Caroline Fasano

Neuroinflammation is associated with changes in glial mGluR5 expression and the development of neonatal excitotoxic lesions

It has been hypothesized that neuroinflammation triggered during brain development can alter brain functions later in life. We investigated the contribution of inflammation to the alteration of normal brain circuitries in the context of neuroexcitotoxicity following neonatal ventral hippocampal lesions in rats with ibotenic acid, an NMDA glutamate receptor agonist. Excitotoxic ibotenic acid lesions led to a significant and persistent astrogliosis and microglial activation, associated with the production of inflammatory mediators. This response was accompanied by a significant increase in metabotropic glutamate receptor type 5 (mGluR5) expression within two distinct neuroinflammatory cell types; astrocytes and microglia. The participation of inflammation to the neurotoxin‐induced lesion was further supported by the prevention of hippocampal neuronal loss, glial mGluR5 expression and some of the behavioral perturbations associated to the excitotoxic lesion by concurrent anti‐inflammatory treatment with minocycline. These results indicate that neuroinflammation significantly contributes to long‐lasting excitotoxic effects of the neurotoxin and to some behavioral phenotypes associated with this model. Thus, the control of the inflammatory response may prevent the deleterious effects of excitotoxic processes that are triggered during brain development, limiting the risk to develop some of the behavioral manifestations related to these processes in adulthood.

Culture of postnatal mesencephalic dopamine neurons on an astrocyte monolayer.

This unit presents a protocol for primary culture of postnatal mesencephalic dopamine neurons grown on an astrocyte monolayer, which can be used to investigate cellular and molecular mechanisms regulating dopamine neuron function. Using this in vitro approach, dopamine neurons survive for an extended period of time and establish functional axon terminals and dendrites that display properties similar to those observed in vivo and in brain slices. An alternate protocol is provided for a microculture system in which astrocytes are grown on a spatially limited surface where single or small groups of dopamine neurons develop. Under such conditions, isolated neurons establish synaptic contacts, or autapses, onto their own somatodendritic compartment, thus facilitating morphological and physiological experiments. Curr. Protoc. Neurosci. 44:3.21.1‐3.21.19.

The endocannabinoid 2-arachidonoylglycerol inhibits long-term potentiation of glutamatergic synapses onto ventral tegmental area dopamine neurons in mice

Drugs of abuse cause changes in the mesocorticolimbic dopamine (DA) system, such as a long‐term potentiation (LTP)‐like phenomenon at glutamatergic synapses onto ventral tegmental area (VTA) DA neurons. Abolishing this LTP interferes with drug‐seeking behavior. Endocannabinoids (ECs) can be released by DA neurons in response to repetitive activation, which can inhibit glutamate release. Therefore, we hypothesized that ECs may act as negative regulators of LTP. Here we tested the induction of LTP in DA neurons of the VTA in mice expressing enhanced green fluorescent protein under the control of the tyrosine hydroxylase promoter. Immunohistochemistry showed colocalization of CB1 receptors with vesicular glutamate transporter (VGLUT)1 in terminals near DA neuron dendrites, with less extensive colocalization with VGLUT2. In addition, a CB1 receptor agonist, as well as trains of stimulation leading to EC production, decreased glutamate release onto DA neurons. We found that blocking CB1 receptors or synthesis of the EC 2‐arachidonoylglycerol (2‐AG) was without effect on basal excitatory postsynaptic potential amplitude; however, it facilitated the induction of LTP. As previously reported, antagonizing γ‐aminobutyric acid (GABA)A transmission also facilitated LTP induction. Combining GABAA and CB1 receptor antagonists did not lead to larger LTP. LTP induced in the presence of CB1 receptor blockade was prevented by an N‐methyl‐d‐aspartate receptor antagonist. Our observations argue in favor of the hypothesis that 2‐AG acts as a negative regulator of LTP in the VTA. Understanding the factors that regulate long‐term synaptic plasticity in this circuit is critical to aid our comprehension of drug addiction in humans.

Chronic activation of the D2 dopamine autoreceptor inhibits synaptogenesis in mesencephalic dopaminergic neurons in vitro

Chronic blockade or activation of dopamine receptors is critical for the pharmacological treatment of diseases like schizophrenia, Parkinson’s or attention deficit and hyperactivity disorder. However, the long‐term impact of such treatments on dopamine neurons is unclear. Chronic blockade of the dopamine D2 receptor in vivo triggers an increase in the axonal arborization of dopamine neurons [ European Journal of Neuroscience, 2002, 16, 787–794 ]. However, the specific involvement of presynaptic (autoreceptors) vs. postsynaptic D2 receptors as well as the molecular mechanisms involved have not been determined. Here, we examined the role of D2 autoreceptors in regulating the ability of mouse dopamine neurons to establish axon terminals. Chronic activation of this receptor with quinpirole, a specific agonist, decreased the number of axon terminals established by isolated dopamine neurons. This effect was accompanied by a decrease in dopamine release and was mediated through inhibition of protein kinase A. The decrease in axon terminal number induced by D2 receptor activation was also occluded when the mammalian Target of Rapamycin pathway of mRNA translation was blocked. Our results suggest that chronic activation of the D2 autoreceptor inhibits synaptogenesis by mesencephalic dopamine neurons through translational regulation of the synthesis of proteins required for synapse formation. This study provides a better understanding of the impact of long‐term pharmacological interventions acting through the D2 receptor.

Somatodendritic Dopamine Release Requires Synaptotagmin 4 and 7 and the Participation of Voltage-gated Calcium Channels

Somatodendritic (STD) dopamine (DA) release is a key mechanism for the autoregulatory control of DA release in the brain. However, its molecular mechanism remains undetermined. We tested the hypothesis that differential expression of synaptotagmin (Syt) isoforms explains some of the differential properties of terminal and STD DA release. Down-regulation of the dendritically expressed Syt4 and Syt7 severely reduced STD DA release, whereas terminal release required Syt1. Moreover, we found that although mobilization of intracellular Ca2+ stores is inefficient, Ca2+ influx through N- and P/Q-type voltage-gated channels is critical to trigger STD DA release. Our findings provide an explanation for the differential Ca2+ requirement of terminal and STD DA release. In addition, we propose that not all sources of intracellular Ca2+ are equally efficient to trigger this release mechanism. Our findings have implications for a better understanding of a fundamental cell biological process mediating transcellular signaling in a system critical for diseases such as Parkinson disease.

Chronic activation of the D2 autoreceptor inhibits both glutamate and dopamine synapse formation and alters the intrinsic properties of mesencephalic dopamine neurons in vitro

Dysfunctional dopamine (DA)‐mediated signaling is implicated in several diseases including Parkinson’s disease, schizophrenia and attention deficit and hyperactivity disorder. Chronic treatment with DA receptor agonists or antagonists is often used in pharmacotherapy, but the consequences of these treatments on DA neuron function are unclear. It was recently demonstrated that chronic D2 autoreceptor (D2R) activation in DA neurons decreases DA release and inhibits synapse formation. Given that DA neurons can establish synapses that release glutamate in addition to DA, we evaluated the synapse specificity of the functional and structural plasticity induced by chronic D2R activation. We show that chronic activation of the D2R with quinpirole in vitro caused a parallel decrease in the number of dopaminergic and glutamatergic axon terminals. The capacity of DA neurons to synthesize DA was not altered, as indicated by the lack of change in protein kinase A‐mediated Ser(40) phosphorylation of tyrosine hydroxylase. However, the spontaneous firing rate of DA neurons was decreased and was associated with altered intrinsic properties as revealed by a prolonged latency to first spike after release from hyperpolarization. Moreover, D2R function was decreased after its chronic activation. Our results demonstrate that chronic activation of the D2R induces a complex neuronal reorganization involving the inhibition of both DA and glutamate synapse formation and an alteration in electrical activity, but not in DA synthesis. A better understanding of D2R‐induced morphological and functional long‐term plasticity may lead to improved pharmacotherapy of DA‐related neurological and psychiatric disorders.