phase, the new neurons express microtubule-associated protein doublecortin (DCX). Here, we show that the entire population of cells expressing DCX is increased after 14 days of treatment with melatonin. As melatonin also affects microtubule polymerization which is important for neuritogenesis and dendritogenesis, we studied the consequences of chronic melatonin administration on dendrite maturation of DCX-positive cells. Treatment with melatonin increased the number of DCX-positive immature neurons with more complex dendrites. Sholl analysis revealed that melatonin treatment lead to greater complexity of the dendritic tree. In addition, melatonin increased the total volume of the granular cell layer. Besides its survival-promoting effect, melatonin thus also increases dendritic maturation in adult neurogenesis. This might open the opportunity of using melatonin as an adjuvant in attempts to extrinsically stimulate adult hippocampal neurogenesis in neuropsychiatric disease, dementia or cognitive ageing.     

Recruitment and replacement of hippocampal neurons in young and adult chickadees: an addition to the theory of hippocampal learning

We used [3H]thymidine to document the birth of neurons and their recruitment into the hippocampal complex (HC) of juvenile (4.5 months old) and adult blackcapped chickadees (Parus atricapillus) living in their natural surroundings. Birds received a single dose of [3H]thymidine in August and were recaptured and killed 6 weeks later, in early October. All brains were stained with Cresyl violet, a Nissl stain. The boundaries of the HC were defined by reference to the ventricular wall, the brain surface, or differences in neuronal packing density. The HC of juveniles was as large as or larger than that of adults and packing density of HC neurons was 31% higher in juveniles than in adults. Almost all of the 3H-labeled HC neurons were found in a 350-m-wide layer of tissue adjacent to the lateral ventricle. Within this layer the fraction of 3H-labeled neurons was 50% higher in juveniles than in adults. We conclude that the HC of juvenile chickadees recruits more neurons and has more neurons than that of adults. We speculate that juveniles encounter greater environmental novelty than adults and that the greater number of HC neurons found in juveniles allows them to learn more than adults. At a more general level, we suggest that (i) long-term learning alters HC neurons irreversibly; (ii) sustained hippocampal learning requires the periodic replacement of HC neurons; (iii) memories coded by hippocampal neurons are transferred elsewhere before the neurons are replaced.     

Deletion of TrkB in adult progenitors alters newborn neuron integration into hippocampal circuits and increases anxiety-like behavior

New neurons in the adult dentate gyrus are widely held to incorporate into hippocampal circuitry via a stereotypical sequence of morphological and physiological transitions, yet the molecular control over this process remains unclear. We studied the role of brain-derived neurotrophic factor (BDNF)/TrkB signaling in adult neurogenesis by deleting the full-length TrkB via Cre expression within adult progenitors in TrkB(lox/lox) mice. By 4 weeks after deletion, the growth of dendrites and spines is reduced in adult-born neurons demonstrating that TrkB is required to create the basic organization of synaptic connections. Later, when new neurons normally display facilitated synaptic plasticity and become preferentially recruited into functional networks, lack of TrkB results in impaired neurogenesis-dependent long-term potentiation and cell survival becomes compromised. Because of the specific lack of TrkB signaling in recently generated neurons a remarkably increased anxiety-like behavior was observed in mice carrying the mutation, emphasizing the contribution of adult neurogenesis in regulating mood-related behavior.     

Adult hippocampal neural stem and progenitor cells regulate the neurogenic niche by secreting VEGF

The adult hippocampus hosts a population of neural stem and progenitor cells (NSPCs) that proliferates throughout the mammalian life span. To date, the new neurons derived from NSPCs have been the primary measure of their functional relevance. However, recent studies show that undifferentiated cells may shape their environment through secreted growth factors. Whether endogenous adult NSPCs secrete functionally relevant growth factors remains unclear. We show that adult hippocampal NSPCs secrete surprisingly large quantities of the essential growth factor VEGF in vitro and in vivo. This self-derived VEGF is functionally relevant for maintaining the neurogenic niche as inducible, NSPC-specific loss of VEGF results in impaired stem cell maintenance despite the presence of VEGF produced from other niche cell types. These findings reveal adult hippocampal NSPCs as an unanticipated source of an essential growth factor and imply an exciting functional role for adult brain NSPCs as secretory cells.In the adult brain, two major neurogenic niches persist throughout the mammalian life span: the subventricular zone (SVZ) and the subgranular zone (SGZ) of the hippocampus. Resident neural stem and progenitor cells (NSPCs) in each of these areas proliferate and give rise to new neurons that migrate and integrate into existing circuitry in the olfactory bulb or dentate gyrus (DG), respectively. Particularly in the DG, where neurogenesis is found in both rodents and humans, newly born neurons play critical roles in facilitating memory function (1, 2). This role of new neurons in memory is currently considered the dominant functional output of adult neurogenesis. However, recent research has revealed that transplanted embryonic stem cells can aid in injury recovery by secreting growth factors while undifferentiated (3, 4). The secretion of functionally relevant growth factors from endogenous adult hippocampal NSPCs has yet to be reported.We recently showed that cultured neonatal hippocampal progenitors secrete surprisingly large quantities of VEGF compared with astrocytes, microglia, and neurons (5), raising the possibility that NSPCs could be an unexpected source of this essential growth factor in the brain. Within the adult brain, VEGF (also known as VEGF-A) is a potent angiogenic and neurogenic growth factor (6–13). Although several studies have previously noted VEGF expression in cultured adult NSPCs (14, 15), the relative quantity and function of this VEGF are not clear, particularly in vivo, where other cellular sources of VEGF abound. We therefore investigated the contribution of NSPCs to hippocampal VEGF production and the functional role of NSPC-derived VEGF in maintaining the neurogenic niche.     

Cranial irradiation-induced inhibition of neurogenesis in hippocampal dentate gyrus of adult mice: attenuation by melatonin pretreatment

Abstract:  Radiation is an important therapeutic tool in the treatment of cancer. The tremendous development in radiotherapeutic techniques and dosimetry has made it possible to augment the patient survival. Therefore, attention has focused on long-range treatment side effects especially in relation to the neurocognitive changes. As cognitive health of an organism is considered to be maintained by the capacity of hippocampal neurogenesis, this study designed to evaluate the delayed effect of cranial irradiation on hippocampal neurogenesis, possible implication of oxidative stress and prophylactic action of melatonin in mice. One month after cranial irradiation (6 Gy, X-ray), changes in the population of immature and proliferating neurons in dentate gyrus were localized through the expression of the microtubule binding protein doublecortin (Dcx) and proliferation marker Ki-67. We found a substantial reduction in the Dcx and Ki-67 positive cells after irradiation. Melatonin pretreatment significantly ameliorated the radiation-induced decline in the Dcx and Ki-67 positive cells. In addition, profound increase in the 4-hydroxynonenal (4-HNE) and 8-hydroxy-2'-deoxyguanosine positive cells were reported in subventricular zone, granular cell layer and hilus after day 30 postirradiation. Immunoreactivity of these oxidative stress markers were significantly inhibited by melatonin pretreatment. To confirm the magnitude of free-radical scavenging potential of melatonin, we measured the in-vitro OH radical scavenging power of melatonin by electron spin resonance. Interestingly, the melatonin was capable of scavenging the OH radicals at very low concentration (IC₅₀ = 214.46 n m ). The findings indicate the possible benefit of melatonin treatment to combat the delayed side effects of cranial radiotherapy.     

Molecular layer perforant path-associated cells contribute to feed-forward inhibition in the adult dentate gyrus

New neurons, which have been implicated in pattern separation, are continually generated in the dentate gyrus in the adult hippocampus. Using a genetically modified rabies virus, we demonstrated that molecular layer perforant pathway (MOPP) cells innervated newborn granule neurons in adult mouse brain. Stimulating the perforant pathway resulted in the activation of MOPP cells before the activation of dentate granule neurons. Moreover, activation of MOPP cells by focal uncaging of glutamate induced strong inhibition of granule cells. Together, these results indicate that MOPP cells located in the molecular layer of the dentate gyrus contribute to feed-forward inhibition of granule cells via perforant pathway activation.     

Radial glia give rise to adult neural stem cells in the subventricular zone

Neural stem cells with the characteristics of astrocytes persist in the subventricular zone (SVZ) of the juvenile and adult brain. These cells generate large numbers of new neurons that migrate through the rostral migratory stream to the olfactory bulb. The developmental origin of adult neural stem cells is not known. Here, we describe a lox-Cre-based technique to specifically and permanently label a restricted population of striatal radial glia in newborn mice. Within the first few days after labeling, these radial glial cells gave rise to neurons, oligodendrocytes, and astrocytes, including astrocytes in the SVZ. Remarkably, the rostral migratory stream contained labeled migratory neuroblasts at all ages examined, including 150-day-old mice. Labeling dividing cells with the S-phase marker BrdUrd showed that new neurons continue to be produced in the adult by precursors ultimately derived from radial glia. Furthermore, both radial glia in neonates and radial glia-derived cells in the adult lateral ventricular wall generated self-renewing, multipotent neurospheres. These results demonstrate that radial glial cells not only serve as progenitors for many neurons and glial cells soon after birth but also give rise to adult SVZ stem cells that continue to produce neurons throughout adult life. This study identifies and provides a method to genetically modify the lineage that links neonatal and adult neural stem cells.     

Selective induction of neocortical GABAergic neurons by the PDK1-Akt pathway through activation of Mash1

Extracellular stimuli regulate neuronal differentiation and subtype specification during brain development, although the intracellular signaling pathways that mediate these processes remain largely unclear. We now show that the PDK1-Akt pathway regulates differentiation of telencephalic neural precursor cells (NPCs). Active Akt promotes differentiation of NPC into γ-aminobutyric acid-containing (GABAergic) but not glutamatergic neurons. Disruption of the Pdk1 gene or expression of dominant-negative forms of Akt suppresses insulin-like growth factor (IGF)-1 enhancement of NPC differentiation into neurons in vitro and production of neocortical GABAergic neurons in vivo. Furthermore, active Akt increased the protein levels and transactivation activity of Mash1, a proneural basic helix-loop-helix protein required for the generation of neocortical GABAergic neurons, and Mash1 was required for Akt-induced neuronal differentiation. These results have unveiled an unexpected role of the PDK1-Akt pathway: a key mediator of extracellular signals regulating the production of neocortical GABAergic neurons.     

Targeting neural precursors in the adult brain rescues injured dopamine neurons

In Parkinson's disease, multiple cell types in many brain regions are afflicted. As a consequence, a therapeutic strategy that activates a general neuroprotective response may be valuable. We have previously shown that Notch ligands support neural precursor cells in vitro and in vivo. Here we show that neural precursors express the angiopoietin receptor Tie2 and that injections of angiopoietin2 activate precursors in the adult brain. Signaling downstream of Tie2 and the Notch receptor regulate blood vessel formation. In the adult brain, angiopoietin2 and the Notch ligand Dll4 activate neural precursors with opposing effects on the density of blood vessels. A model of Parkinson's disease was used to show that angiopoietin2 and Dll4 rescue injured dopamine neurons with motor behavioral improvement. A combination of growth factors with little impact on the vasculature retains the ability to stimulate neural precursors and protect dopamine neurons. The cellular and pharmacological basis of the neuroprotective effects achieved by these single treatments merits further analysis.     

Dopamine controls the firing pattern of dopamine neurons via a network feedback mechanism

Changes in the firing pattern of midbrain dopamine neurons are thought to encode information for certain types of reward-related learning. In particular, the burst pattern of firing is predicted to result in more efficient dopamine release at target loci, which could underlie changes in synaptic plasticity. In this study, the effects of dopamine on the firing patterns of dopaminergic neurons in vivo and their electrophysiological characteristics in vitro were examined by using a genetic dopamine-deficient (DD) mouse model. Extracellular recordings in vivo showed that, although the firing pattern of dopamine neurons in normal mice included bursting activity, DD mice recordings showed only a single-spike pattern of activity with no bursts. Bursting was restored in DD mice after systemic administration of the dopamine precursor, L-3,4-dihydroxyphenylalanine (L-dopa). Whole-cell recordings in vitro demonstrated that the basic electrophysiology and pharmacology of dopamine neurons were identical between DD and control mice, except that amphetamine did not elicit a hyperpolarizing current in slices from DD mice. These data suggest that endogenously released dopamine plays a critical role in the afferent control of dopamine neuron bursting activity and that this control is exerted via a network feedback mechanism.