Cbln1 and the Delta2 Glutamate Receptor—An Orphan Ligand and an Orphan Receptor Find Their Partners

Cerebellin was originally discovered as a Purkinje cell-specific peptide more than two decades ago. Later, its precursor protein precerebellin (Cbln1) was found to be produced in cerebellar granule cells. It has become increasingly clear that although the cerebellin peptide may have certain functions, Cbln1 is an actual signaling molecule that belongs to the C1q family. However, the precise function of Cbln1 has been unresolved. Cbln1 is released from granule cells, and disruption of the cbln1 gene in mice causes a severe reduction in the number of synapses between Purkinje cells and parallel fibers (PFs; axons of granule cells) and results in cerebellar ataxia. The glutamate receptor δ2 (GluD2) is highly expressed on Purkinje cells’ dendritic spines which make synapses with PFs. Although GluD2 was identified as a member of the ionotropic glutamate receptors more than 15 years ago, it has been referred to as an orphan receptor because its endogenous ligands are unclear. Interestingly, GluD2-null mice phenocopy cbln1-null mice precisely. Cbln1 and GluD2 have therefore been thought to participate in a common signaling pathway that is required for the formation of PF synapses. We recently established a direct ligand–receptor relationship between Cbln1 and GluD2. The Cbln1–GluD2 complex is located at the cleft of PF-Purkinje cell synapses and bidirectionally regulates both presynaptic and postsynaptic differentiation.     

Cbln1 downregulates the formation and function of inhibitory synapses in mouse cerebellar Purkinje cells

The formation of excitatory and inhibitory synapses must be tightly coordinated to establish functional neuronal circuitry during development. In the cerebellum, the formation of excitatory synapses between parallel fibers and Purkinje cells is strongly induced by Cbln1, which is released from parallel fibers and binds to the postsynaptic δ2 glutamate receptor (GluD2). However, Cbln1's role, if any, in inhibitory synapse formation has been unknown. Here, we show that Cbln1 downregulates the formation and function of inhibitory synapses between Purkinje cells and interneurons. Immunohistochemical analyses with an anti‐vesicular GABA transporter antibody revealed an increased density of interneuron–Purkinje cell synapses in the cbln1‐null cerebellum. Whole‐cell patch‐clamp recordings from Purkinje cells showed that both the amplitude and frequency of miniature inhibitory postsynaptic currents were increased in cbln1‐null cerebellar slices. A 3‐h incubation with recombinant Cbln1 reversed the increased amplitude of inhibitory currents in Purkinje cells in acutely prepared cbln1‐null slices. Furthermore, an 8‐day incubation with recombinant Cbln1 reversed the increased interneuron–Purkinje cell synapse density in cultured cbln1‐null slices. In contrast, recombinant Cbln1 did not affect cerebellar slices from mice lacking both Cbln1 and GluD2. Finally, we found that tyrosine phosphorylation was upregulated in the cbln1‐null cerebellum, and acute inhibition of Src‐family kinases suppressed the increased inhibitory postsynaptic currents in cbln1‐null Purkinje cells. These findings indicate that Cbln1–GluD2 signaling inhibits the number and function of inhibitory synapses, and shifts the excitatory–inhibitory balance towards excitation in Purkinje cells. Cbln1's effect on inhibitory synaptic transmission is probably mediated by a tyrosine kinase pathway.     

Postnatal development of tyrosine hydroxylase mRNA-expressing neurons in mouse neostriatum

The striatum harbors a small number of tyrosine hydroxylase (TH) mRNA‐containing GABAergic neurons that express TH immunoreactivity after dopamine depletion, some of which reportedly resembled striatal medium spiny projection neurons (MSNs). To clarify whether the TH mRNA‐expressing neurons were a subset of MSNs, we characterized their postnatal development of electrophysiological and morphological properties using a transgenic mouse strain expressing enhanced green fluorescent protein (EGFP) under the control of the rat TH gene promoter. At postnatal day (P)1, EGFP‐TH⁺ neurons were present as clusters in the striatum and, thereafter, gradually scattered ventromedially by P18 without regard to the striatal compartments. They were immunonegative for calbindin, but immunopositive for enkephalin (54.5%) and dynorphin (80.0%). Whole‐cell patch‐clamp recordings revealed at least two distinct neuronal types, termed EGFP‐TH⁺ Type A and B. Whereas Type B neurons were aspiny and negative for the MSN marker dopamine‐ and cyclic AMP‐regulated phosphoprotein of 32 kDa (DARPP‐32), Type A neurons constituted 75% of the EGFP⁺ cells, had dendritic spines (24.6%), contained DARPP‐32 (73.6%) and a proportion acquired TH immunoreactivity after injections of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine and 3‐nitropropionic acid. The membrane properties and N‐methyl‐d ‐aspartate : non‐N‐methyl‐d ‐aspartate excitatory postsynaptic current ratio of Type A neurons were very similar to MSNs at P18. However, their resting membrane potentials and spike widths were statistically different from those of MSNs. In addition, the calbindin‐like, DARPP‐32‐like and dynorphin B‐like immunoreactivity of Type A neurons developed differently from that of MSNs in the matrix. Thus, Type A neurons closely resemble MSNs, but constitute a cell type distinct from classical MSNs.     

Single Exposure to Cocaine Impairs Reinforcement Learning by Potentiating the Activity of Neurons in the Direct Striatal Pathway in Mice

Plasticity in the glutamatergic synapses on striatal medium spiny neurons (MSNs) is not only essential for behavioral adaptation but also extremely vulnerable to drugs of abuse. Modulation on these synapses by even a single exposure to an addictive drug may interfere with the plasticity required by behavioral learning and thus produce impairment. In the present work, we found that the negative reinforcement learning, escaping mild foot-shocks by correct nose-poking, was impaired by a single in vivo exposure to 20 mg/kg cocaine 24 h before the learning in mice. Either a single exposure to cocaine or reinforcement learning potentiates the glutamatergic synapses on MSNs expressing the striatal dopamine 1 (D1) receptor (D1-MSNs). However, 24 h after the cocaine exposure, the potentiation required for reinforcement learning was disrupted. Specific manipulation of the activity of striatal D1-MSNs in D1-cre mice demonstrated that activation of these MSNs impaired reinforcement learning in normal D1-cre mice, but inhibition of these neurons reversed the reinforcement learning impairment induced by cocaine. The results suggest that cocaine potentiates the activity of direct pathway neurons in the dorsomedial striatum and this potentiation might disrupt the potentiation produced during and required for reinforcement learning.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12264-021-00687-8.     

Parvalbumin‐producing striatal interneurons receive excitatory inputs onto proximal dendrites from the motor thalamus in male mice

In rodents, the dorsolateral striatum regulates voluntary movement by integrating excitatory inputs from the motor‐related cerebral cortex and thalamus to produce contingent inhibitory output to other basal ganglia nuclei. Striatal parvalbumin (PV)‐producing interneurons receiving this excitatory input then inhibit medium spiny neurons (MSNs) and modify their outputs. To understand basal ganglia function in motor control, it is important to reveal the precise synaptic organization of motor‐related cortical and thalamic inputs to striatal PV interneurons. To examine which domains of the PV neurons receive these excitatory inputs, we used male bacterial artificial chromosome transgenic mice expressing somatodendritic membrane–targeted green fluorescent protein in PV neurons. An anterograde tracing study with the adeno‐associated virus vector combined with immunodetection of pre‐ and postsynaptic markers visualized the distribution of the excitatory appositions on PV dendrites. Statistical analysis revealed that the density of thalamostriatal appositions along the dendrites was significantly higher on the proximal than distal dendrites. In contrast, there was no positional preference in the density of appositions from axons of the dorsofrontal cortex. Population observations of thalamostriatal and corticostriatal appositions by immunohistochemistry for pathway‐specific vesicular glutamate transporters confirmed that thalamic inputs preferentially, and cortical ones less preferentially, made apposition on proximal dendrites of PV neurons. This axodendritic organization suggests that PV neurons produce fast and reliable inhibition of MSNs in response to thalamic inputs and process excitatory inputs from motor cortices locally and plastically, possibly together with other GABAergic and dopaminergic dendritic inputs, to modulate MSN inhibition.     

Impact of dendritic spine preservation in medium spiny neurons on dopamine graft efficacy and the expression of dyskinesias in parkinsonian rats

Dopamine deficiency associated with Parkinson’s disease (PD) results in numerous changes in striatal transmitter function and neuron morphology. Specifically, there is marked atrophy of dendrites and dendritic spines on striatal medium spiny neurons (MSN), primary targets of inputs from nigral dopamine and cortical glutamate neurons, in advanced PD and rodent models of severe dopamine depletion. Dendritic spine loss occurs via dysregulation of intraspine Cav1.3 L‐type Ca²⁺channels and can be prevented, in animal models, by administration of the calcium channel antagonist, nimodipine. The impact of MSN dendritic spine loss in the parkinsonian striatum on dopamine neuron graft therapy remains unexamined. Using unilaterally parkinsonian Sprague–Dawley rats, we tested the hypothesis that MSN dendritic spine preservation through administration of nimodipine would result in improved therapeutic benefit and diminished graft‐induced behavioral abnormalities in rats grafted with embryonic ventral midbrain cells. Analysis of rotational asymmetry and spontaneous forelimb use in the cylinder task found no significant effect of dendritic spine preservation in grafted rats. However, analyses of vibrissae‐induced forelimb use, levodopa‐induced dyskinesias and graft‐induced dyskinesias showed significant improvement in rats with dopamine grafts associated with preserved striatal dendritic spine density. Nimodipine treatment in this model did not impact dopamine graft survival but allowed for increased graft reinnervation of striatum. Taken together, these results demonstrate that even with grafting suboptimal numbers of cells, maintaining normal spine density on target MSNs results in overall superior behavioral efficacy of dopamine grafts.     

Mapping of Cbln1-like immunoreactivity in adult and developing mouse brain and its localization to the endolysosomal compartment of neurons

Cbln1 is a secreted glycoprotein essential for synapse structure and function in cerebellum that is also expressed in extracerebellar structures where its function is unknown. Furthermore, Cbln1 assembles into homomeric complexes and heteromeric complexes with three family members (Cbln2-Cbln4), thereby influencing each other's degradation and secretion. Therefore, to understand its function, it is essential to establish the location of Cbln1 relative to other family members. The localization of Cbln1 in brain was determined using immunohistochemistry and cbln1-lacZ transgenic mice. Cbln1-like immunoreactivity (CLI) was always punctate and localized to the cytoplasm of neurons. The punctate CLI colocalized with cathepsin D, a lysosomal marker, but not with markers of endoplasmic reticulum or Golgi, indicating that Cbln1 is present in neuronal endosomes/lysosomes. This may represent the cellular mechanism underlying the regulated degradation of Cbln1 observed in vivo. Outside the cerebellum, CLI mapped to multiple brain regions that were frequently synaptically interconnected, warranting their analysis in cbln1-null mice. Furthermore, whereas CLI increased dramatically in the cerebellum of cbln3-null mice it was unchanged in extracerebellar neurons. This opens the possibility that other family members that are coexpressed in these areas control Cbln1 levels, potentially by modulating processing in the endolysosomal pathway. During development of cbln1-lacZ mice, beta-galactosidase staining was first observed in proliferating granule cell precursors prior to synaptogenesis and thereafter in maturing and adult granule cells. As cbln3 is only expressed in post-mitotic, post-migratory granule cells, Cbln1 homomeric complexes in precursors and Cbln1-Cbln3 heteromeric complexes in mature granule cells may have distinct functions and turnover.     

Characterization of striatal phenotypes in heterozygous Disc1 mutant mice,a model of haploinsufficiency

Disrupted-in-Schizophrenia 1 (DISC1) is a susceptibility gene for several psychiatric illnesses. To study the pathogenesis of these disorders, we generated Disc1 mutant mice by introducing the 129S6/SvEv 25-bp deletion Disc1 variants into the C57BL/6J strain. In this study, we used heterozygous Disc1 mutant (Het) mice to evaluate the DISC1 haploinsufficiency model of schizophrenia. No changes in locomotor behaviors were observed in Het mice; however, after amphetamine injection, greater locomotor activity was observed in Het mice compared with wild-type (WT) mice. Moreover, amphetamine-induced elevations of c-Fos expression and dopamine level in the striatum were greater in Het mice than in WT controls, suggesting an altered dopaminergic regulation in the striatum of Het mice. Compared with those in WTs, the striatal protein levels of dopamine transporter and D2 dopamine receptor were increased in Het mice, while D1 dopamine receptor level was decreased. DISC1 interacting proteins, GSK3α and GSK3β, were downregulated in Het mice, whereas the levels of PDE4B and CREB were not altered. Morphologically, the complexities of striatal median spiny neurons (MSNs), parvalbumin-positive interneurons and Iba1-positive microglia were all decreased in Het mice. The density and head diameter of dendritic spines in the MSNs of Het mice were also reduced. Our results indicate that mice lacking one WT Disc1 allele are more sensitive to psychostimulant amphetamine challenge, which might be attributed to the altered structure and function of the striatal dopaminergic system. Here, we demonstrated striatal phenotypes in heterozygous Disc1 mutant mice, which could be a promising model of DISC1 haploinsufficiency.     

Lingual and palatal gustatory afferents each depend on both BDNF and NT‐4, but the dependence is greater for lingual than palatal afferents

Neurons of the geniculate ganglion innervate taste buds located in two spatially distinct targets, the tongue and palate. About 50% of these neurons die in Bdnf^−/− mice and Ntf4/5^−/− mice. Bdnf^−/−/Ntf4/5^−/− double mutants lose 90–95% of geniculate ganglion neurons. To determine whether different subpopulations are differentially influenced by neurotrophins, we quantified neurons from two ganglion subpopulations separately and remaining taste buds at birth within each target field in wild‐type, Bdnf^−/−, Ntf4/5^−/−, and Bdnf^−/−/Ntf4/5^−/− mice. In wild‐type mice the same number of neurons innervated the anterior tongue and soft palate and each target contained the same number of taste buds. Compared to wild‐type mice, Bdnf^−/− mice showed a 50% reduction in geniculate neurons innervating the tongue and a 28% loss in neurons innervating the soft palate. Ntf4/5^−/− mice lost 58% of the neurons innervating the tongue and 41% of the neurons innervating the soft palate. Taste bud loss was not as profound in the NT‐4 null mice compared to BDNF‐null mice. Tongues of Bdnf^−/−/Ntf4/5^−/− mice were innervated by 0 to 4 gustatory neurons and contained 3 to 16 taste buds at birth, indicating that some taste buds remain even when all innervation is lost. Thus, gustatory neurons are equally dependent on BDNF and NT‐4 expression for survival, regardless of what peripheral target they innervate. However, taste buds are more sensitive to BDNF than NT‐4 removal. J. Comp. Neurol. 518:3290–3301, 2010. © 2010 Wiley‐Liss, Inc.     

D1 and D2 dopamine-receptor modulation of striatal glutamatergic signaling in striatal medium spiny neurons

Dopamine shapes a wide variety of psychomotor functions. This is mainly accomplished by modulating cortical and thalamic glutamatergic signals impinging upon principal medium spiny neurons (MSNs) of the striatum. Several lines of evidence suggest that dopamine D1 receptor signaling enhances dendritic excitability and glutamatergic signaling in striatonigral MSNs, whereas D2 receptor signaling exerts the opposite effect in striatopallidal MSNs. The functional antagonism between these two major striatal dopamine receptors extends to the regulation of synaptic plasticity. Recent studies, using transgenic mice in which cells express D1 and D2 receptors, have uncovered unappreciated differences between MSNs that shape glutamatergic signaling and the influence of DA on synaptic plasticity. These studies have also shown that long-term alterations in dopamine signaling produce profound and cell-type-specific reshaping of corticostriatal connectivity and function.     

Cell type-specific distribution of GABAA receptor subtypes in the mouse dorsal striatum

The striatum is the main input nucleus of the basal ganglia, mediating motor and cognitive functions. Striatal projection neurons are GABAergic medium spiny neurons (MSN), expressing either the dopamine receptor type 1 (D₁-R MSN) and forming the direct, movement-promoting pathway, or dopamine receptor type 2 (D₂-R MSN), forming the indirect movement-suppressing pathway. Locally, activity and synchronization of MSN are modulated by several subtypes of GABAergic and cholinergic interneurons. Overall, GABAergic circuits in the striatum remain poorly characterized, and little is known about the intrastriatal connectivity of interneurons and the distribution of GABA_A receptor (GABA_AR) subtypes, distinguished by their subunit composition, in striatal synapses. Here, by using immunofluorescence in mouse tissue, we investigated the distribution of GABA_ARs containing the α₁, α₂, or α₃ subunit in perisomatic synapses of striatal MSN and interneurons, as well as the innervation pattern of D₁R- and D₂R-MSN soma and axonal initial segment (AIS) by GABAergic and cholinergic interneurons. Our results show that perisomatic GABAergic synapses of D₁R- and D₂R-MSN contain the GABA_AR α₁ and/or α₂ subunits, but not the α₃ subunit; D₂R-MSN have significantly more α₁-GABA_ARs on their soma than D₁R-MSN. Further, interneurons have few perisomatic synapses containing α₂-GABA_ARs, whereas α₃-GABA_ARs (along with the α₁-GABA_ARs) are abundant in perisomatic synapses of CCK⁺, NPY⁺/SOM⁺, and vAChT⁺ interneurons. Each MSN and interneuron population analyzed received a distinct pattern of GABAergic and cholinergic innervation, complementing this postsynaptic heterogeneity. In conclusion, intra-striatal GABAergic circuits are distinguished by cell-type specific innervation patterns, differential expression and postsynaptic targeting of GABA_AR subtypes.     

Cholinergic interneurons in the Q140 knockin mouse model of Huntington's disease: Reductions in dendritic branching and thalamostriatal input

We have previously found that thalamostriatal axodendritic terminals are reduced as early as 1 month of age in heterozygous Q140 HD mice (Deng et al. [ 2013 ] Neurobiol Dis 60:89–107). Because cholinergic interneurons are a major target of thalamic axodendritic terminals, we examined the VGLUT2‐immunolabeled thalamic input to striatal cholinergic interneurons in heterozygous Q140 males at 1 and 4 months of age, using choline acetyltransferase (ChAT) immunolabeling to identify cholinergic interneurons. Although blinded neuron counts showed that ChAT⁺ perikarya were in normal abundance in Q140 mice, size measurements indicated that they were significantly smaller. Sholl analysis further revealed the dendrites of Q140 ChAT⁺ interneurons were significantly fewer and shorter. Consistent with the light microscopic data, ultrastructural analysis showed that the number of ChAT⁺ dendritic profiles per unit area of striatum was significantly decreased in Q140 striata, as was the abundance of VGLUT2⁺ axodendritic terminals making synaptic contact with ChAT⁺ dendrites per unit area of striatum. The density of thalamic terminals along individual cholinergic dendrites was, however, largely unaltered, indicating that the reduction in the areal striatal density of axodendritic thalamic terminals on cholinergic neurons was due to their dendritic territory loss. These results show that the abundance of thalamic input to individual striatal cholinergic interneurons is reduced early in the life span of Q140 mice, raising the possibility that this may occur in human HD as well. Because cholinergic interneurons differentially affect striatal direct vs. indirect pathway spiny projection neurons, their reduced thalamic excitatory drive may contribute to early abnormalities in movement in HD. J. Comp. Neurol. 524:3518–3529, 2016. © 2016 Wiley Periodicals, Inc.     

Cerebellins are differentially expressed in selective subsets of neurons throughout the brain

Cerebellins are secreted hexameric proteins that form tripartite complexes with the presynaptic cell‐adhesion molecules neurexins or ‘deleted‐in‐colorectal‐cancer’, and the postsynaptic glutamate‐receptor‐related proteins GluD1 and GluD2. These tripartite complexes are thought to regulate synapses. However, cerebellins are expressed in multiple isoforms whose relative distributions and overall functions are not understood. Three of the four cerebellins, Cbln1, Cbln2, and Cbln4, autonomously assemble into homohexamers, whereas the Cbln3 requires Cbln1 for assembly and secretion. Here, we show that Cbln1, Cbln2, and Cbln4 are abundantly expressed in nearly all brain regions, but exhibit strikingly different expression patterns and developmental dynamics. Using newly generated knockin reporter mice for Cbln2 and Cbln4, we find that Cbln2 and Cbln4 are not universally expressed in all neurons, but only in specific subsets of neurons. For example, Cbln2 and Cbln4 are broadly expressed in largely non‐overlapping subpopulations of excitatory cortical neurons, but only sparse expression was observed in excitatory hippocampal neurons of the CA1‐ or CA3‐region. Similarly, Cbln2 and Cbln4 are selectively expressed, respectively, in inhibitory interneurons and excitatory mitral projection neurons of the main olfactory bulb; here, these two classes of neurons form dendrodendritic reciprocal synapses with each other. A few brain regions, such as the nucleus of the lateral olfactory tract, exhibit astoundingly high Cbln2 expression levels. Viewed together, our data show that cerebellins are abundantly expressed in relatively small subsets of neurons, suggesting specific roles restricted to subsets of synapses.     

Cell type–specific spatial and functional coupling between mammalian brain Kv2.1 K+ channels and ryanodine receptors

The Kv2.1 voltage‐gated K⁺ channel is widely expressed throughout mammalian brain, where it contributes to dynamic activity‐dependent regulation of intrinsic neuronal excitability. Here we show that somatic plasma membrane Kv2.1 clusters are juxtaposed to clusters of intracellular ryanodine receptor (RyR) Ca²⁺‐release channels in mouse brain neurons, most prominently in medium spiny neurons (MSNs) of the striatum. Electron microscopy–immunogold labeling shows that in MSNs, plasma membrane Kv2.1 clusters are adjacent to subsurface cisternae, placing Kv2.1 in close proximity to sites of RyR‐mediated Ca²⁺ release. Immunofluorescence labeling in transgenic mice expressing green fluorescent protein in specific MSN populations reveals the most prominent juxtaposed Kv2.1:RyR clusters in indirect pathway MSNs. Kv2.1 in both direct and indirect pathway MSNs exhibits markedly lower levels of labeling with phosphospecific antibodies directed against the S453, S563, and S603 phosphorylation site compared with levels observed in neocortical neurons, although labeling for Kv2.1 phosphorylation at S563 was significantly lower in indirect pathway MSNs compared with those in the direct pathway. Finally, acute stimulation of RyRs in heterologous cells causes a rapid hyperpolarizing shift in the voltage dependence of activation of Kv2.1, typical of Ca²⁺/calcineurin‐dependent Kv2.1 dephosphorylation. Together, these studies reveal that striatal MSNs are distinct in their expression of clustered Kv2.1 at plasma membrane sites juxtaposed to intracellular RyRs, as well as in Kv2.1 phosphorylation state. Differences in Kv2.1 expression and phosphorylation between MSNs in direct and indirect pathways provide a cell‐ and circuit‐specific mechanism for coupling intracellular Ca²⁺ release to phosphorylation‐dependent regulation of Kv2.1 to dynamically impact intrinsic excitability. J. Comp. Neurol. 522:3555–3574, 2014. © 2014 Wiley Periodicals, Inc.     

Dopamine increases NMDA‐stimulated calcium flux in striatopallidal neurons through a matrix metalloproteinase‐dependent mechanism

Dopamine (DA) is a potent neuromodulator known to influence glutamatergic transmission in striatal medium spiny neurons (MSNs). It acts on D1‐ and D2‐like DA receptors that are expressed on two distinct subpopulations. MSNs projecting to the substantia nigra express D1 receptors (D1Rs), while those projecting to the lateral globus pallidus express D2 receptors (D2Rs). D1R signalling in particular can increase excitatory transmission through varied protein kinase A‐dependent, cell‐autonomous pathways. Mechanisms by which D1R signalling could increase excitatory transmission in D2R‐bearing MSNs have been relatively less explored. Herein, the possibility is considered that D1R agonists increase levels of soluble factors that subsequently influence N‐methyl‐d ‐aspartate (NMDA)‐stimulated calcium flux in D2R neurons. This study focuses on matrix metalloproteinases (MMPs) and MMP‐generated integrin binding ligands, important soluble effectors of glutamatergic transmission that may be elevated in the setting of excess DA. It was observed that DA and a D1R agonist, SKF81297, increase MMP activity in extracts from striatal slices, as determined by cleavage of the substrate β‐dystroglycan. Using mice engineered to express the calcium indicator GCaMP3 in striatopallidal D2R‐bearing neurons, it was also observed that SKF81297 pretreatment of slices (60 min) potentiates NMDA‐stimulated calcium increases in this subpopulation. Effects are diminished by pretreatment with an antagonist of MMP activity or an inhibitor of integrin‐dependent signalling. Together, results suggest that DA signalling can increase excitatory transmission in D2R neurons through an MMP‐dependent mechanism. Future studies may be warranted to determine whether D1R‐stimulated MMP‐dependent processes contribute to behaviours in which increased activity in striatopallidal MSNs plays a role.     

Localization of the M2 muscarinic cholinergic receptor in dendrites,cholinergic terminals,and noncholinergic terminals in the rat basolateral amygdala: An ultrastructural analysis

Activation of M2 muscarinic receptors (M2Rs) in the rat anterior basolateral nucleus (BLa) is critical for the consolidation of memories of emotionally arousing events. The present investigation used immunocytochemistry at the electron microscopic level to determine which structures in the BLa express M2Rs. In addition, dual localization of M2R and the vesicular acetylcholine transporter protein (VAChT), a marker for cholinergic axons, was performed to determine whether M2R is an autoreceptor in cholinergic axons innervating the BLa. M2R immunoreactivity (M2R‐ir) was absent from the perikarya of pyramidal neurons, with the exception of the Golgi complex, but was dense in the proximal dendrites and axon initial segments emanating from these neurons. Most perikarya of nonpyramidal neurons were also M2R–negative. About 95% of dendritic shafts and 60% of dendritic spines were M2 immunoreactive (M2R⁺). Some M2R⁺ dendrites had spines, suggesting that they belonged to pyramidal cells, whereas others had morphological features typical of nonpyramidal neurons. M2R‐ir was also seen in axon terminals, most of which formed asymmetrical synapses. The main targets of M2R⁺ terminals forming asymmetrical (putative excitatory) synapses were dendritic spines, most of which were M2R⁺. The main targets of M2R⁺ terminals forming symmetrical (putative inhibitory or neuromodulatory) synapses were unlabeled perikarya and M2R⁺ dendritic shafts. M2R‐ir was also seen in VAChT⁺ cholinergic terminals, indicating a possible autoreceptor role. These findings suggest that M2R‐mediated mechanisms in the BLa are very complex, involving postsynaptic effects in dendrites as well as regulating release of glutamate, γ‐aminobutyric acid, and acetylcholine from presynaptic axon terminals. J. Comp. Neurol. 524:2400–2417, 2016. © 2016 Wiley Periodicals, Inc.     

Striatal astrocytes transdifferentiate into functional mature neurons following ischemic brain injury

To determine whether reactive astrocytes stimulated by brain injury can transdifferentiate into functional new neurons, we labeled these cells by injecting a glial fibrillary acidic protein (GFAP) targeted enhanced green fluorescence protein plasmid (pGfa2‐eGFP plasmid) into the striatum of adult rats immediately following a transient middle cerebral artery occlusion (MCAO) and performed immunolabeling with specific neuronal markers to trace the neural fates of eGFP‐expressing (GFP⁺) reactive astrocytes. The results showed that a portion of striatal GFP⁺ astrocytes could transdifferentiate into immature neurons at 1 week after MCAO and mature neurons at 2 weeks as determined by double staining GFP‐expressing cells with βIII‐tubulin (GFP⁺‐Tuj‐1⁺) and microtubule associated protein‐2 (GFP⁺‐MAP‐2⁺), respectively. GFP⁺ neurons further expressed choline acetyltransferase, glutamic acid decarboxylase, dopamine receptor D2‐like family proteins, and the N‐methyl‐d ‐aspartate receptor subunit R2, indicating that astrocyte‐derived neurons could develop into cholinergic or GABAergic neurons and express dopamine and glutamate receptors on their membranes. Electron microscopy analysis indicated that GFP⁺ neurons could form synapses with other neurons at 13 weeks after MCAO. Electrophysiological recordings revealed that action potentials and active postsynaptic currents could be recorded in the neuron‐like GFP⁺ cells but not in the astrocyte‐like GFP⁺ cells, demonstrating that new GFP⁺ neurons possessed the capacity to fire action potentials and receive synaptic inputs. These results demonstrated that striatal astrocyte‐derived new neurons participate in the rebuilding of functional neural networks, a fundamental basis for brain repair after injury. These results may lead to new therapeutic strategies for enhancing brain repair after ischemic stroke. GLIA 2015;63:1660–1670     

Effect of optogenetic manipulation of accumbal medium spiny neurons expressing dopamine D2 receptors in cocaine‐induced behavioral sensitization

Repetitive exposure to addictive drugs causes synaptic modification in the mesocorticolimbic dopamine (DA) system. Dopamine D1 receptors (D1R) or D2 receptors (D2R) expressed in the medium spiny neurons (MSNs) of the nucleus accumbens (NAc) play critical roles in the control of addictive behaviors. Optogenetic activation of D2R‐expressing MSNs (D2R‐MSNs) in the NAc previously demonstrated that these neurons play a key role in withdrawal‐induced plasticity. Here, we examined the effect of optogenetic inhibition of D2R‐MSNs in the NAc on cocaine‐induced behavioral sensitization. Adeno‐associated viral vectors encoding archaerhodopsin (ArchT) were delivered into the NAc of D2‐Cre transgenic mice. Activation of ArchT produced photoinhibition of D2R‐MSNs and caused disinhibition of neighboring MSNs in the NAc. However, such optogenetic silencing of D2R‐MSNs in the NAc in vivo affected neither the initiation nor the expression of cocaine‐induced behavioral sensitization. Similarly, photoinhibition of NAc D2R‐MSNs in the NAc during the drug withdrawal period did not affect the expression of cocaine‐induced behavioral sensitization. More detailed analysis of the effects of optogenetic activation of D2R‐MSNs suggests that D2R‐MSNs in the NAc exert important modulatory effects on neighboring MSN neurons, which may control the balanced output of NAc MSNs to control addictive behaviors.