High-frequency head impact causes chronic synaptic adaptation and long-term cognitive impairment in mice.

Repeated head impact exposure can cause memory and behavioral impairments. Here, we report that exposure to non-damaging, but high frequency, head impacts can alter brain function in mice through synaptic adaptation. High frequency head impact mice develop chronic cognitive impairments in the absence of traditional brain trauma pathology, and transcriptomic profiling of mouse and human chronic traumatic encephalopathy brain reveal that synapses are strongly affected by head impact. Electrophysiological analysis shows that high frequency head impacts cause chronic modification of the AMPA/NMDA ratio in neurons that underlie the changes to cognition. To demonstrate that synaptic adaptation is caused by head impact-induced glutamate release, we pretreated mice with memantine prior to head impact. Memantine prevents the development of the key transcriptomic and electrophysiological signatures of high frequency head impact, and averts cognitive dysfunction. These data reveal synapses as a target of high frequency head impact in human and mouse brain, and that this physiological adaptation in response to head impact is sufficient to induce chronic cognitive impairment in mice.

Stress increases GABAergic neurotransmission in CRF neurons of the central amygdala and bed nucleus stria terminalis.

Corticotrophin Releasing Factor (CRF) is a critical stress-related neuropeptide in major output pathways of the amygdala, including the central nucleus (CeA), and in a key projection target of the CeA, the bed nucleus of the stria terminalis (BnST). While progress has been made in understanding the contributions and characteristics of CRF as a neuropeptide in rodent behavior, little attention has been committed to determine the properties and synaptic physiology of specific populations of CRF-expressing (CRF(+)) and non-expressing (CRF(-)) neurons in the CeA and BnST. Here, we fill this gap by electrophysiologically characterizing distinct neuronal subtypes in CeA and BnST. Crossing tdTomato or channelrhodopsin-2 (ChR2-YFP) reporter mice to those expressing Cre-recombinase under the CRF promoter allowed us to identify and manipulate CRF(+) and CRF(-) neurons in CeA and BnST, the two largest areas with fluorescently labeled neurons in these mice. We optogenetically activated CRF(+) neurons to elicit action potentials or synaptic responses in CRF(+) and CRF(-) neurons. We found that GABA is the predominant co-transmitter in CRF(+) neurons within the CeA and BnST. CRF(+) neurons are highly interconnected with CRF(-) neurons and to a lesser extent with CRF(+) neurons. CRF(+) and CRF(-) neurons differentially express tonic GABA currents. Chronic, unpredictable stress increase the amplitude of evoked IPSCs and connectivity between CRF(+) neurons, but not between CRF(+) and CRF(-) neurons in both regions. We propose that reciprocal inhibition of interconnected neurons controls CRF(+) output in these nuclei.

Evidence for glycinergic GluN1/GluN3 NMDA receptors in hippocampal metaplasticity.

Hebbian, or associative, forms of synaptic plasticity are considered the molecular basis of learning and memory. However, associative synaptic modifications, including long-term potentiation (LTP) and depression (LTD), can form positive feedback loops which must be constrained for neural networks to remain stable. One proposed constraint mechanism is metaplasticity, a process whereby synaptic changes shift the threshold for subsequent plasticity. Metaplasticity has been functionally observed but the molecular basis is not well understood. Here, we report that stimulation which induces LTP recruits GluN2B-lacking GluN1/GluN3 NMDA receptors (NMDARs) to excitatory synapses of hippocampal pyramidal neurons. These unconventional receptors may compete against conventional GluN1/GluN2 NMDARs to favor synaptic depotentiation in response to subsequent "LTP-inducing" stimulation. These results implicate glycinergic GluN1/GluN3 NMDAR as molecular brakes on excessive synaptic strengthening, suggesting a role for these receptors in the brain that has previously been elusive.

Utilizing GCaMP transgenic mice to monitor endogenous Gq/11-coupled receptors.

The family of GCaMPs are engineered proteins that contain Ca(2+) binding motifs within a circularly permutated variant of the Aequorea Victoria green fluorescent protein (cp-GFP). The rapidly advancing field of utilizing GCaMP reporter constructs represents a major step forward in our ability to monitor intracellular Ca(2+) dynamics. With the use of these genetically encoded Ca(2+) sensors, investigators have studied activation of endogenous Gq types of G protein-coupled receptors (GPCRs) and subsequent rises in intracellular calcium. Escalations in intracellular Ca(2+) from GPCR activation can be faithfully monitored in space and time as an increase in fluorescent emission from these proteins. Further, transgenic mice are now commercially available that express GCaMPs in a Cre recombinase dependent fashion. These GCaMP reporter mice can be bred to distinct Cre recombinase driver mice to direct expression of this sensor in unique populations of cells. Concerning the central nervous system (CNS), sources of calcium influx, including those arising from Gq activation can be observed in targeted cell types like neurons or astrocytes. This powerful genetic method allows simultaneous monitoring of the activity of dozens of cells upon activation of endogenous Gq-coupled GPCRs. Therefore, in combination with pharmacological tools, this strategy of monitoring GPCR activation is amenable to analysis of orthosteric and allosteric ligands of Gq-coupled receptors in their endogenous environments.

Contrasting actions of group I metabotropic glutamate receptors in distinct mouse striatal neurones.

In mouse striatum, metabotropic glutamate receptor (mGluR) activation leads to several modulatory effects in synaptic transmission. These effects range from dampening of glutamate release from excitatory terminals to depolarization of divergent classes of interneurones. We compared the action of group I mGluR activation on several populations of striatal neurones using a combination of genetic identification, electrophysiology, and Ca(2+) imaging techniques. Patch-clamp recordings from spiny projection neurones (SPNs) and various interneurone populations demonstrated that the group I mGluR agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) robustly depolarizes several interneurone classes that form GABAergic synapses onto SPNs. We further utilized the genetic reporter mouse strain Ai38, which expresses the calcium indicator protein GCaMP3 in a Cre-dependent manner. Breeding Ai38 mice with various neurone selective, promoter-driven Cre recombinase mice resulted in GCaMP3 expression in defined cell populations in striatum. Consistent with our electrophysiological findings, group I agonist applications increased intracellular levels of calcium ([Ca(2+)]i) in all interneurone populations tested. We also found that acute DHPG application evoked a transient, rapid increase in [Ca(2+)]i from only a small percentage of identifiable SPNs. Surprisingly, this fast [Ca(2+)]i response exhibited a robust enhancement or sensitization, in a calcium-dependent fashion. Following several procedures to increase [Ca(2+)]i, the vast majority of SPNs responded with rapid changes in [Ca(2+)]i to mGluR agonists in a time-dependent fashion. These findings extend our understanding on group I mGluR influence of striatal output via powerful, local GABAergic connections in addition to [Ca(2+)]i dynamics that impact on activity or spike-timing-dependent forms of synaptic plasticity.

Distinct roles of synaptic and extrasynaptic GABAAreceptors in striatal inhibition dynamics.

Striatonigral and striatopallidal projecting medium spiny neurons (MSNs) express dopamine D1 (D1+) and D2 receptors (D2+), respectively. Both classes receive extensive GABAergic input via expression of synaptic, perisynaptic, and extrasynaptic GABAA receptors. The activation patterns of different presynaptic GABAergic neurons produce transient and sustained GABAA receptor-mediated conductance that fulfill distinct physiological roles. We performed single and dual whole cell recordings from striatal neurons in mice expressing fluorescent proteins in interneurons and MSNs. We report specific inhibitory dynamics produced by distinct activation patterns of presynaptic GABAergic neurons as source of synaptic, perisynaptic, and extrasynaptic inhibition. Synaptic GABAA receptors in MSNs contain the α2, γ2, and a ß subunit. In addition, there is evidence for the developmental increase of the α1 subunit that contributes to faster inhibitory post-synaptic current (IPSC). Tonic GABAergic currents in MSNs from adult mice are carried by extrasynaptic receptors containing the α4 and δ subunit, while in younger mice this current is mediated by receptors that contain the α5 subunit. Both forms of tonic currents are differentially expressed in D1+ and D2+ MSNs. This study extends these findings by relating presynaptic activation with pharmacological analysis of inhibitory conductance in mice where the ß3 subunit is conditionally removed in fluorescently labeled D2+ MSNs and in mice with global deletion of the δ subunit. Our results show that responses to low doses of gaboxadol (2 µM), a GABAA receptor agonist with preference to δ subunit, are abolished in the δ but not the ß3 subunit knock out mice. This suggests that the ß3 subunit is not a component of the adult extrasynaptic receptor pool, in contrast to what has been shown for tonic current in young mice. Deletion of the ß3 subunit from D2+ MSNs however, removed slow spontaneous IPSCs, implicating its role in mediating synaptic input from striatal neurogliaform interneurons.

Dopamine D2 receptors regulate collateral inhibition between striatal medium spiny neurons.

The principle neurons of the striatum are GABAergic medium spiny neurons (MSNs), whose collateral synapses onto neighboring neurons play critical roles in striatal function. MSNs can be divided by dopamine receptor expression into D1-class and D2-class MSNs, and alterations in D2 MSNs are associated with various pathological states. Despite overwhelming evidence for D2 receptors (D2Rs) in maintaining proper striatal function, it remains unclear how MSN collaterals are specifically altered by D2R activation. Here, we report that chronic D2R stimulation regulates MSN collaterals in vitro by presynaptic and postsynaptic mechanisms. We used corticostriatal cultures from mice in which MSN subtypes were distinguished by fluorophore expression. Quinpirole, an agonist for D2/3 receptors, was used to chronically activate D2Rs. Quinpirole increased the rate and strength of collateral formation onto D2R-containing MSNs as measured by dual whole-cell patch-clamp recordings. Additionally, these neurons were more sensitive to low concentrations of GABA and exhibited an increase in gephyrin puncta density, suggesting increased postsynaptic GABAA receptors. Last, quinpirole treatment increased presynaptic GABA release sites, as shown by increased frequency of sIPSCs and mIPSCs, correlating with increased VGAT (vesicular GABA transporter) puncta. Combined with the observation that there were no detectable differences in sensitivity to specific GABAA receptor modulators, we provide evidence that D2R activation powerfully transforms MSN collaterals via coordinated presynaptic and postsynaptic alterations. As the D2 class of MSNs is highly implicated in Parkinson's disease and other neurological disorders, our findings may contribute to understanding and treating the changes that occur in these pathological states.

Distinct roles for somatically and dendritically synthesized brain-derived neurotrophic factor in morphogenesis of dendritic spines.

Dendritic spines undergo the processes of formation, maturation, and pruning during development. Molecular mechanisms controlling spine maturation and pruning remain largely unknown. The gene for brain-derived neurotrophic factor (BDNF) produces two pools of mRNA, with either a short or long 3' untranslated region (3' UTR). Our previous results show that short 3' UTR Bdnf mRNA is restricted to cell bodies, whereas long 3' UTR Bdnf mRNA is also trafficked to dendrites for local translation. Mutant mice lacking long 3' UTR Bdnf mRNA display normal spines at 3 weeks of age, but thinner and denser spines in adults compared to wild-type littermates. These observations suggest that BDNF translated from long 3' UTR Bdnf mRNA, likely in dendrites, is required for spine maturation and pruning. In this study, using rat hippocampal neuronal cultures, we found that knocking down long 3' UTR Bdnf mRNA blocked spine head enlargement and spine elimination, whereas overexpressing long 3' UTR Bdnf mRNA had the opposite effect. The effect of long 3' UTR Bdnf mRNA on spine head enlargement and spine elimination was diminished by a human single-nucleotide polymorphism (SNP, rs712442) in its 3' UTR that inhibited dendritic localization of Bdnf mRNA. Furthermore, we found that overexpression of either Bdnf mRNA increased spine density at earlier time points. Spine morphological alterations were associated with corresponding changes in density, size, and function of synapses. These results indicate that somatically synthesized BDNF promotes spine formation, whereas dendritically synthesized BDNF is a key regulator of spine head growth and spine pruning.

Direct and GABA-mediated indirect effects of nicotinic ACh receptor agonists on striatal neurones.

Choline acetyltransferase-expressing interneurones (ChAT)(+) of the striatum influence the activity of medium spiny projecting neurones (MSNs) and striatal output via a disynaptic mechanism that involves GABAergic neurotransmission. Using transgenic mice that allow visual identification of MSNs and distinct populations of GABAergic interneurones expressing neuropeptide Y (NPY)(+), parvalbumin (PV)(+) and tyrosine hydroxylase (TH)(+), we further elucidate this mechanism by studying nicotinic ACh receptor (nAChR)-mediated responses. First, we determined whether striatal neurones exhibit pharmacologically induced nicotinic responses by performing patch-clamp recordings. With high [Cl(-)](i), our results showed increased spontaneous IPSC frequency and amplitude in MSNs as well as in the majority of interneurones. However, direct nAChR-mediated activity was observed in interneurones but not MSNs. In recordings with physiological [Cl(-)](i), these responses manifested as inward currents in the presence of tetrodotoxin and bicuculline methobromide. Nicotinic responses in MSNs were primarily mediated through GABA(A) receptors in feedforward inhibition. To identify the GABAergic interneurones that mediate the response, we performed dual recordings from GABAergic interneurones and MSNs. Both TH(+) and neurogliaform subtypes of NPY(+) (NPY(+) NGF) interneurones form synaptic connections with MSNs, although the strength of connectivity, response kinetics and pharmacology differ between and within the two populations. Importantly, both cell types appear to contribute to nAChR-mediated GABAergic responses in MSNs. Our data offer insight into the striatal network activity under cholinergic control, and suggest that subclasses of recently identified TH(+) and NPY(+) interneurones are key mediators of striatal nicotinic responses via GABAergic tonic and phasic currents.

Excitatory and inhibitory synapses in neuropeptide Y-expressing striatal interneurons.

Although rare, interneurons are pivotal in governing striatal output by extensive axonal arborizations synapsing on medium spiny neurons. Using a genetically modified mouse strain in which a green fluorescent protein (GFP) is driven to be expressed under control of the neuropeptide Y (NPY) promoter, we identified NPY interneurons and compared them with striatal principal neurons. We found that the bacteria artificial chromosome (BAC)-npy mouse expresses GFP with high fidelity in the striatum to the endogenous expression of NPY. Patch-clamp analysis from NPY neurons showed a heterogeneous population of striatal interneurons. In the majority of cells, we observed spontaneous firing of action potentials in extracellular recordings. On membrane rupture, most NPY interneurons could be classified as low-threshold spiking interneurons and had high-input resistance. Voltage-clamp recordings showed that both GABA and glutamate gated ion channels mediate synaptic inputs onto these striatal interneurons. AMPA receptor-mediated spontaneous excitatory postsynaptic currents (sEPSCs) were small in amplitude and infrequent in NPY neurons. Evoked EPSCs did not show short-term plasticity but some rectification. Evoked N-methyl-d-aspartate (NMDA) EPSCs had fast decay kinetics and were poorly sensitive to an NR2B subunit containing NMDA receptor blocker. Spontaneous inhibitory postsynaptic currents (sIPSCs) were mediated by GABA(A) receptors and were quite similar among all striatal neurons studied. On the contrary, evoked IPSCs decayed faster in NPY neurons than in other striatal neurons. These data report for the first time specific properties of synaptic transmission to NPY striatal interneurons.

Long-lasting NMDA receptor-mediated EPSCs in mouse striatal medium spiny neurons.

Excitatory postsynaptic currents (EPSCs) from dorsolateral medium spiny neurons (MSNs) were recorded in cortico-striatal slice preparations from postnatal day 6-8 (P6-8) and >P12 wild-type mice and mice that were lacking either the NR2A or the NR2C subunit of the N-methyl-D-aspartate (NMDA) receptor. EPSCs were elicited by stimulation of the excitatory afferents and the NMDA and non-NMDA receptor-mediated components were pharmacologically isolated. The ratio of these components decreased with development and was significantly reduced only between age-matched +/+ and NR2A -/- neurons. In many MSNs, the NMDA-EPSC decay was characterized by the presence of a slow exponential component with a time constant lasting >1 s regardless of genotype or age. In the NR2A -/-, no developmental increase in the decay time (Tw) of the NMDA-EPSCs was observed although it was almost twofold longer than in +/+ MSNs. NR1/NR2B antagonists were ineffective in reducing the slow NMDA-EPSCs at all ages. Input-output studies revealed differences in stimulation threshold sensitivity of MSNs based on stimulus location. High-threshold responders were preferentially identified with stimulation from intracortical locations that produced considerably faster NMDA-EPSCs, whereas low-threshold responders were mainly elicited with stimulation more proximal to the striatum and exhibited slower NMDA-EPSCs. A low-affinity competitive antagonist of NMDA receptors failed to alter the decay of NMDA-EPSCs elicited from either location, suggesting that glutamate spillover is not responsible for the long-lasting NMDA-EPSCs. Our data are consistent with the expression of a unique NMDA receptor complex in MSNs with very slow deactivation kinetics.

Phosducin and phosducin-like protein attenuate G-protein-coupled receptor-mediated inhibition of voltage-gated calcium channels in rat sympathetic neurons.

Phosducin (PDC) has been shown in structural and biochemical experiments to bind the Gbetagamma subunit of heterotrimeric G-proteins. A proposed function of PDC and phosducin-like protein (PDCL) is the sequestration of "free" Gbetagamma from the plasma membrane, thereby terminating signaling by Gbetagamma. The functional impact of heterologously expressed PDC and PDCL on N-type calcium channel (CaV2.2) modulation was examined in sympathetic neurons, isolated from rat superior cervical ganglia, using whole-cell voltage clamp. Expression of PDC and PDCL attenuated voltage-dependent inhibition of N-type calcium channels, a Gbetagamma-dependent process, in a time-dependent fashion. Calcium current inhibition after short-term exposure to norepinephrine was minimally altered by PDC or PDCL expression. However, in the continued presence of norepinephrine, PDC or PDCL relieved calcium channel inhibition compared with control neurons. We observed similar results after activation of heterologously expressed metabotropic glutamate receptors with 100 microM L-glutamate. Neurons expressing PDC or PDCL maintained suppression of inhibition after re-exposure to agonist. Unlike other Gbetagamma sequestering proteins that abolish the short-term inhibition of Ca2+ channels, PDC and PDCL require prolonged agonist exposure before effects on modulation are realized.

Plastic control of striatal glutamatergic transmission by ensemble actions of several neurotransmitters and targets for drugs of abuse.

Long-lasting alterations in the efficacy of glutamatergic synapses, such as long-term potentiation (LTP) and long-term depression (LTD), are prominent models for mechanisms of information storage in the brain. It has been suggested that exposure to drugs of abuse produces synaptic plasticity at glutamatergic synapses that shares many features with LTP and LTD, and that these synaptic changes may play roles in addiction. We have examined the involvement of particular neurotransmitters in synaptic plasticity at glutamatergic synapses within the striatum, a brain region with prominent roles in initiation and sequencing of actions, as well as habit formation. Our studies indicate that multiple neurotransmitters interact to produce striatal synaptic plasticity, and that the relative strength and patterning of the afferent inputs that release the various neurotransmitters determines whether LTP or LTD is activated. Drugs of abuse interact with glutamatergic synaptic plasticity in multiple ways, including alterations in dopamine release and more direct effects on glutamate release and glutamate receptors. We hypothesize that these effects contribute to addiction by facilitating the formation of new, drug-centered habits, and by disruption of more adaptive behaviors.

It could be habit forming: drugs of abuse and striatal synaptic plasticity.

Drug addiction can take control of the brain and behavior, activating behavioral patterns that are directed excessively and compulsively toward drug usage. Such patterns often involve the development of repetitive and nearly automatic behaviors that we call habits. The striatum, a subcortical brain region important for proper motor function as well as for the formation of behavioral habits, is a major target for drugs of abuse. Here, we review recent studies of long-term synaptic plasticity in the striatum, emphasizing that drugs of abuse can exert pronounced influences on these processes, both in the striatum and in the dopaminergic midbrain. Synaptic plasticity in the ventral striatum appears to play a prominent role in early stages of drug use, whereas dopamine- and endocannabinoid-dependent synaptic plasticity in the dorsal striatum could contribute to the formation of persistent drug-related habits when casual drug use progresses towards compulsive drug use and addiction.

Nicotinic acetylcholine receptors interact with dopamine in induction of striatal long-term depression.

The dorsal striatum participates in motor function and stimulus-response or "habit" learning. Acetylcholine (ACh) is a prominent neurotransmitter in the striatum and exerts part of its actions through nicotinic cholinergic receptors. Activation of these receptors has been associated with the enhancement of learning and certainly is instrumental in habitual use of nicotine. Nicotinic receptors have also been suggested to be a possible therapeutic target for disorders of the basal ganglia. In this report we show that the activation of nicotinic acetylcholine receptors in the dorsal striatum contributes to dopamine (DA)- and activity-dependent changes in synaptic efficacy. High-frequency activation of glutamatergic synapses onto striatal neurons results in a long-term depression (LTD) of synaptic efficacy that is dependent on the activation of dopamine receptors. This stimulation also produces robust increases in extracellular dopamine concentration as well as strong activation of cholinergic striatal interneurons. Antagonists of nicotinic acetylcholine receptors inhibit striatal LTD. However, on coapplication of dopamine reuptake inhibitors with nicotinic receptor antagonists, activity-induced striatal LTD is restored. Dopamine release is modulated by activation of nicotinic receptors in the dorsal striatum, and activation of nicotinic receptors during high-frequency synaptic activation appears to be capable of interacting with dopaminergic actions that lead to striatal LTD. Our results suggest that stimulation of mechanisms involved in striatal synaptic plasticity is an important role for striatal nicotinic acetylcholine receptors and that these mechanisms may contribute to the enhancement of learning and habit formation produced by nicotine intake.