Excitatory Glutamatergic Neurons Research Articles

Production and organization of neocortical interneurons

Inhibitory GABA (γ-aminobutyric acid)-ergic interneurons are a vital component of the neocortex responsible for shaping its output through a variety of inhibitions. Consisting of many flavors, interneuron subtypes are predominantly defined by their morphological, physiological, and neurochemical properties that help to determine their functional role within the neocortex. During development, these cells are born in the subpallium where they then tangentially migrate over long distances before being radially positioned to their final location in the cortical laminae. As development progresses into adolescence, these cells mature and form chemical and electrical connections with both glutamatergic excitatory neurons and other interneurons ultimately establishing the cortical network. The production, migration, and organization of these cells are determined by vast array of extrinsic and intrinsic factors that work in concert in order to assemble a proper functioning cortical inhibitory network. Failure of these cells to undergo these processes results in abnormal positioning and cortical function. In humans, this can bring about several neurological disorders including schizophrenia, epilepsy, and autism spectrum disorders. In this article, we will review previous literature that has revealed the framework for interneuron neurogenesis and migratory behavior as well as discuss recent findings that aim to elucidate the spatial and functional organization of interneurons within the neocortex.

The role of cannabinoidergic system in prenatal neurodevelopment.

The role of cannabinoidergic system in prenatal neurodevelopment.

Glutamatergic neuronal populations in the brainstem of the sea lamprey, Petromyzon marinus: An in situ hybridization and immunocytochemical study

Glutamate is the major excitatory neurotransmitter in vertebrates, and glutamatergic cells probably represent a majority of neurons in the brain. Physiological studies have demonstrated a wide presence of excitatory (glutamatergic) neurons in lampreys. The present in situ hybridization study with probes for the lamprey vesicular glutamate transporter (VGLUT) provides an anatomical basis for the general distribution and precise localization of glutamatergic neurons in the sea lamprey brainstem. Most glutamatergic neurons were found within the periventricular gray layer throughout the brainstem, with the following regions being of particular interest: the optic tectum, torus semicircularis, isthmus, dorsal and medial nuclei of the octavolateral area, dorsal column nucleus, solitary tract nucleus, motoneurons, and reticular formation. The reticular population revealed a high degree of cellular heterogeneity including small, medium-sized, large, and giant glutamatergic neurons. We also combined glutamate immunohistochemistry with neuronal tract-tracing methods or γ-aminobutyric acid (GABA) immunohistochemistry to better characterize the glutamatergic populations. Injection of Neurobiotin into the spinal cord revealed that retrogradely labeled small and medium-sized cells of some reticulospinal-projecting groups were often glutamate-immunoreactive, mostly in the hindbrain. In contrast, the large and giant glutamatergic reticulospinal perikarya mostly lacked glutamate immunoreactivity. These results indicate that glutamate immunoreactivity did not reveal the entire set of glutamatergic populations. Some spinal-projecting octaval populations lacked both VGLUT and glutamate. As regards GABA and glutamate, their distribution was largely complementary, but colocalization of glutamate and GABA was observed in some small neurons, suggesting that glutamate immunohistochemistry might also detect non-glutamatergic cells or neurons that co-release both GABA and glutamate.

Posteromedial cortex glutamate and GABA predict intrinsic functional connectivity of the default mode network

The balance between excitatory glutamatergic projection neurons and inhibitory GABAergic interneurons determines the function of cortical microcircuits. How these neurotransmitters relate to the functional status of an entire macro-scale network remains unknown. The posteromedial cortex (PMC) is the default mode network (DMN) node with the greatest functional connectivity; therefore, we hypothesized that PMC glutamate and GABA predict intrinsic functional connectivity (iFC) across the entire DMN. In 20 healthy men, we combined J-resolved magnetic resonance spectroscopy to measure glutamate and GABA in the PMC and resting fMRI followed by group Independent Components Analysis to extract the entire DMN. We showed that, controlling for age and partial GM volume in the MRS voxel, PMC glutamate and GABA explained about half of the variance of DMN iFC (represented by the network's beta coefficient for rest). Glutamate correlated positively and GABA correlated negatively with DMN iFC; in an alternative statistical model which included the glutamate/GABA ratio, the ratio correlated positively with DMN iFC. Age had no independent association with DMN iFC. No other network was associated with PMC glutamate or GABA. We conclude that regional neurotransmitter concentrations in a network node strongly predict network but not global brain iFC.

Guidepost neurons for the lateral olfactory tract: Expression of metabotropic glutamate receptor 1 and innervation by glutamatergic olfactory bulb axons

The guidepost neurons for the lateral olfactory tract, which are called lot cells, are the earliest-generated neurons in the neocortex. They migrate tangentially and ventrally further down this tract, and provide scaffolding for the olfactory bulb axons projecting into this pathway. The molecular profiles of the lot cells are largely uncharacterized. We found that lot cells specifically express metabotropic glutamate receptor subtype-1 at a very early stage of development. This receptor is functionally competent and responds to a metabotropic glutamate receptor agonist with a transient increase in the intracellular calcium ion concentration. When the glutamatergic olfactory bulb axons were electrically stimulated, lot cells responded to the stimulation with a calcium increase mainly via ionotropic glutamate receptors, suggesting potential neurotransmission between the axons and lot cells during early development. Together with the finding that lot cells themselves are glutamatergic excitatory neurons, our results provide another notable example of precocious interactions between the projecting axons and their intermediate targets.

Tbr2 Deficiency in Mitral and Tufted Cells Disrupts Excitatory-Inhibitory Balance of Neural Circuitry in the Mouse Olfactory Bulb

The olfactory bulb (OB) is the first relay station in the brain where odor information from the olfactory epithelium is integrated, processed through its intrinsic neural circuitry, and conveyed to higher olfactory centers. Compared with profound mechanistic insights into olfactory axon wiring from the nose to the OB, little is known about the molecular mechanisms underlying the formation of functional neural circuitry among various types of neurons inside the OB. T-box transcription factor Tbr2 is expressed in various types of glutamatergic excitatory neurons in the brain including the OB projection neurons, mitral and tufted cells. Here we generated conditional knockout mice in which the Tbr2 gene is inactivated specifically in mitral and tufted cells from late embryonic stages. Tbr2 deficiency caused cell-autonomous changes in molecular expression including a compensatory increase of another T-box member, Tbr1, and a concomitant shift of vesicular glutamate transporter (VGluT) subtypes from VGluT1 to VGluT2. Tbr2-deficient mitral and tufted cells also exhibited anatomical abnormalities in their dendritic morphology and projection patterns. Additionally, several non-cell-autonomous phenotypes were observed in parvalbumin-, calbindin-, and 5T4-positive GABAergic interneurons. Furthermore, the number of dendrodendritic reciprocal synapses between mitral/tufted cells and GABAergic interneurons was significantly reduced. Upon stimulation with odorants, larger numbers of mitral and tufted cells were activated in Tbr2 conditional knockout mice. These results suggest that Tbr2 is required for not only the proper differentiation of mitral and tufted cells, but also for the establishment of functional neuronal circuitry in the OB and maintenance of excitatory-inhibitory balance crucial for odor information processing.

Pronociceptive effect of 5-HT1A receptor agonist on visceral pain involves spinal N-methyl-d-aspartate (NMDA) receptor

The functional role of serotonergic 5-HT1A receptors in the modulation of visceral pain is controversial. The objective of this study was to systematically examine the mechanism and site of action of a selective 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)-tetralin (DPAT) on visceral pain. In the behavioral model of visceral pain, systemic injection (5–250μg/kg) of DPAT produced a significant increase in the viscero-motor response (VMR) to colorectal distension (CRD) and this effect was blocked by the selective 5-HT1A receptor antagonist WAY-100135 (5mg/kg, s.c.). Similarly, intrathecal (i.t.) injection (5μmol) of DPAT into the lumbo-sacral (L6–S1) spinal cord produced a significant increase in VMR. The administration of N-methyl d-aspartate (NMDA) receptor antagonist AP5 (50μg/kg) prior to DPAT injection completely blocked the pronociceptive effect of DPAT. Similarly, DPAT failed to increase VMR in rats chronically treated with NR1 subunit-targeted antisense oligonucleotide (ON), whereas the drug increased VMR in rats treated with mismatched-ON. Chronic i.t. injection of allylglycine (AG), a γ-amino decarboxylase (GAD) enzyme inhibitor, produced significant increase in VMRs, suggesting that the inhibition of GABA synthesis produces pronociception. In AG-treated rats, i.t. injection of DPAT failed to further increase in VMR, suggesting that the DPAT action is linked to GABA release. Similarly, WAY-100135 failed to attenuate VMR in AG-treated rats, suggesting that unlike DPAT, AG action is not via the activation of 5-HT1A receptors. In electrophysiology experiments, DPAT (50μg/kg) significantly increased the responses of spinal neurons to CRD, but did not influence the mechanotransduction property of CRD-sensitive pelvic nerve afferent fibers. The effect of DPAT on spinal neurons remained unaffected when tested in spinal-transected (C1–C2) rats. These results indicate that the 5-HT1A receptor agonist DPAT produces pronociceptive effects, primarily via the activation of presynaptic 5-HT1A receptors in GABAergic neuron to restrict GABA release and thereby disinhibits the excitatory glutamatergic neurons in the spinal cord.

The Fraction of Cortical GABAergic Neurons Is Constant from Near the Start of Cortical Neurogenesis to Adulthood

Approximately one in five neurons is GABAergic in many neocortical areas and species, forming a critical balance between inhibition and excitation in adult circuits. During development, cortical GABAergic neurons are generated in ventral telencephalon and migrate up to developing cortex where the excitatory glutamatergic neurons are born. We ask here: when during development is the adult GABAergic/glutamatergic neuron ratio first established? To answer this question, we have determined the fraction of all neocortical GABAergic neurons that will become inhibitory (GAD67(+)) in mice from embryonic day 10.5 (E10.5) to postnatal day 28 (P28). We find that this fraction is close to 1/5, the adult value, starting from early in corticogenesis (E14.5, when GAD67(+) neurons are still migrating tangentially to the cortex) and continuing at the same 1/5 value throughout the remainder of brain development. Thus our data indicate the one-in-five fraction of GABAergic neurons is already established during their neuronal migration and well before significant synapse formation.

Circuit Specific Functions of Cannabinoid CB1 Receptor in the Balance of Investigatory Drive and Exploration

Well balanced novelty seeking and exploration are fundamental behaviours for survival and are found to be dysfunctional in several psychiatric disorders. Recent studies suggest that the endocannabinoid (eCB) system is an important control system for investigatory drive. Pharmacological treatment of rodents with cannabinergic drugs results in altered social and object investigation. Interestingly, contradictory results have been obtained, depending on the treatment, drug concentration and experimental conditions. The cannabinoid type 1 (CB1) receptor, a central component of the eCB system, is predominantly found at the synapses of two opposing neuronal populations, i.e. on inhibitory GABAergic and excitatory glutamatergic neurons. In the present study, using different transgenic mouse lines, we aimed at investigating the impact of CB1 receptor inactivation in glutamatergic or GABAergic neurons on investigatory behaviour. We evaluated animate (interaction partner) and inanimate (object) exploratory behaviour in three different paradigms. We show that exploration was increased when CB1 receptor was deleted from cortical and striatal GABAergic neurons. No effect was observed when CB1 receptor was deleted specifically from dopamine receptor D1-expressing striatal GABAergic medium spiny neurons. In contrast, deletion of CB1 receptor from cortical glutamatergic neurons resulted in a decreased exploration. Thus, our results indicate that exploratory behaviour is accurately balanced in both, the social and non-social context, by the eCB system via CB1 receptor activation on cortical glutamatergic and GABAergic neurons. In addition, the results could explain the contradictory findings of previous pharmacological studies and could further suggest a possibility to readjust an imbalance in exploratory behaviour observed in psychiatric disorders.

In Vivo Optogenetic Stimulation of Neocortical Excitatory Neurons Drives Brain-State-Dependent Inhibition

Synaptic interactions between excitatory and inhibitory neocortical neurons are important for mammalian sensory perception. Synaptic transmission between identified neurons within neocortical microcircuits has mainly been studied in brain slice preparations in vitro. Here, we investigate brain-state-dependent neocortical synaptic interactions in vivo by combining the specificity of optogenetic stimulation with the precision of whole-cell recordings from postsynaptic excitatory glutamatergic neurons and GFP-labeled inhibitory GABAergic neurons targeted through two-photon microscopy. Channelrhodopsin-2 (ChR2) stimulation of excitatory layer 2/3 barrel cortex neurons evoked larger and faster depolarizing postsynaptic potentials and more synaptically driven action potentials in fast-spiking (FS) GABAergic neurons compared to both non-fast-spiking (NFS) GABAergic neurons and postsynaptic excitatory pyramidal neurons located within the same neocortical microcircuit. The number of action potentials evoked in ChR2-expressing neurons showed low trial-to-trial variability, but postsynaptic responses varied strongly with near-linear dependence upon spontaneously driven changes in prestimulus membrane potential. Postsynaptic responses in excitatory neurons had reversal potentials, which were hyperpolarized relative to action potential threshold and were therefore inhibitory. Reversal potentials measured in postsynaptic GABAergic neurons were close to action potential threshold. Postsynaptic inhibitory neurons preferentially fired synaptically driven action potentials from spontaneously depolarized network states, with stronger state-dependent modulation in NFS GABAergic neurons compared to FS GABAergic neurons. Inhibitory neurons appear to dominate neocortical microcircuit function, receiving stronger local excitatory synaptic input and firing more action potentials compared to excitatory neurons. In mouse layer 2/3 barrel cortex, we propose that strong state-dependent recruitment of inhibitory neurons drives competition among excitatory neurons enforcing sparse coding.

Identification of Minimal Neuronal Networks Involved in Flexor-Extensor Alternation in the Mammalian Spinal Cord

Neural networks in the spinal cord control two basic features of locomotor movements: rhythm generation and pattern generation. Rhythm generation is generally considered to be dependent on glutamatergic excitatory neurons. Pattern generation involves neural circuits controlling left-right alternation, which has been described in great detail, and flexor-extensor alternation, which remains poorly understood. Here, we use a mouse model in which glutamatergic neurotransmission has been ablated in the locomotor region of the spinal cord. The isolated invitro spinal cord from these mice produces locomotor-like activity-when stimulated with neuroactive substances-with prominent flexor-extensor alternation. Under these conditions, unlike in control mice, networks of inhibitory interneurons generate the rhythmic activity. In the absence of glutamatergic synaptic transmission, the flexor-extensor alternation appears to be generated by Ia inhibitory interneurons, which mediate reciprocal inhibition from muscle proprioceptors to antagonist motor neurons. Our study defines a minimal inhibitory network that is needed to produce flexor-extensor alternation during locomotion.

Sciatic nerve ligation induces up-regulation of functional 5-HT3 receptor on glutamatergic excitatory neurons in the mouse spinal superficial dorsal horn

Sciatic nerve ligation induces up-regulation of functional 5-HT3 receptor on glutamatergic excitatory neurons in the mouse spinal superficial dorsal horn

Selective GABA release as a mechanistic basis of high-frequency stimulation used for the treatment of neuropsychiatric diseases

Electrical high-frequency stimulation (HFS) is applied in many brain areas to treat various clinical syndromes. The nearly identical constellation of stimulation parameters raises the question of a unique mechanism of action of this therapeutic option. The identification of a single HFS mechanism may help to optimize the HFS technology by targeting this single mechanism. Experimentally, only axonal membranes are targets of HFS, but not other membranes of neurons or glial cells. Within all HFS target regions, axons of excitatory glutamatergic and inhibitory GABAergic neurons are present and play roles in all clinical syndromes treated successfully with HFS. Therefore, glutamatergic or GABAergic fibres are likely candidates as mediators of a unique HFS mode of action. The selective involvement of another neuronal fibre type (e.g. monoaminergic, cholinergic, etc.) in the HFS mode of action is highly unlikely since the regional and syndromal dissimilarity of the clinical HFS applications precludes the assumption of such a fibre type as primary HFS site of action. Our recent experimental finding that HFS of human neocortical slices induces the action potential-mediated release of GABA, but not of glutamate, simplifies the possibilities to explain the HFS mode of action, as the explanation now may concentrate on GABAergic axons only. Thus, we are analysing, on the basis of the pathophysiological grounds of the various syndromes treated with deep brain stimulation, whether a selective GABA release is a collective explanation of the mode of action of HFS. We suggest that selective GABA release indeed may needfully and sufficiently explain efficacy and side effects of HFS.

Perspectives on adult neurogenesis

New neurons are generated throughout life. This discovery has challenged firmly held concepts about the structural plasticity and regenerative capacity of the mammalian brain. In this special issue of the EJN, leaders in the field summarize and review recent advances aiming to understand the molecular mechanisms underlying adult neurogenesis and the impact of new neurons on brain function in health and disease. Below we discuss pivotal yet unsolved aspects of adult neurogenesis as well as potential future directions in the field.

Endogenous SNAP-25 Regulates Native Voltage-gated Calcium Channels in Glutamatergic Neurons

In addition to its primary role as a fundamental component of the SNARE complex, SNAP-25 also modulates voltage-gated calcium channels (VGCCs) in various overexpression systems. Although these studies suggest a potential negative regulatory role of SNAP-25 on VGCC activity, the effects of endogenous SNAP-25 on native VGCC function in neurons are unclear. In the present study, we investigated the VGCC properties of cultured glutamatergic and GABAergic rat hippocampal neurons. Glutamatergic currents were dominated by P/Q-type channels, whereas GABAergic cells had a dominant L-type component. Also, glutamatergic VGCC current densities were significantly lower with enhanced inactivation rates and shifts in the voltage dependence of activation and inactivation curves compared with GABAergic cells. Silencing endogenous SNAP-25 in glutamatergic neurons did not alter P/Q-type channel expression or localization but led to increased VGCC current density without changes in the VGCC subtype proportions. Isolation of the P/Q-type component indicated that increased current in the absence of SNAP-25 was correlated with a large depolarizing shift in the voltage dependence of inactivation. Overexpressing SNAP-25 in GABAergic neurons reduced current density without affecting the VGCC subtype proportion. Accordingly, VGCC current densities in glutamatergic neurons from Snap-25(+/-) mice were significantly elevated compared with wild type glutamatergic neurons. Overall, this study demonstrates that endogenous SNAP-25 negatively regulates native VGCCs in glutamatergic neurons which could have important implications for neurological diseases associated with altered SNAP-25 expression.

Baseline GABA concentration and fMRI response

Coordination between glutamatergic excitatory neurons and γ-aminobutyric acid (GABA)-ergic inhibitory interneurons is fundamental to the regulation of neuronal firing rates and is believed to have relevance to functional magnetic resonance imaging (fMRI) contrast. While much is known regarding the molecular behavior of excitatory and inhibitory processes, comparatively less is known regarding the role of such processes in explaining variations in fMRI and related hemodynamic imaging metrics. The relationship between baseline GABA levels, as measured by MR spectroscopy, and hemodynamic contrasts from four sequences in human visual cortex are investigated (n = 12; field strength = 3.0 T): blood oxygenation level-dependent (BOLD), cerebral blood flow (CBF)-weighted arterial spin labelling (ASL), cerebral blood volume (CBV)-weighted vascular-space-occupancy (VASO), and arterial CBV (aCBV)-weighted inflow VASO (iVASO). Results indicate that baseline GABA levels (GABA+ macromolecules normalized to creatine) inversely correlate with BOLD reactivity (R = −0.70; P = 0.01) and magnitude CBV-weighted VASO reactivity (R = −0.71; P = 0.01). A trend for significance was found between baseline aCBV-weighted iVASO (R = −0.50; P = 0.10) and baseline GABA. A positive correlation was found between baseline CBF-weighted ASL signal and GABA (R = 0.65; P = 0.02) and ASL time-to-peak and baseline GABA (R = 0.58; P = 0.05). These findings demonstrate that both the dominant BOLD fMRI contrast, as well as other emerging MR hemodynamic contrasts, have signal variations that are linked to baseline GABA levels.

A transgenic mouse line for molecular genetic analysis of excitatory glutamatergic neurons

Excitatory glutamatergic neurons are part of most of the neuronal circuits in the mammalian nervous system. We have used BAC-technology to generate a BAC-Vglut2::Cre mouse line where Cre expression is driven by the vesicular glutamate transporter 2 (Vglut2) promotor. This BAC-Vglut2::Cre mouse line showed specific expression of Cre in Vglut2 positive cells in the spinal cord with no ectopic expression in GABAergic or glycinergic neurons. This mouse line also showed specific Cre expression in Vglut2 positive structures in the brain such as thalamus, hypothalamus, superior colliculi, inferior colliculi and deep cerebellar nuclei together with nuclei in the midbrain and hindbrain. Cre-mediated recombination was restricted to Cre expressing cells in the spinal cord and brain and occurred as early as E 12.5. Known Vglut2 positive neurons showed normal electrophysiological properties in the BAC-Vglut2::Cre transgenic mice. Altogether, this BAC-Vglut2::Cre mouse line provides a valuable tool for molecular genetic analysis of excitatory neuronal populations throughout the mouse nervous system.

Last But Not Least: Cortical Interneurons from Caudal Ganglionic Eminence

Mammalian cerebral cortex is composed of excitatory glutamatergic principal neurons (∼80% of the total) and inhibitory GABAergic interneurons (∼20%). Although GABAergic interneurons are less abundant, they have a crucial role in regulating cortical excitability and ultimately, in ensuring the

Ptf1a Directly Controls Expression of Immunoglobulin Superfamily Molecules Nephrin and Neph3 in the Developing Central Nervous System

Ptf1a, a basic helix-loop-helix transcription factor, plays an indispensable role for cell fate specification of subsets of neurons in the developing central nervous system. However, downstream molecules induced by Ptf1a during neural development have not been well characterized. In the present study, we identified immunoglobulin superfamily molecules, Nephrin and Neph3, as direct downstream targets of Ptf1a. First, the expression domains of Nephrin and Neph3 closely resembled those of Ptf1a in the developing retina, hypothalamus, cerebellum, hindbrain, and spinal cord. Second, Ptf1a bound directly to a PTF-binding motif in the 5'-flanking region of Nephrin and Neph3 genes. Third, Ptf1a activated transcription driven by the 5'-flanking region of these genes. Finally, the expression of Nephrin and Neph3 was lost in Ptf1a-null mice, whereas ectopic expression of Nephrin and Neph3 was induced by forced expression of Ptf1a. We provided further evidence that Nephrin and Neph3 could interact homophilically and heterophilically, suggesting that Nephrin and Neph3 might regulate certain developmental aspects of Ptf1a-positive neurons as homo- or heterooligomers.

Inhibitory and excitatory subtypes of cochlear nucleus neurons are defined by distinct bHLH transcription factors, Ptf1a and Atoh1

The cochlear nucleus (CN), which consists of dorsal and ventral cochlear nuclei (DCN and VCN), plays pivotal roles in processing and relaying auditory information to the brain. Although it contains various types of neurons, the origins of the distinct subtypes and their developmental molecular machinery are still elusive. Here we reveal that two basic helix-loop-helix transcription factors play crucial roles in specifying neuron subtypes in the CN. Pancreatic transcription factor 1a (Ptf1a) and atonal homolog 1 (Atoh1) were found to be expressed in discrete dorsolateral regions of the embryonic neuroepithelia of the middle hindbrain (rhombomeres 2-5). Genetic lineage tracing using mice that express Cre recombinase from the Ptf1a locus or under the control of the Atoh1 promoter revealed that inhibitory (GABAergic and glycinergic) or excitatory (glutamatergic) neurons of both DCN and VCN are derived from the Ptf1a- and Atoh1-expressing neuroepithelial regions, respectively. In the Ptf1a or Atoh1 null embryos, production of inhibitory or excitatory neurons, respectively, was severely inhibited in the CN. These findings suggest that inhibitory and excitatory subtypes of CN neurons are defined by Ptf1a and Atoh1, respectively and, furthermore, provide important insights into understanding the machinery of neuron subtype specification in the dorsal hindbrain.