Margaret I. Davis

Dynamic reorganization of striatal circuits during the acquisition and consolidation of a skill

The learning of new skills is characterized by an initial phase of rapid improvement in performance and a phase of more gradual improvements as skills are automatized and performance asymptotes. Using in vivo striatal recordings, we observed region-specific changes in neural activity during the different phases of skill learning, with the associative or dorsomedial striatum being preferentially engaged early in training and the sensorimotor or dorsolateral striatum being engaged later in training. Ex vivo recordings from medium spiny striatal neurons in brain slices of trained mice revealed that the changes observed in vivo corresponded to regional- and training-specific changes in excitatory synaptic transmission in the striatum. Furthermore, the potentiation of glutamatergic transmission observed in dorsolateral striatum after extensive training was preferentially expressed in striatopallidal neurons, rather than striatonigral neurons. These findings demonstrate that region- and pathway-specific plasticity sculpts the circuits involved in the performance of the skill as it becomes automatized.

Loss of GluN2B-Containing NMDA Receptors in CA1 Hippocampus and Cortex Impairs Long-Term Depression, Reduces Dendritic Spine Density, and Disrupts Learning

NMDA receptors (NMDARs) are key mediators of certain forms of synaptic plasticity and learning. NMDAR complexes are heteromers composed of an obligatory GluN1 subunit and one or more GluN2 (GluN2A–GluN2D) subunits. Different subunits confer distinct physiological and molecular properties to NMDARs, but their contribution to synaptic plasticity and learning in the adult brain remains uncertain. Here, we generated mice lacking GluN2B in pyramidal neurons of cortex and CA1 subregion of hippocampus. We found that hippocampal principal neurons of adult GluN2B mutants had faster decaying NMDAR-mediated EPSCs than nonmutant controls and were insensitive to GluN2B but not NMDAR antagonism. A subsaturating form of hippocampal long-term potentiation (LTP) was impaired in the mutants, whereas a saturating form of LTP was intact. An NMDAR-dependent form of long-term depression (LTD) produced by low-frequency stimulation combined with glutamate transporter inhibition was abolished in the mutants. Additionally, mutants exhibited decreased dendritic spine density in CA1 hippocampal neurons compared with controls. On multiple assays for corticohippocampal-mediated learning and memory (hidden platform Morris water maze, T-maze spontaneous alternation, and pavlovian trace fear conditioning), mutants were impaired. These data further demonstrate the importance of GluN2B for synaptic plasticity in the adult hippocampus and suggest a particularly critical role in LTD, at least the form studied here. The finding that loss of GluN2B was sufficient to cause learning deficits illustrates the contribution of GluN2B-mediated forms of plasticity to memory formation, with implications for elucidating NMDAR-related dysfunction in disease-related cognitive impairment.

Ethanol-BDNF interactions: Still More Questions than Answers

Brain-derived neurotrophic factor (BDNF) has emerged as a regulator of development, plasticity and, recently, addiction. Decreased neurotrophic activity may be involved in ethanol-induced neurodegeneration in the adult brain and in the etiology of alcohol-related neurodevelopmental disorders. This can occur through decreased expression of BDNF or through inability of the receptor to transduce signals in the presence of ethanol. In contrast, recent studies implicate region-specific up-regulation of BDNF and associated signaling pathways in anxiety, addiction and homeostasis after ethanol exposure. Anxiety and depression are precipitating factors for substance abuse and these disorders also involve region-specific changes in BDNF in both pathogenesis and response to pharmacotherapy. Polymorphisms in the genes coding for BDNF and its receptor TrkB are linked to affective, substance abuse and appetitive disorders and therefore may play a role in the development of alcoholism. This review summarizes historical and pre-clinical data on BDNF and TrkB as it relates to ethanol toxicity and addiction. Many unresolved questions about region-specific changes in BDNF expression and the precise role of BDNF in neuropsychiatric disorders and addiction remain to be elucidated. Resolution of these questions will require significant integration of the literature on addiction and comorbid psychiatric disorders that contribute to the development of alcoholism.

Metabotropic glutamate receptor 5 activation inhibits microglial associated inflammation and neurotoxicity

The Group I metabotropic glutamate receptor 5 (mGluR5) can modulate addiction, pain, and neuronal cell death. Expression of some mGluRs, such as Group II and III mGluRs, has been reported in microglia and may affect their activation. However, the expression and role of mGluR5 in microglia is unclear. Using immunocytochemistry and Western blot, we demonstrate that mGluR5 protein is expressed in primary microglial cultures. Activation of mGluR5 using the selective agonist (RS)‐2‐chloro‐5‐hydroxyphenylglycine (CHPG) significantly reduces microglial activation in response to lipopolysaccharide, as indicated by a reduction in nitric oxide, reactive oxygen species, and TNFα production. Microglial induced neurotoxicity is also markedly reduced by CHPG treatment. The anti‐inflammatory effects of CHPG are not observed in microglial cultures from mGluR5 knockout mice and are blocked by selective mGluR5 antagonists, suggesting that these actions are mediated by the mGluR5 receptor. Anti‐inflammatory actions of mGluR5 activation are attenuated by phospholipase C and protein kinase C inhibitors, as well as by calcium chelators, suggesting that the mGluR5 activation in microglia involves the Gαq‐protein signal transduction pathway. These data indicate that microglial mGluR5 may represent a novel target for modulating neuroinflammation, an important component of both acute and chronic neurodegenerative disorders.

The role of protein synthesis in striatal long-term depression

Long-term depression (LTD) at the corticostriatal synapse is postsynaptically induced but presynaptically expressed, the depression being a result of retrograde endocannabinoid signaling that activates presynaptic cannabinoid CB1 receptors and reduces the probability of glutamate release. To study the role of protein synthesis in striatal LTD, we used a striatum-only preparation in which the presynaptic cell body is cut off, leaving intact only its axons, whose terminals synapse on medium spiny neurons. LTD (duration >150 min) was induced in this preparation, thus providing evidence that transcription in the presynaptic cell nucleus is not necessary for this form of plasticity. The maintenance of striatal LTD, however, was blocked by bath application of protein translation inhibitors but not by the same inhibitors loaded into the postsynaptic cell. These results suggest that local translation is critical for the expression of striatal LTD, distinguishing this form of mammalian synaptic plasticity from other forms that require postsynaptic protein synthesis. Possible roles of axonal or glial translation in striatal LTD are considered.

GluN2B in corticostriatal circuits governs choice learning and choice shifting

A choice that reliably produces a preferred outcome can be automated to liberate cognitive resources for other tasks. Should an outcome become less desirable, behavior must adapt in parallel or it becomes perseverative. Corticostriatal systems are known to mediate choice learning and flexibility, but the molecular mechanisms of these processes are not well understood. We integrated mouse behavioral, immunocytochemical, in vivo electrophysiological, genetic and pharmacological approaches to study choice. We found that the dorsal striatum (DS) was increasingly activated with choice learning, whereas reversal of learned choice engaged prefrontal regions. In vivo, DS neurons showed activity associated with reward anticipation and receipt that emerged with learning and relearning. Corticostriatal or striatal deletion of Grin2b (encoding the NMDA-type glutamate receptor subunit GluN2B) or DS-restricted GluN2B antagonism impaired choice learning, whereas cortical Grin2b deletion or OFC GluN2B antagonism impaired shifting. Our convergent data demonstrate how corticostriatal GluN2B circuits govern the ability to learn and shift choice behavior.

The Endogenous Brain Constituent N-Arachidonoyl l-Serine Is an Activator of Large Conductance Ca2+-Activated K+ Channels

The novel endocannabinoid-like lipid N-arachidonoyl l-serine (ARA-S) causes vasodilation through both endothelium-dependent and -independent mechanisms. We have analyzed the vasorelaxant effect of ARA-S in isolated vascular preparations and its effects on Ca2+-activated K+ currents in human embryonic kidney cells stably transfected with the α-subunit of the human, large conductance Ca+-activated K+ (BKCa) channel [human embryonic kidney (HEK) 293hSlo cells]. ARA-S caused relaxation of rat isolated, intact and denuded, small mesenteric arteries preconstricted with (R)-(-)-1-(3-hydroxyphenyl)-2-methylaminoethanol hydrochloride (pEC50, 5.49 and 5.14, respectively), whereas it caused further contraction of vessels preconstricted with KCl (pEC50, 5.48 and 4.82, respectively). Vasorelaxation by ARA-S was inhibited by 100 nM iberiotoxin. In human embryonic kidney cells stably transfected with the α-subunit of the human BKCa channel cells, ARA-S and its enantiomer, N-arachidonoyl-d-serine, enhanced the whole-cell outward K+ current with similar potency (pEC50, 5.63 and 5.32, respectively). The potentiation was not altered by the β1 subunit or mediated by ARA-S metabolites, stimulation of known cannabinoid receptors, G proteins, protein kinases, or Ca2+-dependent processes; it was lost after patch excision or after membrane cholesterol depletion but was restored after cholesterol reconstitution. BKCa currents were also enhanced by N-arachidonoyl ethanolamide (pEC50, 5.27) but inhibited by another endocannabinoid, O-arachidonoyl ethanolamine (pIC50, 6.35), or by the synthetic cannabinoid O-1918 [(-)-1,3-dimethoxy-2-(3-3,4-trans-p-menthadien-(1,8)-yl)-orcinol] (pIC50, 6.59), which blocks ARA-S-induced vasodilation. We conclude the following. 1) ARA-S directly activates BKCa channels. 2) This interaction does not involve cannabinoid receptors or cytosolic factors but is dependent on the presence of membrane cholesterol. 3) Direct BKCa channel activation probably contributes to the endothelium-independent component of ARA-S-induced mesenteric vasorelaxation. 4) O-1918 is a BKCa channel inhibitor.

Endocannabinoid- and mGluR5-Dependent Short-Term Synaptic Depression in an Isolated Neuron/Bouton Preparation From the Hippocampal CA1 Region

Endocannabinoids released from the postsynaptic neuronal membrane can activate presynaptic CB1 receptors and inhibit neurotransmitter release. In hippocampal slices, depolarization of the CA1 pyramidal neurons elicits an endocannabinoid-mediated inhibition of gamma-aminobutyric acid release known as depolarization-induced suppression of inhibition (DSI). Using the highly reduced neuron/synaptic bouton preparation from the CA1 region of hippocampus, we have begun to examine endocannabinoid-dependent short-term depression (STD) of inhibitory synaptic transmission under well-controlled physiological and pharmacological conditions in an environment free of other cells. Application of the CB1 synthetic agonist WIN55212-2 and endogenous cannabinoids 2-AG and anandamide produced a decrease in spontaneous inhibitory postsynaptic current (sIPSC) frequency and amplitude, indicating the presence of CB1 receptors at synapses in this preparation. Endocannabinoid-dependent STD is different from DSI found in hippocampal slices and the neuron/bouton preparation from basolateral amygdala (BLA) since depolarization alone was not sufficient to induce suppression of sIPSCs. However, concurrent application of the metabotropic glutamate receptor (mGluR) agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) and postsynaptic depolarization resulted in a transient (30-50 s) decrease in sIPSC frequency and amplitude. Application of DHPG alone had no effect on sIPSCs. The depolarization/DHPG-induced STD was blocked by the CB1 antagonist SR141716A and the mGluR5 antagonist MPEP and was sensitive to intracellular calcium concentration. Comparing the present findings with earlier work in hippocampal slices and BLA, it appears that endocannabinoid release is less robust in isolated hippocampal neurons.

Nr4a1-eGFP is a marker of striosome-matrix architecture, development and activity in the extended striatum.

Transgenic mice expressing eGFP under population specific promoters are widely used in neuroscience to identify specific subsets of neurons in situ and as sensors of neuronal activity in vivo. Mice expressing eGFP from a bacterial artificial chromosome under the Nr4a1 promoter have high expression within the basal ganglia, particularly within the striosome compartments and striatal-like regions of the extended amygdala (bed nucleus of the stria terminalis, striatal fundus, central amygdaloid nucleus and intercalated cells). Grossly, eGFP expression is inverse to the matrix marker calbindin 28K and overlaps with mu-opioid receptor immunoreactivity in the striatum. This pattern of expression is similar to Drd1, but not Drd2, dopamine receptor driven eGFP expression in structures targeted by medium spiny neuron afferents. Striosomal expression is strong developmentally where Nr4a1-eGFP expression overlaps with Drd1, TrkB, tyrosine hydroxylase and phospho-ERK, but not phospho-CREB, immunoreactivity in “dopamine islands”. Exposure of adolescent mice to methylphenidate resulted in an increase in eGFP in both compartments in the dorsolateral striatum but eGFP expression remained brighter in the striosomes. To address the role of activity in Nr4a1-eGFP expression, primary striatal cultures were prepared from neonatal mice and treated with forskolin, BDNF, SKF-83822 or high extracellular potassium and eGFP was measured fluorometrically in lysates. eGFP was induced in both neurons and contaminating glia in response to forskolin but SKF-83822, brain derived neurotrophic factor and depolarization increased eGFP in neuronal-like cells selectively. High levels of eGFP were primarily associated with Drd1+ neurons in vitro detected by immunofluorescence; however ∼15% of the brightly expressing cells contained punctate met-enkephalin immunoreactivity. The Nr4a1-GFP mouse strain will be a useful model for examining the connectivity, physiology, activity and development of the striosome system.

Dopamine dynamics and cocaine sensitivity differ between striosome and matrix compartments of the striatum.

The striatum is typically classified according to its major output pathways, which consist of dopamine D1 and D2 receptor-expressing neurons. The striatum is also divided into striosome and matrix compartments, based on the differential expression of a number of proteins, including the mu opioid receptor, dopamine transporter (DAT), and Nr4a1 (nuclear receptor subfamily 4, group A, member 1). Numerous functional differences between the striosome and matrix compartments are implicated in dopamine-related neurological disorders including Parkinsons disease and addiction. Using Nr4a1-eGFP mice, we provide evidence that electrically evoked dopamine release differs between the striosome and matrix compartments in a regionally-distinct manner. We further demonstrate that this difference is not due to differences in inhibition of dopamine release by dopamine autoreceptors or nicotinic acetylcholine receptors. Furthermore, cocaine enhanced extracellular dopamine in striosomes to a greater degree than in the matrix and concomitantly inhibited dopamine uptake in the matrix to a greater degree than in striosomes. Importantly, these compartment differences in cocaine sensitivity were limited to the dorsal striatum. These findings demonstrate a level of exquisite microanatomical regulation of dopamine by the DAT in striosomes relative to the matrix.