Matthew P. Parsons

Extrasynaptic NMDA Receptor Involvement in Central Nervous System Disorders

NMDA receptor (NMDAR)-induced excitotoxicity is thought to contribute to the cell death associated with certain neurodegenerative diseases, stroke, epilepsy, and traumatic brain injury. Targeting NMDARs therapeutically is complicated by the fact that cell signaling downstream of their activation can promote cell survival and plasticity as well as excitotoxicity. However, research over the past decade has suggested that overactivation of NMDARs located outside of the synapse plays a major role in NMDAR toxicity, whereas physiological activation of those inside the synapse can contribute to cell survival, raising the possibility of therapeutic intervention based on NMDAR subcellular localization. Here, we review the evidence both supporting and refuting this localization hypothesis of NMDAR function and discuss the role of NMDAR localization in disorders of the nervous system. Preventing excessive extrasynaptic NMDAR activation may provide therapeutic benefit, particularly in Alzheimer disease and Huntington disease.

Orexin (hypocretin) innervation of the paraventricular nucleus of the thalamus.

The paraventricular nucleus of the thalamus (PVT) is a midline thalamic nucleus with projections to limbic forebrain areas such as the nucleus accumbens and amygdala. The orexin (hypocretin) peptides are synthesized in hypothalamic neurons that project throughout the CNS. The present experiments were done to describe the extent of orexin fiber innervation of the PVT in comparison to other midline and intralaminar thalamic nuclei and to establish the location and proportion of orexin neurons innervating the PVT. All aspects of the anteroposterior PVT were found to be densely innervated by orexin fibers with numerous enlargements that also stained for synaptophysin, a marker for synaptic vesicle protein associated with pre-synaptic sites. Small discrete injections of cholera toxin B into the PVT of rats resulted in the retrograde labeling of a relatively small number of orexin neurons in the medial and lateral hypothalamus. The results also showed a lack of topographical organization among orexin neurons projecting to the PVT. Previous studies indicate that orexin neurons and neurons in the PVT appear to be most active during periods of arousal. Therefore, orexin neurons and their projections to the PVT may be part of a limbic forebrain arousal system.

Functional and anatomical connection between the paraventricular nucleus of the thalamus and dopamine fibers of the nucleus accumbens.

The shell of the nucleus accumbens (NacSh) receives a dense innervation from dopamine (DA) neurons in the ventral tegmental area (VTA) and from glutamate neurons in the paraventricular nucleus of the thalamus (PVT). The present study examined in urethane‐anesthetized rats the effects of electrical stimulation of the PVT on DA levels in the NacSh as measured with amperometry and chronoamperometry. Stimulation of the PVT (40 Hz, 1.0 ms, 400 μA, 5 seconds) resulted in a brief increase in electrochemical currents detected in the NacSh. Inhibition of DA neurons in the VTA using lidocaine (4%, 500 nL) or intravenous apomorphine (0.15 mg/kg) decreased the resting voltammetric signal but had no effect on PVT‐evoked responses. Blocking of ionotropic glutamate receptors in the NacSh with local administration of kynurenic acid attenuated the PVT‐evoked responses. Anterograde tracing with biotinylated dextran amine demonstrated that PVT targets regions of very dense tyrosine hydroxylase fiber staining in the NacSh. Consistent with the projection pattern of the PVT to the NacSh, stimulation of the PVT evoked the largest oxidation current changes in the NacSh, whereas small or no changes were elicited in other areas of the striatum. This study suggests that glutamate release from PVT terminals can act on ionotropic glutamate receptors in the NacSh to induce DA efflux. Modulation of DA levels in the NacSh by the PVT may be linked to arousal‐induced increases in DA tone and could be involved in the facilitation of specific behavioral patterns associated with arousal or stressful situations. J. Comp. Neurol. 500:1050–1063, 2007.

ATP-Sensitive Potassium Channel-Mediated Lactate Effect on Orexin Neurons: Implications for Brain Energetics during Arousal

Active neurons have a high demand for energy substrate, which is thought to be mainly supplied as lactate by astrocytes. Heavy lactate dependence of neuronal activity suggests that there may be a mechanism that detects and controls lactate levels and/or gates brain activation accordingly. Here, we demonstrate that orexin neurons can behave as such lactate sensors. Using acute brain slice preparations and patch-clamp techniques, we show that the monocarboxylate transporter blocker α-cyano-4-hydroxycinnamate (4-CIN) inhibits the spontaneous activity of orexin neurons despite the presence of extracellular glucose. Furthermore, fluoroacetate, a glial toxin, inhibits orexin neurons in the presence of glucose but not lactate. Thus, orexin neurons specifically use astrocyte-derived lactate. The effect of lactate on firing activity is concentration dependent, an essential characteristic of lactate sensors. Furthermore, lactate disinhibits and sensitizes these neurons for subsequent excitation. 4-CIN has no effect on the activity of some arcuate neurons, indicating that lactate dependency is not universal. Orexin neurons show an indirect concentration-dependent sensitivity to glucose below 1 mm, responding by hyperpolarization, which is mediated by ATP-sensitive potassium channels composed of Kir6.1 and SUR1 subunits. In conclusion, our study suggests that lactate is a critical energy substrate and a regulator of the orexin system. Together with the known effects of orexins in inducing arousal, food intake, and hepatic glucose production, as well as lactate release from astrocytes in response to neuronal activity, our study suggests that orexin neurons play an integral part in balancing brain activity and energy supply.

Innervation of the paraventricular nucleus of the thalamus from cocaine- and amphetamine-regulated transcript (CART) containing neurons of the hypothalamus

The paraventricular nucleus of the thalamus (PVT) is a midline thalamic nucleus with heavy projections to the nucleus accumbens and other limbic regions. Previous studies have shown that the PVT contains fibers immunoreactive for cocaine‐ and amphetamine‐related transcript (CART). The purpose of the present study was to determine the location of CART neurons innervating the PVT of the rat by using retrograde tracing with cholera toxin B (CTb) combined with immunofluorescence for CTb and CART (amino acid sequence 55–102). Immunohistochemical analysis of CART in the dorsal thalamus showed that the PVT is densely innervated by CART fibers whereas adjacent midline and intralaminar thalamic nuclei are unlabeled. Injections of CTb in the dorsal midline thalamus retrogradely labeled neurons in several areas of the hypothalamus and brainstem which also contained CART neurons. The largest number of double‐labeled neurons (CTb/CART) was found in the arcuate nucleus of the hypothalamus. CTb/CART neurons were also found in the lateral hypothalamus, zona incerta, and periventricular hypothalamus. These results indicate that the arcuate nucleus is a major source of CART fibers in the PVT. CART neurons in the arcuate nucleus monitor circulating hormonal signals and may regulate food intake and hypothalamic‐pituitary‐adrenal (HPA) activity. Consequently, CART neurons in the arcuate nucleus may transmit signals to the PVT which in turn may influence limbic regions involved in regulating food intake and the HPA. J. Comp. Neurol. 497:155–165, 2006.

Local network regulation of orexin neurons in the lateral hypothalamus

Obesity and inadequate sleep are among the most common causes of health problems in modern society. Thus, the discovery that orexin (hypocretin) neurons play a pivotal role in sleep/wake regulation, energy balance, and consummatory behaviors has sparked immense interest in understanding the regulatory mechanisms of these neurons. The local network consisting of neurons and astrocytes within the lateral hypothalamus and perifornical area (LH/PFA), where orexin neurons reside, shapes the output of orexin neurons and the LH/PFA. Orexin neurons not only send projections to remote brain areas but also contribute to the local network where they release multiple neurotransmitters to modulate its activity. These neurotransmitters have opposing actions, whose balance is determined by the amount released and postsynaptic receptor desensitization. Modulation and negative feedback regulation of excitatory glutamatergic inputs as well as release of astrocyte-derived factors, such as lactate and ATP, can also affect the excitability of orexin neurons. Furthermore, distinct populations of LH/PFA neurons express neurotransmitters with known electrophysiological actions on orexin neurons, such as melanin-concentrating hormone, corticotropin-releasing factor, thyrotropin-releasing hormone, neurotensin, and GABA. These LH/PFA-specific mechanisms may be important for fine tuning the firing activity of orexin neurons to maintain optimal levels of prolonged output to sustain wakefulness and stimulate consummatory behaviors. Building on these exciting findings should shed further light onto the cellular mechanisms of energy balance and sleep-wake regulation.

Real-time imaging of glutamate clearance reveals normal striatal uptake in Huntington disease mouse models

It has become well accepted that Huntington disease (HD) is associated with impaired glutamate uptake, resulting in a prolonged time-course of extracellular glutamate that contributes to excitotoxicity. However, the data supporting this view come largely from work in synaptosomes, which may overrepresent nerve-terminal uptake over astrocytic uptake. Here, we quantify real-time glutamate dynamics in HD mouse models by high-speed imaging of an intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) and electrophysiological recordings of synaptically activated transporter currents in astrocytes. These techniques reveal a disconnect between the results obtained in synaptosomes and those in situ. Exogenous glutamate uptake is impaired in synaptosomes, whereas real-time measures of glutamate clearance in the HD striatum are normal or even accelerated, particularly in the aggressive R6/2 model. Our results highlight the importance of quantifying glutamate dynamics under endogenous release conditions, and suggest that the widely cited uptake impairment in HD does not contribute to pathogenesis.

Memory and synaptic deficits in Hip14/DHHC17 knockout mice.

Significance Palmitoylation can influence the subcellular localization of various synaptic proteins; this process is therefore increasingly recognized as an important regulator of basal synaptic communication as well as activity-dependent synaptic plasticity. Despite the fact that in vitro experiments indicate that many synaptic proteins can be palmitoylated by more than one palmitoyl acyltransferase (PAT), the enzymes responsible for palmitoylation, we describe in this paper various synaptic, plastic, and cognitive consequences that result from constitutive loss of a single PAT, namely Hip14/DHHC17. This reveals an important functional role for this PAT that cannot be compensated for by other existing PATs. Palmitoylation of neurotransmitter receptors and associated scaffold proteins regulates their membrane association in a rapid, reversible, and activity-dependent fashion. This makes palmitoylation an attractive candidate as a key regulator of the fast, reversible, and activity-dependent insertion of synaptic proteins required during the induction and expression of long-term plasticity. Here we describe that the constitutive loss of huntingtin interacting protein 14 (Hip14, also known as DHHC17), a single member of the broad palmitoyl acyltransferase (PAT) family, produces marked alterations in synaptic function in varied brain regions and significantly impairs hippocampal memory and synaptic plasticity. The data presented suggest that, even though the substrate pool is overlapping for the 23 known PAT family members, the function of a single PAT has marked effects upon physiology and cognition. Moreover, an improved understanding of the role of PATs in synaptic modification and maintenance highlights a potential strategy for intervention against early cognitive impairments in neurodegenerative disease.

Striatal synaptic dysfunction and hippocampal plasticity deficits in the Hu97/18 mouse model of Huntington disease.

Huntington disease (HD) is a fatal neurodegenerative disorder caused by a CAG repeat expansion in the gene (HTT) encoding the huntingtin protein (HTT). This mutation leads to multiple cellular and synaptic alterations that are mimicked in many current HD animal models. However, the most commonly used, well-characterized HD models do not accurately reproduce the genetics of human disease. Recently, a new ‘humanized’ mouse model, termed Hu97/18, has been developed that genetically recapitulates human HD, including two human HTT alleles, no mouse Hdh alleles and heterozygosity of the HD mutation. Previously, behavioral and neuropathological testing in Hu97/18 mice revealed many features of HD, yet no electrophysiological measures were employed to investigate possible synaptic alterations. Here, we describe electrophysiological changes in the striatum and hippocampus of the Hu97/18 mice. At 9 months of age, a stage when cognitive deficits are fully developed and motor dysfunction is also evident, Hu97/18 striatal spiny projection neurons (SPNs) exhibited small changes in membrane properties and lower amplitude and frequency of spontaneous excitatory postsynaptic currents (sEPSCs); however, release probability from presynaptic terminals was unaltered. Strikingly, these mice also exhibited a profound deficiency in long-term potentiation (LTP) at CA3-to-CA1 synapses. In contrast, at 6 months of age we found only subtle alterations in SPN synaptic transmission, while 3-month old animals did not display any electrophysiologically detectable changes in the striatum and CA1 LTP was intact. Together, these data reveal robust, progressive deficits in synaptic function and plasticity in Hu97/18 mice, consistent with previously reported behavioral abnormalities, and suggest an optimal age (9 months) for future electrophysiological assessment in preclinical studies of HD.

GIRK channel-mediated inhibition of melanin-concentrating hormone neurons by nociceptin/orphanin FQ

Targeting the melanin-concentrating hormone (MCH) system has been suggested as a potential treatment for obesity, anxiety disorders, as well as addiction. Despite the therapeutic potential of MCH agonists and antagonists, the endogenous factors regulating MCH activity, in particular those implicated in anxiety and reward, are ill-defined. The present study investigated the cellular effects of nociceptin/orphanin FQ (N/OFQ), an endogenous opioid with anxiolytic and antireward properties, on MCH neurons. We found that N/OFQ induced a concentration-dependent reversible outward current in MCH neurons (EC(50) = 50.7 nM), an effect that was blocked by the competitive antagonist of the nociceptin opioid peptide (NOP) receptor UFP-101. N/OFQ-induced outward currents persisted in TTX, reversed near the potassium equilibrium potential, and displayed inward rectification, suggesting direct postsynaptic potassium channel activation. Tertiapin-Q completely abolished the N/OFQ effect, whereas glibenclamide did not, implicating protein G-dependent inwardly rectifying potassium (GIRK) and not ATP-sensitive potassium (K(ATP)) channels as the effector ion channel. The N/OFQ-induced outward current desensitized during repeated applications and occluded the inhibitory effect of dynorphin, suggesting that dynorphin and N/OFQ activate the same pathway. N/OFQ also reversibly inhibited voltage-gated calcium currents in MCH neurons. In conclusion, our study indicates N/OFQ as a robust endogenous regulator of MCH neurons, which may play a role in anxiety and drug addiction.