Loss of Depalmitoylation Disrupts Homeostatic Plasticity of AMPARs in a Mouse Model of Infantile Neuronal Ceroid Lipofuscinosis.

Protein palmitoylation is the only reversible post-translational lipid modification. Palmitoylation is held in delicate balance by depalmitoylation to precisely regulate protein turnover. While over 20 palmitoylation enzymes are known, depalmitoylation is conducted by fewer enzymes. Of particular interest is the lack of the depalmitoylating enzyme palmitoyl-protein thioesterase 1 (PPT1) that causes the devastating pediatric neurodegenerative condition infantile neuronal ceroid lipofuscinosis (CLN1). While most of the research on Ppt1 function has centered on its role in the lysosome, recent findings demonstrated that many Ppt1 substrates are synaptic proteins, including the AMPA receptor (AMPAR) subunit GluA1. Still, the impact of Ppt1-mediated depalmitoylation on synaptic transmission and plasticity remains elusive. Thus, the goal of the present study was to use the Ppt1 -/- mouse model (both sexes) to determine whether Ppt1 regulates AMPAR-mediated synaptic transmission and plasticity, which are crucial for the maintenance of homeostatic adaptations in cortical circuits. Here, we found that basal excitatory transmission in the Ppt1 -/- visual cortex is developmentally regulated and that chemogenetic silencing of the Ppt1 -/- visual cortex excessively enhanced the synaptic expression of GluA1. Furthermore, triggering homeostatic plasticity in Ppt1 -/- primary neurons caused an exaggerated incorporation of GluA1-containing, calcium-permeable AMPARs, which correlated with increased GluA1 palmitoylation. Finally, Ca2+ imaging in awake Ppt1 -/- mice showed visual cortical neurons favor a state of synchronous firing. Collectively, our results elucidate a crucial role for Ppt1 in AMPAR trafficking and show that impeded proteostasis of palmitoylated synaptic proteins drives maladaptive homeostatic plasticity and abnormal recruitment of cortical activity in CLN1.SIGNIFICANCE STATEMENT Neuronal communication is orchestrated by the movement of receptors to and from the synaptic membrane. Protein palmitoylation is the only reversible post-translational lipid modification, a process that must be balanced precisely by depalmitoylation. The significance of depalmitoylation is evidenced by the discovery that mutation of the depalmitoylating enzyme palmitoyl-protein thioesterase 1 (Ppt1) causes severe pediatric neurodegeneration. In this study, we found that the equilibrium provided by Ppt1-mediated depalmitoylation is critical for AMPA receptor (AMPAR)-mediated plasticity and associated homeostatic adaptations of synaptic transmission in cortical circuits. This finding complements the recent explosion of palmitoylation research by emphasizing the necessity of balanced depalmitoylation.

Endothelial Cell APOE3 Regulates Neurovascular, Neuronal, and Behavioral Function.

BACKGROUND: Specialized brain endothelial cells and human APOE3 are independently important for neurovascular function, yet whether APOE3 expression by endothelial cells contributes to brain function is currently unknown. In the present study, we determined whether the loss of endothelial cell APOE3 impacts brain vascular and neural function. METHODS: We developed APOE3fl/fl/Cdh5(PAC)-CreERT2+/- (APOE3Cre+/-) and APOE3fl/fl/Cdh5(PAC)-CreERT2-/- (APOE3Cre-/-, control) mice and induced endothelial cell APOE3 knockdown with tamoxifen at ≈4 to 5 weeks of age. Neurovascular and neuronal function were evaluated by biochemistry, immunohistochemistry, behavioral testing, and electrophysiology at 9 months of age. RESULTS: We found that the loss of endothelial APOE3 expression was sufficient to cause neurovascular dysfunction including higher permeability and lower vessel coverage in tandem with deficits in spatial memory and fear memory extinction and a disruption of cortical excitatory/inhibitory balance. CONCLUSIONS: Our data collectively support the novel concept that endothelial APOE3 plays a critical role in the regulation of the neurovasculature, neural circuit function, and behavior.

Synaptic Integration of Subquantal Neurotransmission by Colocalized G Protein-Coupled Receptors in Presynaptic Terminals.

In presynaptic terminals, membrane-delimited Gi/o-mediated presynaptic inhibition is ubiquitous and acts via Gßγ to inhibit Ca2+ entry, or directly at SNARE complexes to inhibit Ca2+-dependent synaptotagmin-SNARE complex interactions. At CA1-subicular presynaptic terminals, 5-HT1B and GABAB receptors colocalize. GABAB receptors inhibit Ca2+ entry, whereas 5-HT1B receptors target SNARE complexes. We demonstrate in male and female rats that GABAB receptors alter Pr, whereas 5-HT1B receptors reduce evoked cleft glutamate concentrations, allowing differential inhibition of AMPAR and NMDAR EPSCs. This reduction in cleft glutamate concentration was confirmed by imaging glutamate release using a genetic sensor (iGluSnFR). Simulations of glutamate release and postsynaptic glutamate receptor currents were made. We tested effects of changes in vesicle numbers undergoing fusion at single synapses, relative placement of fusing vesicles and postsynaptic receptors, and the rate of release of glutamate from a fusion pore. Experimental effects of Pr changes, consistent with GABAB receptor effects, were straightforwardly represented by changes in numbers of synapses. The effects of 5-HT1B receptor-mediated inhibition are well fit by simulated modulation of the release rate of glutamate into the cleft. Colocalization of different actions of GPCRs provides synaptic integration within presynaptic terminals. Train-dependent presynaptic Ca2+ accumulation forces frequency-dependent recovery of neurotransmission during 5-HT1B receptor activation. This is consistent with competition between Ca2+-synaptotagmin and Gßγ at SNARE complexes. Thus, stimulus trains in 5-HT1B receptor agonist unveil dynamic synaptic modulation and a sophisticated hippocampal output filter that itself is modulated by colocalized GABAB receptors, which alter presynaptic Ca2+ In combination, these pathways allow complex presynaptic integration.SIGNIFICANCE STATEMENT Two G protein-coupled receptors colocalize at presynaptic sites, to mediate presynaptic modulation by Gßγ, but one (a GABAB receptor) inhibits Ca2+ entry whereas another (a 5-HT1B receptor) competes with Ca2+-synaptotagmin binding to the synaptic vesicle machinery. We have investigated downstream effects of signaling and integrative properties of these receptors. Their effects are profoundly different. GABAB receptors alter Pr leaving synaptic properties unchanged, whereas 5-HT1B receptors fundamentally change properties of synaptic transmission, modifying AMPAR but sparing NMDAR responses. Coactivation of these receptors allows synaptic integration because of convergence of GABAB receptor alteration on Ca2+ and the effect of this altered Ca2+ signal on 5-HT1B receptor signaling. This presynaptic convergence provides a novel form of synaptic integration.

MK-801 Exposure during Adolescence Elicits Enduring Disruption of Prefrontal E-I Balance and Its Control of Fear Extinction Behavior.

Understanding how disruption of prefrontal cortex (PFC) maturation during adolescence is crucial to reveal which neural processes could contribute to the onset of psychiatric disorders that display frontal cortical deficits. Of particular interest is the gain of GABAergic function in the PFC during adolescence and its susceptibility to the impact of transient blockade of NMDA receptor function. Here we assessed whether exposure to MK-801 during adolescence in male rats triggers a state of excitatory-inhibitory imbalance in the PFC that limits its functional capacity to regulate behavior in adulthood. Recordings from PFC brain slices revealed that MK-801 exposure during adolescence preferentially reduces the presynaptic functionality of GABAergic activity over that of excitatory synapses. As a result, an imbalance of excitatory-inhibitory synaptic activity emerges in the PFC that correlates linearly with the GABAergic deficit. Notably, the data also suggest that the diminished prefrontal GABAergic function could arise from a deficit in the recruitment of fast-spiking interneurons by excitatory inputs during adolescence. At the behavioral level, MK-801 exposure during adolescence did not disrupt the acquisition of trace fear conditioning, but markedly increased the level of freezing response during extinction testing. Infusion of the GABAA receptor-positive allosteric modulator Indiplon into the PFC before extinction testing reduced the level of freezing response in MK-801-treated rats to control levels. Collectively, the results indicate NMDA receptor signaling during adolescence enables the gain of prefrontal GABAergic function, which is required for maintaining proper excitatory-inhibitory balance in the PFC and its control of behavioral responses.SIGNIFICANCE STATEMENT A developmental disruption of prefrontal cortex maturation has been implicated in the pathophysiology of cognitive deficits in psychiatric disorders. Of particular interest is the susceptibility of the local GABAergic circuit to the impact of transient disruption of NMDA receptors. Here we found that NMDA receptor signaling is critical to enable the gain of prefrontal GABAergic transmission during adolescence for maintaining proper levels of excitatory-inhibitory balance in the PFC and its control of behavior.

Downregulation of parvalbumin expression in the prefrontal cortex during adolescence causes enduring prefrontal disinhibition in adulthood.

The expression of the calcium binding protein parvalbumin (PV) has been observed in several cortical regions during development in a temporal pattern consistent with increased afferent-dependent activity. In the prefrontal cortex (PFC), PV expression appears last and continues to substantially increase throughout adolescence, yet the significance of this increase remains unclear. Because of the expression of PV in fast-spiking GABAergic interneurons, we hypothesized that PV upregulation during adolescence is necessary to sustain the increase in GABAergic activity observed in the PFC during this period. To test this hypothesis, we utilized an RNAi strategy to directly downregulate PV levels in the PFC during adolescence and examined its impact on prefrontal GABAergic function, plasticity, and associated behaviors during adulthood. The data indicate that a mere 25% reduction of adult PV levels in the PFC was sufficient to reduce local GABAergic transmission onto pyramidal neurons, disrupt prefrontal excitatory-inhibitory balance, and alter processing of afferent information from the ventral hippocampus. Accordingly, these animals displayed an impairment in the level of extinction learning of a trace fear conditioning response, a behavioral paradigm that requires intact PFC-ventral hippocampus connectivity. These results indicate the PV upregulation observed in the PFC during adolescence is necessary for refinement of prefrontal GABAergic function, the absence of which results in immature afferent processing and a hypofunctional state. Importantly, these results suggest there is a critical window of plasticity during which PV upregulation supports the acquisition of mature GABAergic phenotype necessary to sustain adult PFC functions.

EGF Treatment Improves Motor Behavior and Cortical GABAergic Function in the R6/2 Mouse Model of Huntington's Disease.

Recent evidence indicates that disruption of epidermal growth factor (EGF) signaling by mutant huntingtin (polyQ-htt) may contribute to the onset of behavioral deficits observed in Huntington's disease (HD) through a variety of mechanisms, including cerebrovascular dysfunction. Yet, whether EGF signaling modulates the development of HD pathology and the associated behavioral impairments remain unclear. To gain insight on this issue, we used the R6/2 mouse model of HD to assess the impact of chronic EGF treatment on behavior, and cerebrovascular and cortical neuronal functions. We found that bi-weekly treatment with a low dose of EGF (300 µg/kg, i.p.) for 6 weeks was sufficient to effectively improve motor behavior in R6/2 mice and diminish mortality, compared to vehicle-treated littermates. These beneficial effects of EGF treatment were dissociated from changes in cerebrovascular leakiness, a result that was surprising given that EGF ameliorates this deficit in other neurodegenerative diseases. Rather, the beneficial effect of EGF on R6/2 mice behavior was concomitant with a marked amelioration of cortical GABAergic function. As GABAergic transmission in cortical circuits is disrupted in HD, these novel data suggest a potential mechanistic link between deficits in EGF signaling and GABAergic dysfunction in the progression of HD.

Preferential Disruption of Prefrontal GABAergic Function by Nanomolar Concentrations of the α7nACh Negative Modulator Kynurenic Acid.

Increased concentrations of kynurenic acid (KYNA) in the prefrontal cortex (PFC) are thought to contribute to the development of cognitive deficits observed in schizophrenia. Although this view is consistent with preclinical studies showing a negative impact of prefrontal KYNA elevation on executive function, the mechanism underlying such a disruption remains unclear. Here, we measured changes in local field potential (LFP) responses to ventral hippocampal stimulation in vivo and conducted whole-cell patch-clamp recordings in brain slices to reveal how nanomolar concentrations of KYNA alter synaptic transmission in the PFC of male adult rats. Our data show that prefrontal infusions of KYNA attenuated the inhibitory component of PFC LFP responses, a disruption that resulted from local blockade of α7-nicotinic acetylcholine receptors (α7nAChR). At the cellular level, we found that the inhibitory action exerted by KYNA in the PFC occurred primarily at local GABAergic synapses through an α7nAChR-dependent presynaptic mechanism. As a result, the excitatory-inhibitory ratio of synaptic transmission becomes imbalanced in a manner that correlates highly with the level of GABAergic suppression by KYNA. Finally, prefrontal infusion of a GABAAR positive allosteric modulator was sufficient to overcome the disrupting effect of KYNA and normalized the pattern of LFP inhibition in the PFC. Thus, the preferential inhibitory effect of KYNA on prefrontal GABAergic transmission could contribute to the onset of cognitive deficits observed in schizophrenia because proper GABAergic control of PFC output is one key mechanism for supporting such cortical functions.SIGNIFICANCE STATEMENT Brain kynurenic acid (KYNA) is an astrocyte-derived metabolite and its abnormal elevation in the prefrontal cortex (PFC) is thought to impair cognitive functions in individuals with schizophrenia. However, the mechanism underlying the disrupting effect of KYNA remains unclear. Here we found that KYNA biases the excitatory-inhibitory balance of prefrontal synaptic activity toward a state of disinhibition. Such disruption emerges as a result of a preferential suppression of local GABAergic transmission by KYNA via presynaptic inhibition of α7-nicotinic acetylcholine receptor signaling. Therefore, the degree of GABAergic dysregulation in the PFC could be a clinically relevant contributing factor for the onset of cognitive deficits resulting from abnormal increases of cortical KYNA.

Frequency-Dependent Corticostriatal Disinhibition Resulting from Chronic Dopamine Depletion: Role of Local Striatal cGMP and GABA-AR Signaling.

The onset of motor deficits in parkinsonism is thought to result from dopamine (DA) loss-induced corticostriatal disruption and the development of excessive cortico-basal ganglia synchronization. To gain insights into the mechanisms underlying such corticostriatal dysfunction, we conducted local field potential (LFP) recordings in rats and measured how striatal manipulations of DA, cyclic guanosine monophosphate (cGMP), and gamma-aminobutyric acid- A receptor (GABA-AR) signaling impact corticostriatal transmission at specific oscillatory frequencies. Results indicate that the degree of 6-hydroxydopamine-induced DA lesion and subsequent changes in striatal DA, cGMP, and GABA-AR signaling contribute to impair LFP suppression such that the DA-depleted striatum becomes more permissive to cortically driven oscillations at 10-20 Hz, and to a lesser extent, at 40 Hz. Notably, the corticostriatal dysfunction at 40 Hz emerged only when the degree of chronic DA lesion surpassed 90%, which coincides with the appearance of severe forelimb stepping deficits. Collectively, these results indicate that several mechanisms contribute to suppress LFP within the 10-20 Hz range, yet a critical level of striatal GABAergic activity is required for sustaining corticostriatal inhibition at 40 Hz. Both the degree and chronicity of DA lesion are major contributing factors to the severity of motor and striatal GABAergic deficits that could only be reversed by strengthening local GABA-AR function.

Decrease of a Current Mediated by Kv1.3 Channels Causes Striatal Cholinergic Interneuron Hyperexcitability in Experimental Parkinsonism.

The mechanism underlying a hypercholinergic state in Parkinson's disease (PD) remains uncertain. Here, we show that disruption of the Kv1 channel-mediated function causes hyperexcitability of striatal cholinergic interneurons in a mouse model of PD. Specifically, our data reveal that Kv1 channels containing Kv1.3 subunits contribute significantly to the orphan potassium current known as IsAHP in striatal cholinergic interneurons. Typically, this Kv1 current provides negative feedback to depolarization that limits burst firing and slows the tonic activity of cholinergic interneurons. However, such inhibitory control of cholinergic interneuron excitability by Kv1.3-mediated current is markedly diminished in the parkinsonian striatum, suggesting that targeting Kv1.3 subunits and their regulatory pathways may have therapeutic potential in PD therapy. These studies reveal unexpected roles of Kv1.3 subunit-containing channels in the regulation of firing patterns of striatal cholinergic interneurons, which were thought to be largely dependent on KCa channels.

Synaptic depression via mGluR1 positive allosteric modulation suppresses cue-induced cocaine craving.

Cue-induced cocaine craving is a major cause of relapse in abstinent addicts. In rats, cue-induced craving progressively intensifies (incubates) during withdrawal from extended-access cocaine self-administration. After ~1 month of withdrawal, incubated craving is mediated by Ca(2+)-permeable AMPA receptors (CP-AMPARs) that accumulate in the nucleus accumbens (NAc). We found that decreased mGluR1 surface expression in the NAc preceded and enabled CP-AMPAR accumulation. Thus, restoring mGluR1 transmission by administering repeated injections of an mGluR1 positive allosteric modulator (PAM) prevented CP-AMPAR accumulation and incubation, whereas blocking mGluR1 transmission at even earlier withdrawal times accelerated CP-AMPAR accumulation. In studies conducted after prolonged withdrawal, when CP-AMPAR levels and cue-induced craving are high, we found that systemic administration of an mGluR1 PAM attenuated the expression of incubated craving by reducing CP-AMPAR transmission in the NAc to control levels. These results suggest a strategy in which recovering addicts could use a systemically active compound to protect against cue-induced relapse.

Emergence of GABAergic-dependent regulation of input-specific plasticity in the adult rat prefrontal cortex during adolescence.

OBJECTIVE: The prefrontal cortex (PFC) receives multiple cortical and subcortical afferents that regulate higher order cognitive functions, many of which emerge late in adolescence. However, it remains unclear how these afferents influence PFC processing, especially in light of the protracted, late adolescent maturation of prefrontal GABAergic function. Here we investigated the role of PFC GABAergic transmission in regulating plasticity elicited from the ventral hippocampus and basolateral amygdala, and how such modulation undergoes functional changes during adolescence in rats. METHODS: In vivo local field potential recordings, combined with prefrontal microinfusion of the GABA-A receptor antagonist picrotoxin, were employed to study the impact of ventral hippocampal and basolateral amygdala high-frequency stimulation on PFC plasticity. RESULTS: Ventral hippocampal-induced PFC plasticity begins to appear only by postnatal days (P) 45-55 with a transient suppression of the evoked response. A switch from transient to long-lasting depression (LTD) of the PFC response emerges after P55 and throughout adulthood (P65-120). Recordings conducted in the presence of picrotoxin revealed that PFC GABAergic transmission is critical for the expression of LTD. In contrast, basolateral amygdala stimulation resulted in PFC long-term potentiation, a form of plasticity that is already enabled by P30 and is insensitive to picrotoxin. CONCLUSIONS: The development of ventral hippocampal-dependent PFC LTD is contingent upon the recruitment of local prefrontal GABAergic transmission during adolescence whereas plasticity elicited from the basolateral amygdala is not. Thus, different mechanisms contribute to the refinement of prefrontal plasticity during adolescence as inputs from these two regions are critical for shaping PFC functions.

Differential regulation of parvalbumin and calretinin interneurons in the prefrontal cortex during adolescence.

Determining the normal developmental trajectory of individual GABAergic components in the prefrontal cortex (PFC) during the adolescent transition period is critical because local GABAergic interneurons are thought to play an important role in the functional maturation of cognitive control that occurs in this developmental window. Based on the expression of calcium-binding proteins, three distinctive subtypes of interneurons have been identified in the PFC: parvalbumin (PV)-, calretinin (CR)-, and calbindin (CB)-positive cells. Using biochemical and histochemical measures, we found that the protein level of PV is lowest in juveniles [postnatal days (PD) 25-35] and increases during adolescence (PD 45-55) to levels similar to those observed in adulthood (PD 65-75). In contrast, the protein expression of CR is reduced in adults compared to juvenile and adolescent animals, whereas CB levels remain mostly unchanged across the developmental window studied here. Semi-quantitative immunostaining analyses revealed that the periadolescent upregulation of PV and the loss of the CR signal appear to be attributable to changes in PV- and CR-positive innervation, which are dissociable from the trajectory of PV- and CR-positive cell number. At the synaptic level, our electrophysiological data revealed that a developmental facilitation of spontaneous glutamatergic synaptic inputs onto PV-positive/fast-spiking interneurons parallels the increase in prefrontal PV signal during the periadolescent transition. In contrast, no age-dependent changes in glutamatergic transmission were observed in PV-negative/non fast-spiking interneurons. Together, these findings emphasize that GABAergic inhibitory interneurons in the PFC undergo a dynamic, cell type-specific remodeling during adolescence and provide a developmental framework for understanding alterations in GABAergic circuits that occur in psychiatric disorders.

Late adolescent expression of GluN2B transmission in the prefrontal cortex is input-specific and requires postsynaptic protein kinase A and D1 dopamine receptor signaling.

BACKGROUND: Refinement of mature cognitive functions, such as working memory and decision making, typically takes place during adolescence. The acquisition of these functions is linked to the protracted development of the prefrontal cortex (PFC) and dopamine facilitation of glutamatergic transmission. However, the mechanisms that support these changes during adolescence remain elusive. METHODS: Electrophysiological recordings (in vitro and in vivo) combined with pharmacologic manipulations were employed to determine how N-methyl-D-aspartate transmission in the medial PFC changes during the adolescent transition to adulthood. The relative contribution of GluN2B transmission and its modulation by postsynaptic protein kinase A and D1 receptor signaling were determined in two distinct age groups of rats: postnatal day (P)25 to P40 and P50 to P80. RESULTS: We found that only N-methyl-D-aspartate receptor transmission onto the apical dendrite of layer V pyramidal neurons undergoes late adolescent remodeling due to a functional emergence of GluN2B function after P40. Both protein kinase A and dopamine D1 receptor signaling are required for the functional expression of GluN2B transmission and to sustain PFC plasticity in response to ventral hippocampal, but not basolateral amygdala, inputs. CONCLUSIONS: Thus, the late adolescent acquisition of GluN2B function provides a mechanism for dopamine D1-mediated regulation of PFC responses in an input-specific manner.

Duration differences of corticostriatal responses in striatal projection neurons depend on calcium activated potassium currents.

The firing of striatal projection neurons (SPNs) exhibits afterhyperpolarizing potentials (AHPs) that determine discharge frequency. They are in part generated by Ca(2+)-activated K(+)-currents involving BK and SK components. It has previously been shown that suprathreshold corticostriatal responses are more prolonged and evoke more action potentials in direct pathway SPNs (dSPNs) than in indirect pathway SPNs (iSPNs). In contrast, iSPNs generate dendritic autoregenerative responses. Using whole cell recordings in brain slices, we asked whether the participation of Ca(2+)-activated K(+)-currents plays a role in these responses. Secondly, we asked if these currents may explain some differences in synaptic integration between dSPNs and iSPNs. Neurons obtained from BAC D1 and D2 GFP mice were recorded. We used charybdotoxin and apamin to block BK and SK channels, respectively. Both antagonists increased the depolarization and delayed the repolarization of suprathreshold corticostriatal responses in both neuron classes. We also used NS 1619 and NS 309 (CyPPA), to enhance BK and SK channels, respectively. Current enhancers hyperpolarized and accelerated the repolarization of corticostriatal responses in both neuron classes. Nevertheless, these drugs made evident that the contribution of Ca(2+)-activated K(+)-currents was different in dSPNs as compared to iSPNs: in dSPNs their activation was slower as though calcium took a diffusion delay to activate them. In contrast, their activation was fast and then sustained in iSPNs as though calcium flux activates them at the moment of entry. The blockade of Ca(2+)-activated K(+)-currents made iSPNs to look as dSPNs. Conversely, their enhancement made dSPNs to look as iSPNs. It is concluded that Ca(2+)-activated K(+)-currents are a main intrinsic determinant causing the differences in synaptic integration between corticostriatal polysynaptic responses between dSPNs and iSPNs.

Contribution of different classes of glutamate receptors in the corticostriatal polysynaptic responses from striatal direct and indirect projection neurons.

BACKGROUND: Previous work showed differences in the polysynaptic activation of GABAergic synapses during corticostriatal suprathreshold responses in direct and indirect striatal projection neurons (dSPNs and iSPNs). Here, we now show differences and similarities in the polysynaptic activation of cortical glutamatergic synapses on the same responses. Corticostriatal contacts have been extensively studied. However, several questions remain unanswered, e.g.: what are the differences and similarities in the responses to glutamate in dSPNs and iSPNs? Does glutamatergic synaptic activation exhibits a distribution of latencies over time in vitro? That would be a strong suggestion of polysynaptic cortical convergence. What is the role of kainate receptors in corticostriatal transmission? Current-clamp recordings were used to answer these questions. One hypothesis was: if prolonged synaptic activation distributed along time was present, then it would be mainly generated from the cortex, and not from the striatum. RESULTS: By isolating responses from AMPA-receptors out of the complex suprathreshold response of SPNs, it is shown that a single cortical stimulus induces early and late synaptic activation lasting hundreds of milliseconds. Prolonged responses depended on cortical stimulation because they could not be elicited using intrastriatal stimulation, even if GABAergic transmission was blocked. Thus, the results are not explained by differences in evoked inhibition. Moreover, inhibitory participation was larger after cortical than after intrastriatal stimulation. A strong activation of interneurons was obtained from the cortex, demonstrating that polysynaptic activation includes the striatum. Prolonged kainate (KA) receptor responses were also elicited from the cortex. Responses of dSPNs and iSPNs did not depend on the cortical area stimulated. In contrast to AMPA-receptors, responses from NMDA- and KA-receptors do not exhibit early and late responses, but generate slow responses that contribute to plateau depolarizations. CONCLUSIONS: As it has been established in previous physiological studies in vivo, synaptic invasion over different latencies, spanning hundreds of milliseconds after a single stimulus strongly indicates convergent polysynaptic activation. Interconnected cortical neurons converging on the same SPNs may explain prolonged corticostriatal responses. Glutamate receptors participation in these responses is described as well as differences and similarities between dSPNs and iSPNs.

Dopaminergic modulation of corticostriatal responses in medium spiny projection neurons from direct and indirect pathways.

Suprathreshold corticostriatal responses recorded from medium spiny neurons (MSNs) from the direct and indirect pathways of the basal ganglia are different. Their differences readily distinguish D(1)- and D(2)-type receptor expressing MSNs in both bacterial artificial chromosome-transgenic mice and their control littermates as well as in rats: indirect pathway neurons are more excitable than direct pathway neurons revealing autoregenerative spikes underlying their spike trains, whereas direct pathway neurons exhibit more prolonged plateau potentials and spike trains. SFK 81297, a selective agonist for D(1)-class receptors enhanced corticostriatal responses in direct pathway neurons, while quinelorane, a selective agonist for D(2)-class receptors reduced orthodromic and autoregenerative responses in indirect pathway neurons thus making both neuron classes similarly excitable. Because dopaminergic postsynaptic actions target Ca(V)1 (L) class voltage-gated calcium channels in MSNs, we hypothesized that these channels are involved and can explain a part of the dopaminergic actions on corticostriatal integration. Both 2.5 µM nicardipine and 400 nM calciseptine, selective Ca(V)1 channel blockers, reduced corticostriatal responses in both D(1)- and D(2)-receptor expressing neurons, respectively. A previous blockade of Ca(V)1 channels occluded the actions of dopamine agonists in both neuronal classes. In contrast, a Ca(V)1 (L) channel activator, 2.5 µM Bay K 8644, enhanced corticostriatal responses in neurons from both pathways. It is concluded that Ca(V)1 intrinsic currents mediate a part of the dopaminergic modulation during orthodromic synaptic integration of cortical inputs in both classes of MSNs.

Different corticostriatal integration in spiny projection neurons from direct and indirect pathways.

The striatum is the principal input structure of the basal ganglia. Major glutamatergic afferents to the striatum come from the cerebral cortex and make monosynaptic contacts with medium spiny projection neurons (MSNs) and interneurons. Also: glutamatergic afferents to the striatum come from the thalamus. Despite differences in axonal projections, dopamine (DA) receptors expression and differences in excitability between MSNs from "direct" and "indirect" basal ganglia pathways, these neuronal classes have been thought as electrophysiologically very similar. Based on work with bacterial artificial chromosome (BAC) transgenic mice, here it is shown that corticostriatal responses in D(1)- and D(2)-receptor expressing MSNs (D(1)- and D(2)-MSNs) are radically different so as to establish an electrophysiological footprint that readily differentiates between them. Experiments in BAC mice allowed us to predict, with high probability (P > 0.9), in rats or non-BAC mice, whether a recorded neuron, from rat or mouse, was going to be substance P or enkephalin (ENK) immunoreactive. Responses are more prolonged and evoke more action potentials in D(1)-MSNs, while they are briefer and exhibit intrinsic autoregenerative responses in D(2)-MSNs. A main cause for these differences was the interaction of intrinsic properties with the inhibitory contribution in each response. Inhibition always depressed corticostriatal depolarization in D(2)-MSNs, while it helped in sustaining prolonged depolarizations in D(1)-MSNs, in spite of depressing early discharge. Corticostriatal responses changed dramatically after striatal DA depletion in 6-hydroxy-dopamine (6-OHDA) lesioned animals: a response reduction was seen in substance P (SP)+ MSNs whereas an enhanced response was seen in ENK+ MSNs. The end result was that differences in the responses were greatly diminished after DA depletion.

Dopaminergic modulation of spiny neurons in the turtle striatum.

Intracellular recordings were obtained from brain slice preparation in neurons of the striatum of the turtle Trachemys scripta elegans, analogous to the mammalian striatum in its topographic organization, synaptic connectivity, cytoarchitecture, and neurochemistry. Here we show that these similarities extend to the electrophysiological properties of its neurons. Biocytin staining revealed that 85% of the recorded neurons were medium spiny neurons while 15% were aspiny neurons. Spiny neurons of the turtle resembled those found in the mammalian and avian striatum and express dopaminergic D(1) and D(2) class receptors. Because the striatum of the turtle receives a dense dopaminergic innervation from tegmental dopaminergic neurons we investigated the postsynaptic actions of selective dopamine receptor agonists in the excitability of spiny neurons. As in mammals and birds, activation of D(1)-receptors enhances, whereas activation of D(2)-receptors decreases the evoked discharge. Apparently, actions of dopamine agonists occur via the modulation of L-type (Ca(V)1) Ca2+-conductances. Strong cellular evidence suggests that the role of dopamine in the modulation of motor networks is preserved along vertebrate evolution.

Inhibitory contribution to suprathreshold corticostriatal responses: an experimental and modeling study.

Neostriatal neurons may undergo events of spontaneous synchronization as those observed in recurrent networks of excitatory neurons, even when cortical afferents are transected. It is necessary to explain these events because the neostriatum is a recurrent network of inhibitory neurons. Synchronization of neuronal activity may be caused by plateau-like depolarizations. Plateau-like orthodromic depolarizations that resemble up-states in medium spiny neostriatal neurons (MSNs) may be induced by a single corticostriatal suprathreshold stimulus. Slow synaptic depolarizations may last hundreds of milliseconds, decay slower than the monosynaptic glutamatergic synaptic potentials that induce them, and sustain repetitive firing. Because inhibitory inputs impinging onto MSNs have a reversal potential above the resting membrane potential but below the threshold for firing, they conform a type of "shunting inhibition". This work asks if shunting GABAergic inputs onto MSNs arrive asynchronously enough as to help in sustaining the plateau-like corticostriatal response after a single cortical stimulus. This may help to begin explaining autonomous processing in the striatal micro-circuitry in the presence of a tonic excitatory drive and independently of spatio-temporally organized inputs. It is shown here that besides synaptic currents from AMPA/KA- and NMDA-receptors, as well as L-type intrinsic Ca(2+)- currents, inhibitory synapses help in maintaining the slow depolarization, although they accomplish the role of depressing firing at the beginning of the response. We then used a NEURON model of spiny cells to show that inhibitory synapses arriving asynchronously on the dendrites can help to simulate a plateau potential similar to that observed experimentally.