Neuropilin 2/Plexin-A3 Receptors Regulate the Functional Connectivity and the Excitability in the Layers 4 and 5 of the Cerebral Cortex.

The functions of cortical networks are progressively established during development by series of events shaping the neuronal connectivity. Synaptic elimination, which consists of removing the supernumerary connections generated during the earlier stages of cortical development, is one of the latest stages in neuronal network maturation. The semaphorin 3F coreceptors neuropilin 2 (Nrp2) and plexin-A3 (PlxnA3) may play an important role in the functional maturation of the cerebral cortex by regulating the excess dendritic spines on cortical excitatory neurons. Yet, the identity of the connections eliminated under the control of Nrp2/PlxnA3 signaling is debated, and the importance of this synaptic refinement for cortical functions remains poorly understood. Here, we show that Nrp2/PlxnA3 controls the spine densities in layer 4 (L4) and on the apical dendrite of L5 neurons of the sensory and motor cortices. Using a combination of neuroanatomical, ex vivo electrophysiology, and in vivo functional imaging techniques in Nrp2 and PlxnA3 KO mice of both sexes, we disprove the hypothesis that Nrp2/PlxnA3 signaling is required to maintain the ectopic thalamocortical connections observed during embryonic development. We also show that the absence of Nrp2/PlxnA3 signaling leads to the hyperexcitability and excessive synchronization of the neuronal activity in L5 and L4 neuronal networks, suggesting that this system could participate in the refinement of the recurrent corticocortical connectivity in those layers. Altogether, our results argue for a role of semaphorin-Nrp2/PlxnA3 signaling in the proper maturation and functional connectivity of the cerebral cortex, likely by controlling the refinement of recurrent corticocortical connections.SIGNIFICANCE STATEMENT The function of a neuronal circuit is mainly determined by the connections that neurons establish with one another during development. Understanding the mechanisms underlying the establishment of the functional connectivity is fundamental to comprehend how network functions are implemented, and to design treatments aiming at restoring damaged neuronal circuits. Here, we show that the cell surface receptors for the family of semaphorin guidance cues neuropilin 2 (Nrp2) and plexin-A3 (PlxnA3) play an important role in shaping the functional connectivity of the cerebral cortex likely by trimming the recurrent connections in layers 4 and 5. By removing the supernumerary inputs generated during early development, Nrp2/PlxnA3 signaling reduces the neuronal excitability and participates in the maturation of the cortical network functions.

Striatal cholinergic transmission. Focus on nicotinic receptors' influence in striatal circuits.

The critical role of acetylcholine (ACh) in the basal ganglia is evident from the effect of cholinergic agents in patients suffering from several related neurological disorders, such as Parkinson's disease, Tourette syndrome, or dystonia. The striatum possesses the highest density of ACh markers in the basal ganglia underlying the importance of ACh in this structure. Striatal cholinergic interneurons (CINs) are responsible for the bulk of striatal ACh, although extrinsic cholinergic afferents from brainstem structures may also play a role. CINs are tonically active, and synchronized pause in their activity occurs following the presentation of salient stimuli during behavioral conditioning. However, the synaptic mechanisms involved are not fully understood in this physiological response. ACh modulates striatal circuits by acting on muscarinic and nicotinic receptors existing in several combinations both presynaptically and postsynaptically. While the effects of ACh in the striatum through muscarinic receptors have received particular attention, nicotinic receptors function has been less studied. Here, after briefly reviewing relevant results regarding muscarinic receptors expression and function, I will focus on striatal nicotinic receptor expressed presynaptically on glutamatergic and dopaminergic afferents and postsynaptically on diverse striatal interneurons populations. I will also review recent evidence suggesting the involvement of different GABAergic sources in two distinct nicotinic-receptor-mediated striatal circuits: the disynaptic inhibition of striatal projection neurons and the recurrent inhibition among CINs. A better understanding of striatal nicotinic receptors expression and function may help to develop targeted pharmacological interventions to treat brain disorders such as Parkinson's disease, Tourette syndrome, dystonia, or nicotine addiction.

Pedunculopontine Glutamatergic Neurons Provide a Novel Source of Feedforward Inhibition in the Striatum by Selectively Targeting Interneurons.

The main excitatory inputs to the striatum arising from the cortex and the thalamus innervate both striatal spiny projection neurons and interneurons. These glutamatergic inputs to striatal GABAergic interneurons have been suggested to regulate the spike timing of striatal projection neurons via feedforward inhibition. Understanding how different excitatory inputs are integrated within the striatal circuitry and how they regulate striatal output is crucial for understanding basal ganglia function and related behaviors. Here, using VGLUT2 mice from both sexes, we report the existence of a glutamatergic projection from the mesencephalic locomotor region to the striatum that avoids the spiny neurons and selectively innervates interneurons. Specifically, optogenetic activation of glutamatergic axons from the pedunculopontine nucleus induced monosynaptic excitation in most recorded striatal cholinergic interneurons and GABAergic fast-spiking interneurons. Optogenetic stimulation in awake head-fixed mice consistently induced an increase in the firing rate of putative cholinergic interneurons and fast-spiking interneurons. In contrast, this stimulation did not induce excitatory responses in spiny neurons but rather disynaptic inhibitory responses ex vivo and a decrease in their firing rate in vivo, suggesting a feedforward mechanism mediating the inhibition of spiny projection neurons through the selective activation of striatal interneurons. Furthermore, unilateral stimulation of pedunculopontine nucleus glutamatergic axons in the striatum induced ipsilateral head rotations consistent with the inhibition of striatal output neurons. Our results demonstrate the existence of a unique interneuron-specific midbrain glutamatergic input to the striatum that exclusively recruits feedforward inhibition mechanisms.SIGNIFICANCE STATEMENT Glutamatergic inputs to the striatum have been shown to target both striatal projection neurons and interneurons and have been proposed to regulate spike timing of the projection neurons in part through feedforward inhibition. Here, we reveal the existence of a midbrain source of glutamatergic innervation to the striatum, originating in the pedunculopontine nucleus. Remarkably, this novel input selectively targets striatal interneurons, avoiding the projection neurons. Furthermore, we show that this selective innervation of interneurons can regulate the firing of the spiny projection neurons and inhibit the striatal output via feedforward inhibition. Together, our results describe a unique source of excitatory innervation to the striatum which selectively recruits feedforward inhibition of spiny neurons without any accompanying excitation.

Neuropilin 2 Signaling Mediates Corticostriatal Transmission, Spine Maintenance, and Goal-Directed Learning in Mice.

The striatum represents the main input structure of the basal ganglia, receiving massive excitatory input from the cortex and the thalamus. The development and maintenance of cortical input to the striatum is crucial for all striatal function including many forms of sensorimotor integration, learning, and action control. The molecular mechanisms regulating the development and maintenance of corticostriatal synaptic transmission are unclear. Here we show that the guidance cue, Semaphorin 3F and its receptor Neuropilin 2 (Nrp2), influence dendritic spine maintenance, corticostriatal short-term plasticity, and learning in adult male and female mice. We found that Nrp2 is enriched in adult layer V pyramidal neurons, corticostriatal terminals, and in developing and adult striatal spiny projection neurons (SPNs). Loss of Nrp2 increases SPN excitability and spine number, reduces short-term facilitation at corticostriatal synapses, and impairs goal-directed learning in an instrumental task. Acute deletion of Nrp2 selectively in adult layer V cortical neurons produces a similar increase in the number of dendritic spines and presynaptic modifications at the corticostriatal synapse in the Nrp2-/- mouse, but does not affect the intrinsic excitability of SPNs. Furthermore, conditional loss of Nrp2 impairs sensorimotor learning on the accelerating rotarod without affecting goal-directed instrumental learning. Collectively, our results identify Nrp2 signaling as essential for the development and maintenance of the corticostriatal pathway and may shed novel insights on neurodevelopmental disorders linked to the corticostriatal pathway and Semaphorin signaling.SIGNIFICANCE STATEMENT The corticostriatal pathway controls sensorimotor, learning, and action control behaviors and its dysregulation is linked to neurodevelopmental disorders, such as autism spectrum disorder (ASD). Here we demonstrate that Neuropilin 2 (Nrp2), a receptor for the axon guidance cue semaphorin 3F, has important and previously unappreciated functions in the development and adult maintenance of dendritic spines on striatal spiny projection neurons (SPNs), corticostriatal short-term plasticity, intrinsic physiological properties of SPNs, and learning in mice. Our findings, coupled with the association of Nrp2 with ASD in human populations, suggest that Nrp2 may play an important role in ASD pathophysiology. Overall, our work demonstrates Nrp2 to be a key regulator of corticostriatal development, maintenance, and function, and may lead to better understanding of neurodevelopmental disease mechanisms.

Emergence of novel functions in striatal low-threshold spike interneurons (Commentary on Gazan et al., 2019).

LTSIs selectively target distal dendrites of spiny projection neurons (SPNs, top). In this article by Gazan et al., the authors performed a selective ablation of LTSIs using Cre-dependent diphtheria toxin in SST-Cre mice, injected in the striatum. They demonstrate that the ablation of LTSIs (bottom) induced a decrease in the number of distal dendritic spines on SPNs associated with an increased intrinsic excitability of these cells. Behaviorally, this is associated with a potentiation of cocaine effect on locomotion. Whether these behavioral effects are the direct result of the loss of LTSIs or whether it involves rearrangements in glutamatergic (Glu) or dopaminergic (DA) synapses occurring normally on these dendritic spines remains to be explored. Commentary on Gazan A, Rial D, Schiffmann SN. Ablation of striatal somatostatin interneurons affects MSN morphology and electrophysiological properties, and increases cocaine induced hyperlocomotion in mice. Eur J Neurosci. 2020;51:1388-1402. https://doi.org/10.1111/ejn.14581.

Identification and Characterization of a Novel Spontaneously Active Bursty GABAergic Interneuron in the Mouse Striatum.

The recent availability of different transgenic mouse lines coupled with other modern molecular techniques has led to the discovery of an unexpectedly large cellular diversity and synaptic specificity in striatal interneuronal circuitry. Prior research has described three spontaneously active interneuron types in mouse striatal slices: the cholinergic interneuron, the neuropeptide Y-low threshold spike interneuron, and the tyrosine hydroxylase interneurons (THINs). Using transgenic Htr3a-Cre mice, we now characterize a fourth population of spontaneously active striatal GABAergic interneurons termed spontaneously active bursty interneurons (SABIs) because of their unique burst-firing pattern in cell-attached recordings. Although they bear some qualitative similarity in intrinsic electrophysiological properties to THINs in whole-cell recordings, detailed analysis revealed significant differences in many intrinsic properties and in their morphology. Furthermore, all previously identified striatal GABAergic interneurons have been shown to innervate striatal spiny projection neurons (SPNs), contributing to the suggestion that the principal function of striatal GABAergic interneurons is to provide feedforward inhibition to SPNs. Here, very surprisingly, paired recordings show that SABIs do not innervate SPNs significantly. Further, optogenetic inhibition of striatal Htr3a-Cre interneurons triggers barrages of IPSCs in SPNs. We hypothesize that these IPSCs result from disinhibition of a population of GABAergic interneurons with activity that is constitutively suppressed by the SABIs. We suggest that the SABIs represent the first example of a striatal interneuron-selective interneuron and, further, that their existence, along with previously defined interneuronal networks, may participate in the formation of SPN ensembles observed by others.SIGNIFICANCE STATEMENT Before ∼2010, the main function of the three known subtypes of striatal GABAergic interneurons was assumed to mediate feedforward inhibition of the spiny neurons (SPNs). During the past decade, we and others have described several novel populations of striatal GABAergic interneurons and their synaptic connections and have shown that striatal interneurons and SPNs interact through extensive and highly cell-type-specific connections that form specialized networks. Here, we describe a novel population of striatal GABAergic interneuron and provide several lines of evidence suggesting that it represents the first interneuron-selective interneuron in striatum. Striatal interneurons and their synaptic connections are suggested to play an important role in the formation of ensembles of striatal SPNs interconnected by inhibitory axon collaterals.

Excitatory extrinsic afferents to striatal interneurons and interactions with striatal microcircuitry.

The striatum constitutes the main input structure of the basal ganglia and receives two major excitatory glutamatergic inputs, from the cortex and the thalamus. Excitatory cortico- and thalamostriatal connections innervate the principal neurons of the striatum, the spiny projection neurons (SPNs), which constitute the main cellular input as well as the only output of the striatum. In addition, corticostriatal and thalamostriatal inputs also innervate striatal interneurons. Some of these inputs have been very well studied, for example the thalamic innervation of cholinergic interneurons and the cortical innervation of striatal fast-spiking interneurons, but inputs to most other GABAergic interneurons remain largely unstudied, due in part to the relatively recent identification and characterization of many of these interneurons. In this review, we will discuss and reconcile some older as well as more recent data on the extrinsic excitatory inputs to striatal interneurons. We propose that the traditional feed-forward inhibitory model of the cortical input to the fast-spiking interneuron then inhibiting the SPN, often assumed to be the prototype of the main functional organization of striatal interneurons, is incomplete. We provide evidence that the extrinsic innervation of striatal interneurons is not uniform but shows great cell-type specificity. In addition, we will review data showing that striatal interneurons are themselves interconnected in a highly cell-type-specific manner. These data suggest that the impact of the extrinsic inputs on striatal activity critically depends on synaptic interactions within interneuronal circuitry.

Cortical and thalamic inputs exert cell type-specific feedforward inhibition on striatal GABAergic interneurons.

The classical view of striatal GABAergic interneuron function has been that they operate as largely independent, parallel, feedforward inhibitory elements providing inhibitory inputs to spiny projection neurons (SPNs). Much recent evidence has shown that the extrinsic innervation of striatal interneurons is not indiscriminate but rather very specific, and that striatal interneurons are themselves interconnected in a cell type-specific manner. This suggests that the ultimate effect of extrinsic inputs on striatal neuronal activity depends critically on synaptic interactions within interneuronal circuitry. Here, we compared the cortical and thalamic input to two recently described subtypes of striatal GABAergic interneurons, tyrosine hydroxylase-expressing interneurons (THINs), and spontaneously active bursty interneurons (SABIs) using transgenic TH-Cre and Htr3a-Cre mice of both sexes. Our results show that both THINs and SABIs receive strong excitatory input from the motor cortex and the thalamic parafascicular nucleus. Cortical optogenetic stimulation also evokes disynaptic inhibitory GABAergic responses in THINs but not in SABIs. In contrast, optogenetic stimulation of the parafascicular nucleus induces disynaptic inhibitory responses in both interneuron populations. However, the short-term plasticity of these disynaptic inhibitory responses is different suggesting the involvement of different intrastriatal microcircuits. Altogether, our results point to highly specific interneuronal circuits that are selectively engaged by different excitatory inputs.

Neostriatal GABAergic Interneurons Mediate Cholinergic Inhibition of Spiny Projection Neurons.

UNLABELLED: Synchronous optogenetic activation of striatal cholinergic interneurons ex vivo produces a disynaptic inhibition of spiny projection neurons composed of biophysically distinct GABAAfast and GABAAslow components. This has been shown to be due, at least in part, to activation of nicotinic receptors on GABAergic NPY-neurogliaform interneurons that monosynaptically inhibit striatal spiny projection neurons. Recently, it has been proposed that a significant proportion of this inhibition is actually mediated by activation of presynaptic nicotinic receptors on nigrostriatal terminals that evoke GABA release from the terminals of the dopaminergic nigrostriatal pathway. To disambiguate these the two mechanisms, we crossed mice in which channelrhodopsin is endogenously expressed in cholinergic neurons with Htr3a-Cre mice, in which Cre is selectively targeted to several populations of striatal GABAergic interneurons, including the striatal NPY-neurogliaform interneuron. Htr3a-Cre mice were then virally transduced to express halorhodopsin to allow activation of channelrhodopsin and halorhodopsin, individually or simultaneously. Thus we were able to optogenetically disconnect the interneuron-spiny projection neuron (SPN) cell circuit on a trial-by-trial basis. As expected, optogenetic activation of cholinergic interneurons produced inhibitory currents in SPNs. During simultaneous inhibition of GABAergic interneurons with halorhodopsin, we observed a large, sometimes near complete reduction in both fast and slow components of the cholinergic-evoked inhibition, and a delay in IPSC latency. This demonstrates that the majority of cholinergic-evoked striatal GABAergic inhibition is derived from GABAergic interneurons. These results also reinforce the notion that a semiautonomous circuit of striatal GABAergic interneurons is responsible for transmitting behaviorally relevant cholinergic signals to spiny projection neurons. SIGNIFICANCE STATEMENT: The circuitry between neurons of the striatum has been recently described to be far more complex than originally imagined. One example of this phenomenon is that striatal cholinergic interneurons have been shown to provide intrinsic nicotinic excitation of local GABAergic interneurons, which then inhibit the projection neurons of the striatum. As deficits of cholinergic interneurons are reported in patients with Tourette syndrome, the normal functions of these interneurons are of great interest. Whether this novel route of nicotinic input constitutes a major output of cholinergic interneurons remains unknown. The study addressed this question using excitatory and inhibitory optogenetic technology, so that cholinergic interneurons could be selectively activated and GABAergic interneurons selectively inhibited to determine the causal relationship in this circuit.

Novel fast adapting interneurons mediate cholinergic-induced fast GABAA inhibitory postsynaptic currents in striatal spiny neurons.

Previous work suggests that neostriatal cholinergic interneurons control the activity of several classes of GABAergic interneurons through fast nicotinic receptor-mediated synaptic inputs. Although indirect evidence has suggested the existence of several classes of interneurons controlled by this mechanism, only one such cell type, the neuropeptide-Y-expressing neurogliaform neuron, has been identified to date. Here we tested the hypothesis that in addition to the neurogliaform neurons that elicit slow GABAergic inhibitory responses, another interneuron type exists in the striatum that receives strong nicotinic cholinergic input and elicits conventional fast GABAergic synaptic responses in projection neurons. We obtained in vitro slice recordings from double transgenic mice in which Channelrhodopsin-2 was natively expressed in cholinergic neurons and a population of serotonin receptor-3a-Cre-expressing GABAergic interneurons were visualized with tdTomato. We show that among the targeted GABAergic interneurons a novel type of interneuron, termed the fast-adapting interneuron, can be identified that is distinct from previously known interneurons based on immunocytochemical and electrophysiological criteria. We show using optogenetic activation of cholinergic inputs that fast-adapting interneurons receive a powerful supra-threshold nicotinic cholinergic input in vitro. Moreover, fast adapting neurons are densely connected to projection neurons and elicit fast, GABAA receptor-mediated inhibitory postsynaptic current responses. The nicotinic receptor-mediated activation of fast-adapting interneurons may constitute an important mechanism through which cholinergic interneurons control the activity of projection neurons and perhaps the plasticity of their synaptic inputs when animals encounter reinforcing or otherwise salient stimuli.

Progressive Parkinsonism by acute dysfunction of excitatory amino acid transporters in the rat substantia nigra.

Parkinson's disease (PD) is characterized by the progressive degeneration of substantia nigra (SN) dopamine neurons, involving a multifactorial cascade of pathogenic events. Here we explored the hypothesis that dysfunction of excitatory amino acid transporters (EAATs) might be involved. Acutely-induced dysfunction of EAATs in the rat SN, by single unilateral injection of their substrate inhibitor l-trans-pyrrolidine-2,4-dicarboxylate (PDC), triggers a neurodegenerative process mimicking several PD features. Dopamine neurons are selectively affected, consistent with their sustained excitation by PDC measured by slice electrophysiology. The anti-oxidant N-acetylcysteine and the NMDA receptor antagonists ifenprodil and memantine provide neuroprotection. Besides oxidative stress and NMDA receptor-mediated excitotoxicity, glutathione depletion and neuroinflammation characterize the primary insult. Most interestingly, the degeneration progresses overtime with unilateral to bilateral and caudo-rostral evolution. Transient adaptive changes in dopamine function markers in SN and striatum accompany cell loss and axonal dystrophy, respectively. Motor deficits appear when neuron loss exceeds 50% in the most affected SN and striatal dopamine tone is dramatically reduced. These findings outline a functional link between EAAT dysfunction and several PD pathogenic mechanisms/pathological hallmarks, and provide a novel acutely-triggered model of progressive Parkinsonism.

Cholinergic control of striatal GABAergic microcircuits.

Cholinergic interneurons (CINs) are essential elements of striatal circuits and functions. Although acetylcholine signaling via muscarinic receptors (mAChRs) has been well studied, more recent data indicate that postsynaptic nicotinic receptors (nAChRs) located on striatal GABAergic interneurons (GINs) are equally critical. One example is that CIN stimulation induces large disynaptic inhibition of striatal projection neurons (SPNs) mediated by nAChR activation of GINs. Although these circuits are ideally positioned to modulate striatal output, the neurons involved are not definitively identified because of an incomplete mapping of CINs-GINs interconnections. Here, we show that CINs modulate four GINs populations via an intricate mechanism involving co-activation of presynaptic and postsynaptic mAChRs and nAChRs. Using optogenetics, we demonstrate the participation of tyrosine hydroxylase-expressing GINs in the disynaptic inhibition of SPNs via heterotypic electrical coupling with neurogliaform interneurons. Altogether, our results highlight the importance of CINs in regulating GINs microcircuits via complex synaptic/heterosynaptic mechanisms.

Heterogeneity and Diversity of Striatal GABAergic Interneurons: Update 2018.

Our original review, "Heterogeneity and Diversity of Striatal GABAergic Interneurons," to which this is an invited update, was published in December, 2010 in Frontiers is Neuroanatomy. In that article, we reviewed several decades' worth of anatomical and electrophysiological data on striatal parvalbumin (PV)-, neuropeptide Y (NPY)- and calretinin(CR)-expressing GABAergic interneurons from many laboratories including our own. In addition, we reported on a recently discovered novel tyrosine hydroxylase (TH) expressing GABAergic interneuron class first revealed in transgenic TH EGFP reporter mouse line. In this review, we report on further advances in the understanding of the functional properties of previously reported striatal GABAergic interneurons and their synaptic connections. With the application of new transgenic fluorescent reporter and Cre-driver/reporter lines, plus optogenetic, chemogenetic and viral transduction methods, several additional subtypes of novel striatal GABAergic interneurons have been discovered, as well as the synaptic networks in which they are embedded. These findings make it clear that previous hypotheses in which striatal GABAergic interneurons modulate and/or control the firing of spiny neurons principally by simple feedforward and/or feedback inhibition are at best incomplete. A more accurate picture is one in which there are highly selective and specific afferent inputs, synaptic connections between different interneuron subtypes and spiny neurons and among different GABAergic interneurons that result in the formation of functional networks and ensembles of spiny neurons.

Differential processing of thalamic information via distinct striatal interneuron circuits.

Recent discoveries of striatal GABAergic interneurons require a new conceptualization of the organization of intrastriatal circuitry and their cortical and thalamic inputs. We investigated thalamic inputs to the two populations of striatal neuropeptide Y (NPY) interneurons, plateau low threshold spike (PLTS) and NPY-neurogliaform (NGF) cells. Optogenetic activation of parafascicular inputs evokes suprathreshold monosynaptic glutamatergic excitation in NGF interneurons and a disynaptic, nicotinic excitation through cholinergic interneurons. In contrast, the predominant response of PLTS interneurons is a disynaptic inhibition dependent on thalamic activation of striatal tyrosine hydroxylase interneurons (THINs). In contrast, THINs do not innervate NGF or fast spiking interneurons, showing significant specificity in THINs outputs. Chemospecific ablation of THINs impairs prepulse inhibition of the acoustic startle response suggesting an important behavioural role of this disynaptic pathway. Our findings demonstrate that the impact of the parafascicular nucleus on striatal activity and some related behaviour critically depend on synaptic interactions within interneuronal circuits.

Segregated cholinergic transmission modulates dopamine neurons integrated in distinct functional circuits.

Dopamine neurons in the ventral tegmental area (VTA) receive cholinergic innervation from brainstem structures that are associated with either movement or reward. Whereas cholinergic neurons of the pedunculopontine nucleus (PPN) carry an associative/motor signal, those of the laterodorsal tegmental nucleus (LDT) convey limbic information. We used optogenetics and in vivo juxtacellular recording and labeling to examine the influence of brainstem cholinergic innervation of distinct neuronal subpopulations in the VTA. We found that LDT cholinergic axons selectively enhanced the bursting activity of mesolimbic dopamine neurons that were excited by aversive stimulation. In contrast, PPN cholinergic axons activated and changed the discharge properties of VTA neurons that were integrated in distinct functional circuits and were inhibited by aversive stimulation. Although both structures conveyed a reinforcing signal, they had opposite roles in locomotion. Our results demonstrate that two modes of cholinergic transmission operate in the VTA and segregate the neurons involved in different reward circuits.