Single rodent mesohabenular axons release glutamate and GABA.
ABSTRACT: The lateral habenula (LHb) is involved in reward, aversion, addiction and depression through descending interactions with several brain structures, including the ventral tegmental area (VTA). The VTA provides reciprocal inputs to LHb, but their actions are unclear. Here we show that the majority of rat and mouse VTA neurons innervating LHb coexpress markers for both glutamate signaling (vesicular glutamate transporter 2; VGluT2) and GABA signaling (glutamic acid decarboxylase; GAD, and vesicular GABA transporter; VGaT). A single axon from these mesohabenular neurons coexpresses VGluT2 protein and VGaT protein and, surprisingly, establishes symmetric and asymmetric synapses on LHb neurons. In LHb slices, light activation of mesohabenular fibers expressing channelrhodopsin2 driven by VGluT2 (Slc17a6) or VGaT (Slc32a1) promoters elicits release of both glutamate and GABA onto single LHb neurons. In vivo light activation of mesohabenular terminals inhibits or excites LHb neurons. Our findings reveal an unanticipated type of VTA neuron that cotransmits glutamate and GABA and provides the majority of mesohabenular inputs.
Project description:For decades, it has been thought that glutamate and GABA are released by distinct neurons. However, some mouse neurons innervating the lateral habenula (LHb) co-release glutamate and GABA. Here, we mapped the distribution of neurons throughout the rat brain that co-express vesicular transporters for the accumulation of glutamate (VGluT2) or GABA (VGaT) and for GABA synthesis (GAD). We found concentrated groups of neurons that co-express VGluT2, VGaT, and GAD mRNAs within subdivisions of the ventral tegmental area (VTA), entopeduncular (EPN), and supramammillary (SUM) nuclei. Single axon terminals established by VTA, EPN, or SUM neurons form a common synaptic architecture involving asymmetric (putative excitatory) and symmetric (putative inhibitory) synapses. Within the LHb, which receives co-transmitted glutamate and GABA from VTA and EPN, VGluT2 and VGaT are distributed on separate synaptic vesicles. We conclude that single axon terminals from VGluT2 and VGaT co-expressing neurons co-transmit glutamate and GABA from distinct synaptic vesicles at independent synapses.
Project description:The lateral habenula (LHb) sends complex projections to several areas of the mesopontine tegmentum, the raphe, and the hypothalamus. However, few markers have been available to distinguish subsets of LHb neurons that may serve these pathways. In order to address this complexity, we examined the mouse and rat LHb for neurons that express the GABA biosynthesis enzymes glutamate decarboxylase 1 (GAD1) and GAD2, and the vesicular GABA transporter (VGAT). The mouse LHb contains a population of neurons that express GAD2, while the rat LHb contains discrete populations of neurons that express GAD1 and VGAT. However, we could not detect single neurons in either species that co-express a GABA synthetic enzyme and VGAT, suggesting that these LHb neurons do not use GABA for conventional synaptic transmission. Instead, all of the neuronal types expressing a GABAergic marker in both species showed co-expression of the glutamate transporter VGluT2. Anterograde tract-tracing of the projections of GAD2-expressing LHb neurons in Gad2Cre mice, combined with retrograde tracing from selected downstream nuclei, show that LHb-GAD2 neurons project selectively to the midline structures in the mesopontine tegmentum, including the median raphe (MnR) and nucleus incertus (NI), and only sparsely innervate the hypothalamus, rostromedial tegmental nucleus (RMTg), and ventral tegmental area (VTA). Postsynaptic recording of LHb-GAD2 neuronal input to tegmental neurons confirms that glutamate, not GABA, is the fast neurotransmitter in this circuit. Thus, GAD2 expression can serve as a marker for functional studies of excitatory neurons serving specific LHb output pathways in mice.
Project description:Encoding and predicting aversive events are critical functions of circuits that support survival and emotional well-being. Maladaptive circuit changes in emotional valence processing can underlie the pathophysiology of affective disorders. The lateral habenula (LHb) has been linked to aversion and mood regulation through modulation of the dopamine and serotonin systems. We have defined the identity and function of glutamatergic (Vglut2) control of the LHb, comparing the role of inputs originating in the globus pallidus internal segment (GPi), and lateral hypothalamic area (LHA), respectively. We found that LHb-projecting LHA neurons, and not the proposed GABA/glutamate co-releasing GPi neurons, are responsible for encoding negative value. Monosynaptic rabies tracing of the presynaptic organization revealed a predominantly limbic input onto LHA Vglut2 neurons, while sensorimotor inputs were more prominent onto GABA/glutamate co-releasing GPi neurons. We further recorded the activity of LHA Vglut2 neurons, by imaging calcium dynamics in response to appetitive versus aversive events in conditioning paradigms. LHA Vglut2 neurons formed activity clusters representing distinct reward or aversion signals, including a population that responded to mild foot shocks and predicted aversive events. We found that the LHb-projecting LHA Vglut2 neurons encode negative valence and rapidly develop a prediction signal for negative events. These findings establish the glutamatergic LHA-LHb circuit as a critical node in value processing.
Project description:The lateral habenula (LHb) has a key role in integrating a variety of neural circuits associated with reward and aversive behaviors. There is limited information about how the different cell types and neuronal circuits within the LHb coordinate physiological and motivational states. Here, we report a cell type in the medial division of the LHb (LHbM) in male rats that is distinguished by: (1) a molecular signature for GABAergic neurotransmission (Slc32a1/VGAT) and estrogen receptor (Esr1/ER?) expression, at both mRNA and protein levels, as well as the mRNA for vesicular glutamate transporter Slc17a6/VGLUT2, which we term the GABAergic estrogen-receptive neuron (GERN); (2) its axonal projection patterns, identified by in vivo juxtacellular labeling, to both local LHb and to midbrain modulatory systems; and (3) its somatic expression of receptors for vasopressin, serotonin and dopamine, and mRNA for orexin receptor 2. This cell type is anatomically located to receive afferents from midbrain reward (dopamine and serotonin) and hypothalamic water and energy homeostasis (vasopressin and orexin) circuits. These afferents shared the expression of estrogen synthase (aromatase) and VGLUT2, both in their somata and axon terminals. We demonstrate dynamic changes in LHbM VGAT+ cell density, dependent upon gonadal functional status, that closely correlate with motivational behavior in response to predator and forced swim stressors. The findings suggest that the homeostasis and reward-related glutamatergic convergent projecting pathways to LHbMC employ a localized neurosteroid signaling mechanism via axonal expression of aromatase, to act as a switch for GERN excitation/inhibition output prevalence, influencing depressive or motivated behavior.
Project description:Hypothalamic proopiomelanocortin (POMC) neurons have traditionally been defined by their peptide transmitters, which are important regulators of energy balance and reward. Recent work shows that POMC neurons can also release the amino acid transmitters ?-aminobutyric acid (GABA) and glutamate, although studying GABAergic and glutamatergic populations of POMC neurons has been hindered by the difficulty in reliably identifying amino acid (AA) transmitter phenotypes. In the present study, fluorescent in situ hybridization and immunohistochemistry were used to identify POMC neurons and to detect the presence of mRNA for the transporters responsible for packaging either GABA (vesicular GABA transporter [vGAT]) or glutamate (vesicular glutamate transporter [vGLUT]) into vesicles, as well as the enzymes responsible for GABA synthesis, glutamic acid decarboxylase (GAD)65 and GAD67. Approximately 7% of POMC neurons expressed vGlut2 and the highest percentage of vGlut2-positive POMC cells were located in the rostral arcuate nucleus. Despite the reports of GABA release from POMC neurons, vGat was not detected in POMC neurons, although Gad65 and Gad67 were present in ~40% of POMC neurons. Approximately half of the vGlut2-expressing POMC cells also expressed Gad65. Markers of neurotransmitter phenotype were better detected by using in situ hybridization techniques rather than transgenic expression of fluorophores under the control of the vGat or Gad67 promoters. It is now clear that the expression of markers of AA phenotype provides a useful means to identify distinct subpopulations of POMC neurons. Additionally, the method described will be useful to explore the possibility that plasticity of AA phenotype is an important aspect of POMC neuron function.
Project description:Dopaminergic (DAergic) neurons in the ventral tegmental area (VTA) play crucial roles in motivational control of behaviors, and their activity is regulated directly or indirectly via GABAergic neurons by extrinsic afferents from various sources, including the bed nucleus of the stria terminalis (BST). Here, the neurochemical composition of VTA-projecting BST neurons and their outputs to the VTA were studied in adult mouse brains. By combining retrograde tracing with fluorescence in situ hybridization for 67 kDa glutamate decarboxylase (GAD67) and vesicular glutamate transporters (VGluTs), VTA-targeting BST neurons were classified into GAD67-positive (GAD67(+))/VGluT3-negative (VGluT3(-)), GAD67(+)/VGluT3(+), and VGluT2(+) neurons, of which GAD67(+)/VGluT3(-) neurons constituted the majority (?90%) of VTA-projecting BST neurons. GABAergic efferents from the BST formed symmetrical synapses on VTA neurons, which were mostly GABAergic neurons, and expressed GABA(A) receptor ?1 subunit on their synaptic and extrasynaptic membranes. In the VTA, VGluT3 was detected in terminals expressing vesicular inhibitory amino acid transporter (VIAAT), plasmalemmal serotonin transporter, or neither. Of these, VIAAT(+)/VGluT3(+) terminals, which should include those from GAD67(+)/VGluT3(+) BST neurons, formed symmetrical synapses. When single axons from VGluT3(+) BST neurons were examined, almost all terminals were labeled for VIAAT, whereas VGluT3 was often absent from terminals with high VIAAT loads. VGluT2(+) terminals in the VTA exclusively formed asymmetrical synapses, which expressed AMPA receptors on postsynaptic membrane. Therefore, the major mode of the BST-VTA projection is GABAergic, and its activation is predicted to disinhibit VTA DAergic neurons. VGluT2(+) and VGluT3(+) BST neurons further supply additional projections, which may principally convey excitatory or inhibitory inputs, respectively, to the VTA.
Project description:It is generally assumed that the inhibitory neurotransmitter GABA and the stimulatory neurotransmitter glutamate are released from different neurons in adults. However, this tenet has made it difficult to explain how the same afferent signals can cause opposite changes in GABA and glutamate release. Such reciprocal release is a central mechanism in the neural control of many physiological processes including activation of gonadotropin-releasing hormone (GnRH) neurons, the neural signal for ovulation. Activation of GnRH neurons requires simultaneous suppression of GABA and stimulation of glutamate release, each of which occurs in response to a daily photoperiodic signal, but only in the presence of estradiol (E2). In rodents, E2 and photoperiodic signals converge in the anteroventral periventricular nucleus (AVPV), but it is unclear how these signals differentially regulate GABA and glutamate secretion. We now report that nearly all neurons in the AVPV of female rats express both vesicular glutamate transporter 2 (VGLUT2), a marker of hypothalamic glutamatergic neurons, as well as glutamic acid decarboxylase and vesicular GABA transporter (VGAT), markers of GABAergic neurons. These dual-phenotype neurons are the main targets of E2 in the region and are more than twice as numerous in females as in males. Moreover, dual-phenotype synaptic terminals contact GnRH neurons, and at the time of the surge, VGAT-containing vesicles decrease and VGLUT2-containing vesicles increase in these terminals. Thus, we propose a new model for ovulation that includes dual-phenotype GABA/glutamate neurons as central transducers of hormonal and neural signals to GnRH neurons.
Project description:The importance of neuropeptides in the hypothalamus has been experimentally established. Due to difficulties in assessing function in vivo, the roles of the fast-acting neurotransmitters glutamate and GABA are largely unknown. Synaptic vesicular transporters (VGLUTs for glutamate and VGAT for GABA) are required for vesicular uptake and, consequently, synaptic release of neurotransmitters. Ventromedial hypothalamic (VMH) neurons are predominantly glutamatergic and express VGLUT2. To evaluate the role of glutamate release from VMH neurons, we generated mice lacking VGLUT2 selectively in SF1 neurons (a major subset of VMH neurons). These mice have hypoglycemia during fasting secondary to impaired fasting-induced increases in the glucose-raising pancreatic hormone glucagon and impaired induction in liver of mRNAs encoding PGC-1alpha and the gluconeogenic enzymes PEPCK and G6Pase. Similarly, these mice have defective counterregulatory responses to insulin-induced hypoglycemia and 2-deoxyglucose (an antimetabolite). Thus, glutamate release from VMH neurons is an important component of the neurocircuitry that functions to prevent hypoglycemia.
Project description:The ventral tegmental area (VTA) comprises dopamine (DA), ?-aminobutyric acid (GABA) and glutamate (Glu) neurons. Some rat VTA Glu neurons, expressing vesicular glutamate transporter 2 (VGluT2), co-express tyrosine hydroxylase (TH). While transgenic mice are now being used in attempts to determine the role of VGluT2/TH neurons in reward and neuronal signaling, such neurons have not been characterized in mouse tissue. By cellular detection of VGluT2 mRNA and TH immunoreactivity (TH-IR), we determined the cellular expression of VGluT2 mRNA within VTA TH-IR neurons in the mouse. We found that some mouse VGluT2 neurons coexpressed TH-IR, but their frequency was lower than in the rat. To determine whether low expression of TH mRNA or TH-IR accounts for this low frequency, we evaluated VTA cellular coexpression of TH transcripts and TH protein. Within the medial aspects of the VTA, some neurons expressed TH mRNA but lacked TH-IR; among them a subset coexpressed VGluT2 mRNA. To determine if lack of VTA TH-IR was due to TH trafficking, we tagged VTA TH neurons by Cre-inducible expression of mCherry in TH::Cre mice. By dual immunofluorescence, we detected axons containing mCherry, but lacking TH-IR, in the lateral habenula, indicating that low frequency of VGluT2 mRNA (+)/TH-IR (+) neurons in the mouse is due to lack of synthesis of TH protein, rather than TH protein trafficking. In conclusion, VGluT2 neurons are present in the rat and mouse VTA, but they differ in the populations of VGluT2/TH and TH neurons. Under normal conditions, the translation of TH protein is suppressed in the mouse mesohabenular TH neurons.
Project description:The ventral tegmental area (VTA) contributes to reward and motivation signaling. In addition to the well established populations of dopamine (DA) or GABA VTA neurons, glutamatergic neurons were recently discovered in the VTA. These glutamatergic neurons express the vesicular glutamate transporter 2, VGluT2. To investigate whether VTA glutamatergic neurons establish local synapses, we tagged axon terminals from resident VTA neurons by intra-VTA injection of Phaseolus vulgaris leucoagglutinin (PHA-L) or an adeno-associated virus encoding wheat germ agglutinin (WGA) and by immunoelectron microscopy determined the presence of VGluT2 in PHA-L- or WGA-positive terminals. We found that PHA-L- or WGA-positive terminals from tagged VTA cells made asymmetric or symmetric synapses within the VTA. VGluT2 immunoreactivity was detected in the vast majority of PHA-L- or WGA-positive terminals forming asymmetric synapses. These results indicate that both VTA glutamatergic and nonglutamatergic (likely GABAergic) neurons establish local synapses. To examine the possible DAergic nature of postsynaptic targets of VTA glutamatergic neurons, we did triple immunolabeling with antibodies against VGluT2, tyrosine hydroxylase (TH), and PHA-L. From triple-labeled tissue, we found that double-labeled PHA-L (+)/VGluT2 (+) axon terminals formed synaptic contacts on dendrites of both TH-positive and TH-negative cells. Consistent with these anatomical observations, in whole-cell slice recordings of VTA neurons we observed that blocking action potential activity significantly decreased the frequency of synaptic glutamatergic events in DAergic and non-DAergic neurons. These observations indicate that resident VTA glutamatergic neurons are likely to affect both DAergic and non-DAergic neurotransmission arising from the VTA.