The roles of endocannabinoid signaling during central nervous system development are unknown. We report that CB(1) cannabinoid receptors (CB(1)Rs) are enriched in the axonal growth cones of gamma-aminobutyric acid-containing (GABAergic) interneurons in the rodent cortex during late gestation. Endocannabinoids trigger CB(1)R internalization and elimination from filopodia and induce chemorepulsion and collapse of axonal growth cones of these GABAergic interneurons by activating RhoA. Similarly, endocannabinoids diminish the galvanotropism of Xenopus laevis spinal neurons. These findings, together with the impaired target selection of cortical GABAergic interneurons lacking CB(1)Rs, identify endocannabinoids as axon guidance cues and demonstrate that endocannabinoid signaling regulates synaptogenesis and target selection in vivo.

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In utero exposure to ⌬ 9 -tetrahydrocannabinol (⌬ 9 -THC), the active component from marijuana, induces cognitive deficits enduring into adulthood. Although changes in synaptic structure and plasticity may underlie ⌬ 9 -THC-induced cognitive impairments, the neuronal basis of ⌬ 9 -THC-related developmental deficits remains unknown. Using a Boyden chamber assay, we show that agonist stimulation of the CB 1 cannabinoid receptor (CB1R) on cholecystokinin-expressing interneurons induces chemotaxis that is additive with brain-derived neurotrophic factor (BDNF)-induced interneuron migration. We find that Src kinase-dependent TrkB receptor transactivation mediates endocannabinoid (eCB)-induced chemotaxis in the absence of BDNF. Simultaneously, eCBs suppress the BDNF-dependent morphogenesis of interneurons, and this suppression is abolished by Src kinase inhibition in vitro. Because sustained prenatal ⌬ 9 -THC stimulation of CB1Rs selectively increases the density of cholecystokinin-expressing interneurons in the hippocampus in vivo, we conclude that prenatal CB 1R activity governs proper interneuron placement and integration during corticogenesis. Moreover, eCBs use TrkB receptor-dependent signaling pathways to regulate subtype-selective interneuron migration and specification.corticogenesis ͉ drug abuse ͉ neurotrophin E ndogenous cannabinoids, such as anandamide (AEA) and 2-arachidonoylglycerol (2-AG), modulate synaptic plasticity by the retrograde control of neurotransmitter release (1). Accordingly, a compartmentalization of endocannabinoid (eCB) synthesis and action has been demonstrated in adult brain (1-3). eCBs are predominantly synthesized in dendritic compartments and signal through presynaptic G i/o protein-coupled CB 1 cannabinoid receptors (CB 1 Rs) (4-6). The adult phenotype of cannabinoid systems is achieved through a series of developmentally regulated events culminating in high CB 1 R expression on cholecystokinin (CCK)-containing GABAergic interneurons (CB 1 R ϩ cells) in the hippocampus and neocortex (3,5,6).Recent studies have demonstrated the existence of functional CB 1 Rs in developing cortical neurons (7). A coincidence of eCB synthesis and release and CB 1 R activation within axonal growth cones was postulated to provide an eCB-driven reinforcement loop that regulates axonal growth and guidance (8). Although the functional significance of CB 1 Rs during assembly of cortical neuronal circuitries is unknown, their developmental impact is illustrated by long-lasting cognitive, motor, and social disturbances in offspring exposed prenatally to cannabis (9, 10).The majority of cortical interneurons are derived from extracortical precursor pools and undergo long-distance migration before inhabiting specific cortical layers (11, 12). Interneuron specification is in part governed by epigenetic cues within the neocortex, including brain-derived neurotrophic factor (BDNF) (12-14). Considering that pyramidal cells in the juvenile neocortex harbor the capacity of eCB synthesis and release (15), we hypothesiz...

Subsets of GABAergic neurons are able to maintain high frequency discharge patterns, which requires efficient replenishment of the releasable pool of GABA. Although glutamine is considered a preferred precursor of GABA, the identity of transporters involved in glutamine uptake by GABAergic neurons remains elusive. Molecular analyses revealed that SAT1 (Slc38a1) features system A characteristics with a preferential affinity for glutamine, and that SAT1 mRNA expression is associated with GABAergic neurons. By generating specific antibodies against SAT1 we show that this glutamine carrier is particularly enriched in GABAergic neurons. Cellular SAT1 distribution resembles that of GAD67, an essential GABA synthesis enzyme, suggesting that SAT1 can be involved in translocating glutamine into GABAergic neurons to facilitate inhibitory neurotransmitter generation.

Basal forebrain cholinergic neurons project to diverse cortical and hippocampal areas and receive reciprocal projections therefrom. Maintenance of a fine-tuned synaptic communication between pre- and postsynaptic cells in neuronal circuitries also requires feedback mechanisms to control the probability of neurotransmitter release from the presynaptic terminal. Release of endocannabinoids or glutamate from a postsynaptic neuron has been identified as a means of retrograde synaptic signalling. Presynaptic action of endocannabinoids is largely mediated by type 1 cannabinoid (CB1) receptors, while fatty-acid amide hydrolase (FAAH) is involved in inactivating some endocannabinoids postsynaptically. Alternatively, vesicular glutamate transporter 3 (VGLUT3) controls release of glutamate from postsynaptic cells. Here, we studied the distribution of CB1 receptors, FAAH and VGLUT3 in cholinergic basal forebrain nuclei of mouse and rat. Cholinergic neurons were devoid of CB1 receptor immunoreactivity. A fine CB1 receptor-immunoreactive (ir) fibre meshwork was present in medial septum, diagonal bands and nucleus basalis. In contrast, the ventral pallidum and substantia innominata received dense CB1 receptor-ir innervation and cholinergic neurons received CB1 receptor-ir presumed synaptic contacts. Consistent with CB1 receptor distribution, FAAH-ir somata were abundant in basal forebrain and appeared in contact with CB1 receptor-containing terminals. Virtually all cholinergic neurons were immunoreactive for FAAH. A significant proportion of cholinergic cells exhibited VGLUT3 immunoreactivity in medial septum, diagonal bands and nucleus basalis, and were in close apposition to VGLUT3-ir terminals. VGLUT3 immunoreactivity was largely absent in ventral pallidum and substantia innominata. We propose that specific subsets of cholinergic neurons may utilize endocannabinoids or glutamate for retrograde control of the efficacy of input synapses, and the mutually exclusive complementary distribution pattern of CB1 receptor-ir and VGLUT3-ir fibres in basal forebrain suggests segregated input-specific signalling mechanisms by cholinergic neurons.

Recent studies implicate dendritic endocannabinoid release from subsynaptic dendrites and subsequent inhibition of neurotransmitter release from nerve terminals as a means of retrograde signaling in multiple brain regions. Here we show that type 1 cannabinoid receptor-mediated endocannabinoid signaling is not involved in the retrograde control of synaptic efficacy at inhibitory synapses between fast-spiking interneurons and pyramidal cells in layer 2/3 of the neocortex.Vesicular neurotransmitter transporters, such as vesicular glutamate transporters (VGLUTs) 1 and 2, are localized to presynaptic terminals and accumulate neurotransmitters into synaptic vesicles. A third subtype of VGLUTs (VGLUT3) was recently identified and found localized to dendrites of various cell types. We demonstrate, using multiple immunofluorescence labeling and confocal laserscanning microscopy, that VGLUT3-like immunoreactivity is present in dendrites of layer 2/3 pyramidal neurons in the rat neocortex. Electron microscopy analysis confirmed that VGLUT3-like labeling is localized to vesicular structures, which show a tendency to accumulate in close proximity to postsynaptic specializations in dendritic shafts of pyramidal cells. Dual whole-cell recordings revealed that retrograde signaling between fast-spiking interneurons and pyramidal cells was enhanced under conditions of maximal efficacy of VGLUT3-mediated glutamate uptake, whereas it was reduced when glutamate uptake was inhibited by incrementing concentrations of the nonselective VGLUT inhibitor Evans blue (0.5-5.0 M) or intracellular Cl Ϫ concentrations (4 -145 mM). Our results present further evidence that dendritic vesicular glutamate release, controlled by novel VGLUT isoforms, provides fast negative feedback at inhibitory neocortical synapses, and demonstrate that glutamate can act as a retrograde messenger in the CNS.

Glutamate mediates several modes of neurotransmission in the central nervous system including recently discovered retrograde signaling from neuronal dendrites. We have previously identified the system N transporter SN1 as being responsible for glutamine efflux from astroglia and proposed a system A transporter (SAT) in subsequent transport of glutamine into neurons for neurotransmitter regeneration. Here, we demonstrate that SAT2 expression is primarily confined to glutamatergic neurons in many brain regions with SAT2 being predominantly targeted to the somatodendritic compartments in these neurons. SAT2 containing dendrites accumulate high levels of glutamine. Upon electrical stimulation in vivo and depolarization in vitro, glutamine is readily converted to glutamate in activated dendritic subsegments, suggesting that glutamine sustains release of the excitatory neurotransmitter via exocytosis from dendrites. The system A inhibitor MeAIB (alpha-methylamino-iso-butyric acid) reduces neuronal uptake of glutamine with concomitant reduction in intracellular glutamate concentrations, indicating that SAT2-mediated glutamine uptake can be a prerequisite for the formation of glutamate. Furthermore, MeAIB inhibited retrograde signaling from pyramidal cells in layer 2/3 of the neocortex by suppressing inhibitory inputs from fast-spiking interneurons. In summary, we demonstrate that SAT2 maintains a key metabolic glutamine/glutamate balance underpinning retrograde signaling by dendritic release of the neurotransmitter glutamate.

GABAergic interneurons with high-frequency firing, fast-spiking (FS) cells, form synapses on perisomatic regions of principal cells in the neocortex and hippocampus to control the excitability of cortical networks. Brain-derived neurotrophic factor (BDNF) is essential for the differentiation of multiple interneuron subtypes and the formation of their synaptic contacts. Here, we examined whether BDNF, alone or in conjunction with sustained KCl-induced depolarization, drives functional FS cell differentiation and the formation of inhibitory microcircuits. Homogeneous FS cell cultures were established by target-specific isolation using the voltage-gated potassium channel 3.1b subunit as the selection marker. Isolated FS cells expressed parvalbumin, were surrounded by perineuronal nets, formed immature inhibitory connections and generated slow action potentials at 12 days in vitro. Brain-derived neurotrophic factor (BDNF) promoted FS cell differentiation by increasing the somatic diameter, dendritic branching and the frequency of action potential firing. In addition, BDNF treatment led to a significant up-regulation of synaptophysin and vesicular GABA transporter expression, components of the synaptic machinery critical for GABA release, which was paralleled by an increase in synaptic strength. Long-term membrane depolarization alone was detrimental to dendritic branching. However, we observed that BDNF and KCl exerted additive effects, as reflected by the significantly accelerated maturation of synaptic contacts and high discharge frequencies, and was required for the formation of reciprocal connections between FS cells. Our results show that BDNF, along with membrane depolarization, is critical for FS cells to establish inhibitory circuitries during corticogenesis.