ß-asarone prolongs sleep via regulating the level of glutamate in the PVN.

People of all ages could suffer from sleep disorders, which are increasingly recognized as common manifestations of neurologic disease. Acorus tatarinowii is a herb that has been used in traditional medicine to promote sleep. ß-asarone, as the main component of volatile oil obtained from Acorus tatarinowii, may be the main contributor to the sleeping-promoting efficacy of Acorus tatarinowii. In the study, adult male C57BL/6 mice were administered ß-asarone at 12.5 mg/kg, 25 mg/kg, and 50 mg/kg. Behavioral experiments showed that ß-asarone at 25 mg/kg could significantly improve sleep duration. It was also observed that the proportion of NREM (Non-Rapid Eye Movement) sleep increased considerably after administration of ß-asarone. In the PVN (paraventricular nucleus of hypothalamus) region of the hypothalamus, it was observed that the glutamate content decreased after ß-asarone treatment. At the same time, the expression of VGLUT2 (vesicular glutamate transporters 2) decreased while the expression of GAD65 (glutamic acid decarboxylase 65) and GABARAP (GABA Type A Receptor-Associated Protein) increased in the hypothalamus, suggesting that ß-asarone may suppress arousal by reducing glutamate and promoting transformation of glutamate to the inhibitory neurotransmitter GABA (γ-aminobutyric acid). This study is the first to focus on the association between ß-asarone and sleep, shedding perspectives for pharmacological applications of ß-asarone and providing a new direction for future research.

Toward a dissection of ß-Amyloid localized effects on glutamatergic hippocampal repertoire: An Editorial Highlight for "Amyloid-beta1-42 induced glutamatergic receptor and transporter expression changes in the mouse hippocampus"on https://doi.org/10.1111/jnc.15099.

Hippocampal excitatory glutamatergic transmission is critically involved in cognitive functions such as learning and memory. A severe impairment of spatial memory is associated with the Alzheimer's disease characteristic augmentation of soluble Amyloid-beta1-42 which in turn leads to glutamatergic neurotransmission dysfunction. As the molecular basis of such correlations has not been completely understood, this Editorial highlights a study in the current issue of the Journal of Neurochemistry in which Yeung and coworkers provide an elegant anatomical study that sheds light into this problematic. Through a rigorous immunohistochemical approach, a sub-regional expression pattern of ionotropic glutamate receptors and vesicular transporters was determined in control and beta amyloid-injected mouse hippocampus. The selected areas participate in information processing and thus, in memory formation. Furthermore, the authors discuss their findings in the context of cognitive deficits present in Alzheimer's disease patients delivering an intuitive analysis of plausible molecular events that disturb proper glutamate signaling. This study takes an important step toward a better understanding of the complexity of Amyloid-beta1-42 and glutamatergic neurotransmission interactions.

Neurokinin-1 Receptor-Immunopositive Neurons in the Medullary Dorsal Horn Provide Collateral Axons to both the Thalamus and Parabrachial Nucleus in Rats.

It has been suggested that the trigemino-thalamic and trigemino-parabrachial projection neurons in the medullary dorsal horn (MDH) are highly implicated in the sensory-discriminative and emotional/affective aspects of orofacial pain, respectively. In previous studies, some neurons were reported to send projections to both the thalamus and parabrachial nucleus by way of collaterals in the MDH. However, little is known about the chemoarchitecture of this group of neurons. Thus, in the present study, we determined whether the neurokinin-1 (NK-1) receptor, which is crucial for primary orofacial pain signaling, was expressed in MDH neurons co-innervating the thalamus and parabrachial nucleus. Vesicular glutamate transporter 2 (VGLUT2) mRNA, a biomarker for the subgroup of glutamatergic neurons closely related to pain sensation, was assessed in trigemino-parabrachial projection neurons in the MDH. After stereotactic injection of fluorogold (FG) and cholera toxin subunit B (CTB) into the ventral posteromedial thalamic nucleus (VPM) and parabrachial nucleus (PBN), respectively, triple labeling with fluorescence dyes for FG, CTB and NK-1 receptor (NK-1R) revealed that approximately 76 % of the total FG/CTB dually labeled neurons were detected as NK-1R-immunopositive, and more than 94 % of the triple-labeled neurons were distributed in lamina I. In addition, by FG retrograde tract-tracing combined with fluorescence in situ hybridization (FISH) for VGLUT2 mRNA, 54, 48 and 70 % of FG-labeled neurons in laminae I, II and III, respectively, of the MDH co-expressed FG and VGLUT2 mRNA. Thus, most of the MDH neurons co-innervating the thalamus and PBN were glutamatergic. Most MDH neurons providing the collateral axons to both the thalamus and parabrachial nucleus in rats were NK-1R-immunopositive and expressed VGLUT2 mRNA. NK-1R and VGLUT2 in MDH neurons may be involved in both sensory-discriminative and emotional/affective aspects of orofacial pain processing.

Alzheimer's Disease-associated Region-specific Decrease of Vesicular Glutamate Transporter Immunoreactivity inthe Medial Temporal Lobe and Superior Temporal Gyrus.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder for which there are very limited treatment options. Dysfunction of the excitatory neurotransmitter system is thought to play a major role in the pathogenesis of this condition. Vesicular glutamate transporters (VGLUTs) are key to controlling the quantal release of glutamate. Thus, expressional changes in disease can have implications for aberrant neuronal activity, raising the possibility of a therapeutic target. There is no information regarding the expression of VGLUTs in the human medial temporal lobe in AD, one of the earliest and most severely affected brain regions. This study aimed to quantify and compare the layer-specific expression of VGLUT1 and VGLUT2 between control and AD cases in the hippocampus, subiculum, entorhinal cortex, and superior temporal gyrus. Free-floating fluorescent immunohistochemistry was used to label VGLUT1 and VGLUT2 in the hippocampus, subiculum, entorhinal cortex, and superior temporal gyrus. Sections were imaged using laser-scanning confocal microscopy and transporter densitometric analysis was performed. VGLUT1 density was not significantly different in AD tissue, except lower staining density observed in the dentate gyrus stratum moleculare (p = 0.0051). VGLUT2 expression was not altered in the hippocampus and entorhinal cortex of AD cases but was significantly lower in the subiculum (p = 0.015) and superior temporal gyrus (p = 0.0023). This study indicates a regionally specific vulnerability of VGLUT1 and VGLUT2 expression in the medial temporal lobe and superior temporal gyrus in AD. However, the causes and functional consequences of these disturbances need to be further explored to assess VGLUT1 and VGLUT2 as viable therapeutic targets.

Inhibition of Vesicular Glutamate Transporters (VGLUTs) with Chicago Sky Blue 6B Before Focal Cerebral Ischemia Offers Neuroprotection.

Brain ischemia is one of the leading causes of death and long-term disability in the world. Interruption of the blood supply to the brain is a direct stimulus for many pathological events. The massive vesicular release of glutamate (Glu) after ischemia onset induces excitotoxicity, which is a potent stress on neurons. Loading of presynaptic vesicles with Glu is the first step of glutamatergic neurotransmission. Vesicular glutamate transporters 1, 2, and 3 (VGLUT1, 2, and 3) are the main players involved in filling presynaptic vesicles with Glu. VGLUT1 and VGLUT2 are expressed mainly in glutamatergic neurons. Therefore, the possibility of pharmacological modulation to prevent ischemia-related brain damage is attractive. In this study, we aimed to determine the effect of focal cerebral ischemia on the spatiotemporal expression of VGLUT1 and VGLUT2 in rats. Next, we investigated the influence of VGLUT inhibition with Chicago Sky Blue 6B (CSB6B) on Glu release and stroke outcome. The effect of CSB6B pretreatment on infarct volume and neurological deficit was compared with a reference model of ischemic preconditioning. The results of this study indicate that ischemia upregulated the expression of VGLUT1 in the cerebral cortex and in the dorsal striatum 3 days after ischemia onset. The expression of VGLUT2 was elevated in the dorsal striatum and in the cerebral cortex 24 h and 3 days after ischemia, respectively. Microdialysis revealed that pretreatment with CSB6B significantly reduced the extracellular Glu concentration. Altogether, this study shows that inhibition of VGLUTs might be a promising therapeutic strategy for the future.

A mini-review of the role of vesicular glutamate transporters in Parkinson's disease.

Parkinson's disease (PD) is a common neurodegenerative disease implicated in multiple interacting neurotransmitter pathways. Glutamate is the central excitatory neurotransmitter in the brain and plays critical influence in the control of neuronal activity. Impaired Glutamate homeostasis has been shown to be closely associated with PD. Glutamate is synthesized in the cytoplasm and stored in synaptic vesicles by vesicular glutamate transporters (VGLUTs). Following its exocytotic release, Glutamate activates Glutamate receptors (GluRs) and mediates excitatory neurotransmission. While Glutamate is quickly removed by excitatory amino acid transporters (EAATs) to maintain its relatively low extracellular concentration and prevent excitotoxicity. The involvement of GluRs and EAATs in the pathophysiology of PD has been widely studied, but little is known about the role of VGLUTs in the PD. In this review, we highlight the role of VGLUTs in neurotransmitter and synaptic communication, as well as the massive alterations in Glutamate transmission and VGLUTs levels in PD. Among them, adaptive changes in the expression level and function of VGLUTs may exert a crucial role in excitatory damage in PD, and VGLUTs are considered as novel potential therapeutic targets for PD.

Corrigendum: Cerebellar and basal ganglia inputs define three main nuclei in the mouse ventral motor thalamus.

[This corrects the article DOI: 10.3389/fnana.2023.1242839.].

Cerebellar and basal ganglia inputs define three main nuclei in the mouse ventral motor thalamus.

The thalamus is a central link between cortical and subcortical brain motor systems. Axons from the deep nuclei of the cerebellum (DCN), or the output nuclei of the basal ganglia system (substantia nigra reticulata, SNr; and internal pallidum GPi/ENT) monosynaptically innervate the thalamus, prominently some nuclei of the ventral nuclear group. In turn, axons from these ventral nuclei innervate the motor and premotor areas of the cortex, where their input is critical for planning, execution and learning of rapid and precise movements. Mice have in recent years become a widely used model in motor system research. However, information on the distribution of cerebellar and basal ganglia inputs in the rodent thalamus remains poorly defined. Here, we mapped the distribution of inputs from DCN, SNr, and GPi/ENT to the ventral nuclei of the mouse thalamus. Immunolabeling for glutamatergic and GABAergic neurotransmission markers delineated two distinct main territories, characterized each by the presence of large vesicular glutamate transporter type 2 (vGLUT2) puncta or vesicular GABA transporter (vGAT) puncta. Anterograde labeling of axons from DCN revealed that they reach virtually all parts of the ventral nuclei, albeit its axonal varicosities (putative boutons) in the vGAT-rich sector are consistently smaller than those in the vGLUT2-rich sector. In contrast, the SNr axons innervate the whole vGAT-rich sector, but not the vGLUT2-rich sector. The GPi/ENT axons were found to innervate only a small zone of the vGAT-rich sector which is also targeted by the other two input systems. Because inputs fundamentally define thalamic cell functioning, we propose a new delineation of the mouse ventral motor nuclei that is consistent with the distribution of DCN, SNr and GPi/ENT inputs and resembles the general layout of the ventral motor nuclei in primates.

Glutamatergic pathways in the brains of turtles: A comparative perspective among reptiles, birds, and mammals.

Glutamate acts as the main excitatory neurotransmitter in the brain and plays a vital role in physiological and pathological neuronal functions. In mammals, glutamate can cause detrimental excitotoxic effects under anoxic conditions. In contrast, Trachemys scripta, a freshwater turtle, is one of the most anoxia-tolerant animals, being able to survive up to months without oxygen. Therefore, turtles have been investigated to assess the molecular mechanisms of neuroprotective strategies used by them in anoxic conditions, such as maintaining low levels of glutamate, increasing adenosine and GABA, upregulating heat shock proteins, and downregulating K ATP channels. These mechanisms of anoxia tolerance of the turtle brain may be applied to finding therapeutics for human glutamatergic neurological disorders such as brain injury or cerebral stroke due to ischemia. Despite the importance of glutamate as a neurotransmitter and of the turtle as an ideal research model, the glutamatergic circuits in the turtle brain remain less described whereas they have been well studied in mammalian and avian brains. In reptiles, particularly in the turtle brain, glutamatergic neurons have been identified by examining the expression of vesicular glutamate transporters (VGLUTs). In certain areas of the brain, some ionotropic glutamate receptors (GluRs) have been immunohistochemically studied, implying that there are glutamatergic target areas. Based on the expression patterns of these glutamate-related molecules and fiber connection data of the turtle brain that is available in the literature, many candidate glutamatergic circuits could be clarified, such as the olfactory circuit, hippocampal-septal pathway, corticostriatal pathway, visual pathway, auditory pathway, and granule cell-Purkinje cell pathway. This review summarizes the probable glutamatergic pathways and the distribution of glutamatergic neurons in the pallium of the turtle brain and compares them with those of avian and mammalian brains. The integrated knowledge of glutamatergic pathways serves as the fundamental basis for further functional studies in the turtle brain, which would provide insights on physiological and pathological mechanisms of glutamate regulation as well as neural circuits in different species.

Insights into the Cellular Interactions and Molecular Mechanisms of Ketogenic Diet for Comprehensive Management of Epilepsy.

A high-fat diet with appropriate protein and low carbohydrate content, widely known as the ketogenic diet (KD), is considered as an effective non-pharmacotherapeutic treatment option for certain types of epilepsies. Several preclinical and clinical studies have been carried out to elucidate its mechanism of antiepileptic action. Ketone bodies produced after KD's breakdown interact with cellular excito-inhibitory processes and inhibit abnormal neuronal firing. The generated ketone bodies decrease glutamate release by inhibiting the vesicular glutamate transporter 1 and alter the transmembrane potential by hyperpolarization. Apart from their effect on the well-known pathogenic mechanisms of epilepsy, some recent studies have shown the interaction of KD metabolites with novel neuronal targets, particularly adenosine receptors, adenosine triphosphate-sensitive potassium channel, mammalian target of rapamycin, histone deacetylase, hydroxycarboxylic acid receptors, and the NLR family pyrin domain containing 3 inflammasomes to suppress seizures. The role of KD in augmenting gut microbiota as a potential mechanism for epileptic seizure suppression has been established. Furthermore, some recent findings also support the beneficial effect of KD against epilepsy- associated comorbidities. Despite several advantages of the KD in epilepsy management, its use is also associated with a wide range of side effects. Hypoglycemia, excessive ketosis, acidosis, renal stones, cardiomyopathies, and other metabolic disturbances are the primary adverse effects observed with the use of KD. However, in some recent studies, modified KD has been tested with lesser side effects and better tolerability. The present review discusses the molecular mechanism of KD and its role in managing epilepsy and its associated comorbidities.

Leveraging VGLUT3 Functions to Untangle Brain Dysfunctions.

Vesicular glutamate transporters (VGLUTs) were long thought to be specific markers of glutamatergic excitatory transmission. The discovery, two decades ago, of the atypical VGLUT3 has thoroughly modified this oversimplified view. VGLUT3 is strategically expressed in discrete populations of glutamatergic, cholinergic, serotonergic, and even GABAergic neurons. Recent reports show the subtle, but critical, implications of VGLUT3-dependent glutamate co-transmission and its roles in the regulation of diverse brain functions and dysfunctions. Progress in the neuropharmacology of VGLUT3 could lead to decisive breakthroughs in the treatment of Parkinson's disease (PD), addiction, eating disorders, anxiety, presbycusis, or pain. This review summarizes recent findings on VGLUT3 and its vesicular underpinnings as well as on possible ways to target this atypical transporter for future therapeutic strategies.

Glutamic Acid Transporters: Targets for Neuroprotective Therapies in Parkinson's Disease.

Parkinson's disease (PD) is a common neurodegenerative disease in middle-aged and elderly individuals. At present, no effective drug has been developed to treat PD. Although a variety of drugs exist for the symptomatic treatment of PD, they all have strong side effects. Most studies on PD mainly focus on dopaminergic neurons. This review highlights the function of glutamic acid transporters (GLTs), including excitatory amino acid transporters (EAATs) and vesicular glutamate transporters (VGLUTs), during the development of PD. In addition, using bioinformatics, we compared the expression of different types of glutamate transporter genes in the cingulate gyrus of PD patients and healthy controls. More importantly, we suggest that the functional roles of glutamate transporters may prove beneficial in the treatment of PD. In summary, VGLUTs and EAATs may be potential targets in the treatment of PD. VGLUTs and EAATs can be used as clinical drug targets to achieve better efficacy. Through this review article, we hope to enable future researchers to improve the condition of PD patients.

Evolution of glutamatergic signaling and synapses.

Glutamate (Glu) is the primary excitatory transmitter in the mammalian brain. But, we know little about the evolutionary history of this adaptation, including the selection of l-glutamate as a signaling molecule in the first place. Here, we used comparative metabolomics and genomic data to reconstruct the genealogy of glutamatergic signaling. The origin of Glu-mediated communications might be traced to primordial nitrogen and carbon metabolic pathways. The versatile chemistry of L-Glu placed this molecule at the crossroad of cellular biochemistry as one of the most abundant metabolites. From there, innovations multiplied. Many stress factors or injuries could increase extracellular glutamate concentration, which led to the development of modular molecular systems for its rapid sensing in bacteria and archaea. More than 20 evolutionarily distinct families of ionotropic glutamate receptors (iGluRs) have been identified in eukaryotes. The domain compositions of iGluRs correlate with the origins of multicellularity in eukaryotes. Although L-Glu was recruited as a neuro-muscular transmitter in the early-branching metazoans, it was predominantly a non-neuronal messenger, with a possibility that glutamatergic synapses evolved more than once. Furthermore, the molecular secretory complexity of glutamatergic synapses in invertebrates (e.g., Aplysia) can exceed their vertebrate counterparts. Comparative genomics also revealed 15+ subfamilies of iGluRs across Metazoa. However, most of this ancestral diversity had been lost in the vertebrate lineage, preserving AMPA, Kainate, Delta, and NMDA receptors. The widespread expansion of glutamate synapses in the cortical areas might be associated with the enhanced metabolic demands of the complex brain and compartmentalization of Glu signaling within modular neuronal ensembles.

LSP5-2157 a new inhibitor of vesicular glutamate transporters.

Vesicular glutamate transporters (VGLUT1-3) mediate the uptake of glutamate into synaptic vesicles. VGLUTs are pivotal actors of excitatory transmission and of almost all brain functions. Their implication in various pathologies has been clearly documented. Despite their functional importance, the pharmacology of VGLUTs is limited to a few dyes such as Trypan Blue, Rose Bengal or Brilliant Yellow type. Here, we report the design and evaluation of new potent analogs based on Trypan Blue scaffold. Our best compound, named LSP5-2157, has an EC50 of 50 nM on glutamate vesicular uptake. Using a 3D homology model of VGLUT1 and docking experiments, we determined its putative binding subdomains within vesicular glutamate transporters and validated the structural requirement for VGLUT inhibition. To better estimate the specificity and potency of LSP5-2157, we also investigated its ability to block glutamatergic transmission in autaptic hippocampal cells. Neither glutamate receptors nor GABAergic transmission or transmission machinery were affected by LSP5-2157. Low doses of compound reversibly reduce glutamatergic neurotransmission in hippocampal autpases. LSP5-2157 had a low and depressing effect on synaptic efficacy in hippocampal slice. Furthermore, LSP5-2157 had no effect on NMDA-R- mediated fEPSP but reduce synaptic plasticity induced by 3 trains of 100 Hz. Finally, LSP5-2157 had the capacity to inhibit VGLUT3-dependent auditory synaptic transmission in the guinea pig cochlea. In this model, it abolished the compound action potential of auditory nerve at high concentration showing the limited permeation of LSP5-2157 in an in-vivo model. In summary, the new ligand LSP5-2157, has a high affinity and specificity for VGLUTs and shows some permeability in isolated neuron, tissue preparations or in vivo in the auditory system. These findings broaden the field of VGLUTs inhibitors and open the way to their use to assess glutamatergic functions in vitro and in vivo.

Molecular, Structural, Functional, and Pharmacological Sites for Vesicular Glutamate Transporter Regulation.

Vesicular glutamate transporters (VGLUTs) control quantal size of glutamatergic transmission and have been the center of numerous studies over the past two decades. VGLUTs contain two independent transport modes that facilitate glutamate packaging into synaptic vesicles and phosphate (Pi) ion transport into the synaptic terminal. While a transmembrane proton electrical gradient established by a vacuolar-type ATPase powers vesicular glutamate transport, recent studies indicate that binding sites and flux properties for chloride, potassium, and protons within VGLUTs themselves regulate VGLUT activity as well. These intrinsic ionic binding and flux properties of VGLUTs can therefore be modulated by neurophysiological conditions to affect levels of glutamate available for release from synapses. Despite their extraordinary importance, specific and high-affinity pharmacological compounds that interact with these sites and regulate VGLUT function, distinguish between the various modes of transport, and the different isoforms themselves, are lacking. In this review, we provide an overview of the physiologic sites for VGLUT regulation that could modulate glutamate release in an over-active synapse or in a disease state.

The Claustrum in the Squirrel Monkey.

The claustrum (CLA) is a subcortical structure that is reciprocally and topographically connected with the cerebral cortex. The complexity of the cerebral cortex varies dramatically across mammals, raising the question of whether there might also be differences in CLA organization, circuitry, and function. Species variations in the shape of the CLA are well documented. Studies in multiple species have identified subsets of neurochemically distinct interneurons; some data suggest species variations in the nature, distribution, and numbers of different neurochemically identified neuronal types. We have studied the CLA in a smooth-brained primate, the squirrel monkey, using Nissl-stained sections and immunohistochemistry. We found that the shape of the CLA is different from that in other primates. We found several different neurochemically defined populations of neurons equally distributed throughout the CLA. Immunoreactivity to GAD65/67 and GABAA receptors suggest that GABAergic interneurons provide widespread inhibitory input to CLA neurons. Immunoreactivity to glutamate transporters suggests widespread and overlapping excitatory input from cortical and possibly subcortical sources. Comparison of CLA organization in different species suggests that there may be major species differences both in the organization and in the functions of the CLA. Anat Rec, 303:1439-1454, 2020. © 2019 American Association for Anatomy.

A Mini-Review of the Role of Glutamate Transporter in Drug Addiction.

Goals: The development of new treatment for drug abuse requires identification of targetable molecular mechanisms. The pathology of glutamate neurotransmission system in the brain reward circuit is related to the relapse of multiple drugs. Glutamate transporter regulates glutamate signaling by removing excess glutamate from the synapse. And the mechanisms between glutamate transporter and drug addiction are still unclear. Methods: A systematic review of the literature searched in Pubmed and reporting drug addiction in relation to glutamate transporter. Studies were screened by title, abstract, and full text. Results: This review is to highlight the effects of drug addiction on glutamate transporter and glutamate uptake, and targeting glutamate transporter as an addictive drug addiction treatment. We focus on the roles of glutamate transporter in different brain regions in drug addiction. More importantly, we suggest the functional roles of glutamate transporter may prove beneficial in the treatment of drug addiction. Conclusion: Overall, understanding how glutamate transporter impacts central nervous system may provide a new insight for treatment of drug addiction.

VGLUT1 or VGLUT2 mRNA-positive neurons in spinal trigeminal nucleus provide collateral projections to both the thalamus and the parabrachial nucleus in rats.

The trigemino-thalamic (T-T) and trigemino-parabrachial (T-P) pathways are strongly implicated in the sensory-discriminative and affective/emotional aspects of orofacial pain, respectively. These T-T and T-P projection fibers originate from the spinal trigeminal nucleus (Vsp). We previously determined that many vesicular glutamate transporter (VGLUT1 and/or VGLUT2) mRNA-positive neurons were distributed in the Vsp of the adult rat, and most of these neurons sent their axons to the thalamus or cerebellum. However, whether VGLUT1 or VGLUT2 mRNA-positive projection neurons exist that send their axons to both the thalamus and the parabrachial nucleus (PBN) has not been reported. Thus, in the present study, dual retrograde tract tracing was used in combination with fluorescence in situ hybridization (FISH) for VGLUT1 or VGLUT2 mRNA to identify the existence of VGLUT1 or VGLUT2 mRNA neurons that send collateral projections to both the thalamus and the PBN. Neurons in the Vsp that send collateral projections to both the thalamus and the PBN were mainly VGLUT2 mRNA-positive, with a proportion of 90.3%, 93.0% and 85.4% in the oral (Vo), interpolar (Vi) and caudal (Vc) subnucleus of the Vsp, respectively. Moreover, approximately 34.0% of the collateral projection neurons in the Vc showed Fos immunopositivity after injection of formalin into the lip, and parts of calcitonin gene-related peptide (CGRP)-immunopositive axonal varicosities were in direct contact with the Vc collateral projection neurons. These results indicate that most collateral projection neurons in the Vsp, particularly in the Vc, which express mainly VGLUT2, may relay orofacial nociceptive information directly to the thalamus and PBN via axon collaterals.

Neurochemical fingerprinting of amygdalostriatal and intra-amygdaloid projections: a tracing-immunofluorescence study in the rat.

Amygdalostriatal and intra-amygdaloid fiber connectivity was studied in rats via injections of one of the tracers Phaseolus vulgaris leucoagglutinin (PHA-L) or biotinylated dextran amine (BDA) into various amygdaloid nuclei. To determine the neurotransmitter identity of labeled fibers we combined tracer detection with immunofluorescence staining, using antibodies against vesicular transporters (VTs) associated with glutamatergic (VGluT1, VGluT2) or GABAergic (VGAT) neurotransmission. High-magnification confocal laser scanning images were screened for overlap: occurrence inside tracer labeled fibers or axon terminals of immunofluorescence signal associated with one of the VTs. Labeled amygdalostriatal fibers were seen when tracer had been injected into the magnocellular and parvicellular portions of the basal amygdaloid nucleus and the lateral amygdaloid nucleus (nuclei belonging to 'cortical type' amygdaloid nuclei). Intra-amygdaloidal projection fibers were mostly found after tracer injections in the central and medial amygdaloid nuclei ('striatal type' amygdaloid nuclei). Terminals of tracer-labeled amygdalostriatal fibers contained immunofluorescence signal associated mostly with VGluT1 and to a lesser degree with VGluT2 or VGAT. Intra-amygdaloid labeled fibers showed colocalization mostly of VGluT1, followed by VGAT. VGluT2 co-occurred in a minority of intra-amygdaloid tracer-containing fiber terminals. We conclude from our observations that both amygdalostriatal and intra-amygdaloid projections, arising from, respectively, 'cortical type' and 'striatal type' amygdaloid nuclei contain strong glutamatergic and modest GABAergic components. The glutamatergic fibers express either VGluT1 or VGluT2. The absence in large numbers of tracer labeled fibers of expression of one of the selected VTs leads us to suspect that amygdalostriatal projection fibers may contain hitherto neglected neurotransmitters in these connections, e.g., aspartate.