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issn:0953-816
1.  Heterogeneous presynaptic distribution of monoacylglycerol lipase, a multipotent regulator of nociceptive circuits in the mouse spinal cord 
Monoacylglycerol lipase (MGL) is a multifunctional serine hydrolase, which terminates antinociceptive endocannabinoid signaling and promotes pronociceptive prostaglandin signaling. Accordingly, both acute nociception and its sensitization in chronic pain models are prevented by systemic or focal spinal inhibition of MGL activity. Despite its analgesic potential, the neurobiological substrates of beneficial MGL blockade have remained unexplored. Therefore, we examined the regional, cellular, and subcellular distribution of MGL in spinal circuits involved in nociceptive processing. All immunohistochemical findings obtained with light, confocal, or electron microscopy were validated in MGL-knockout mice. Immunoperoxidase staining revealed a highly concentrated accumulation of MGL in the dorsal horn, especially in superficial layers. Further electron microscopic analysis uncovered that the majority of MGL-immunolabeling is found in axon terminals forming either asymmetric glutamatergic, or symmetric GABA/glycinergic synapses in laminae I/IIo. In line with this presynaptic localization, analysis of double immunofluorescence staining by confocal microscopy showed that MGL colocalizes with neurochemical markers of peptidergic and non-peptidergic nociceptive terminals, and also with markers of local excitatory or inhibitory interneurons. Interestingly, the ratio of MGL-immunolabeling was highest in calcitonin gene-related peptide-positive peptidergic primary afferents and the staining intensity of nociceptive terminals was significantly reduced in MGL-knockout mice. These observations highlight the spinal nociceptor synapse as a potential anatomical site for the analgesic effects of MGL blockade. Moreover, the presence of MGL in additional terminal types raises the possibility that MGL may play distinct regulatory roles in synaptic endocannabinoid or prostaglandin signaling according to its different cellular locations in the dorsal horn pain circuitry.
doi:10.1111/ejn.12470
PMCID: PMC3979158  PMID: 24494682
pain; endocannabinoid; prostaglandin; monoglyceride lipase; MAGL
2.  Estrogen-related receptor β deletion modulates whole-body energy balance via estrogen-related receptor γ and attenuates neuropeptide Y gene expression 
The European journal of neuroscience  2013;37(7):1033-1047.
Estrogen-related receptors (ERRs) α, β, and γ are orphan nuclear hormone receptors with no known ligands. Little is known concerning the role of ERRβ in energy homeostasis, since complete ERRβ-null mice die mid-gestation. We generated two viable conditional ERRβ-null mouse models to address its metabolic function. Whole-body deletion of ERRβ in Sox2-Cre:ERRβlox/lox mice resulted in major alterations in body composition, metabolic rate, meal patterns, and voluntary physical activity levels. Nestin-Cre:ERRβlox/lox mice exhibited decreased expression of ERRβ in hindbrain neurons, the predominant site of expression, decreased Neuropeptide Y (NPY) gene expression in the hindbrain, increased lean body mass, insulin sensitivity, increased energy expenditure, decreased satiety, and decreased time between meals. In the absence of ERRβ, increased ERRγ signaling decreased satiety and the duration of time between meals, similar to meal patterns observed for both the Sox2-Cre:ERRβlox/lox and Nestin-Cre:ERRβlox/lox strains of mice. Central and/or peripheral ERRγ signaling may modulate these phenotypes by decreasing NPY gene expression. Overall, the relative expression ratio between ERRβ and ERRγ may be important in modulating ingestive behavior, specifically satiety, gene expression, as well as whole-body energy balance.
doi:10.1111/ejn.12122
PMCID: PMC3618562  PMID: 23360481
meal patterns; metabolism; hindbrain; medulla; mice
3.  Motor cortex electrical stimulation promotes axon outgrowth to brain stem and spinal targets that control the forelimb impaired by unilateral corticospinal injury 
The European journal of neuroscience  2013;37(7):1090-1102.
We previously showed that electrical stimulation of motor cortex (M1) after unilateral pyramidotomy in the rat increased corticospinal tract (CST) axon length, strengthened spinal connections, and restored forelimb function. Here, we tested: 1) if M1 stimulation only increases spinal axon length or if it also promotes connections to brain stem forelimb control centers, especially magnocellular red nucleus; and 2) if stimulation-induced increase in axon length depends on whether pyramidotomy denervated the structure. After unilateral pyramidotomy, we electrically stimulated the forelimb area of intact M1, to activate the intact CST and other corticofugal pathways, for 10 days. We anterogradely labeled stimulated M1 and measured axon length using stereology. Stimulation increased axon length in both the spinal cord and magnocellular red nucleus, even though the spinal cord is denervated by pyramidotomy and the red nucleus is not. Stimulation also promoted outgrowth in the cuneate and parvocellular red nuclei. In the spinal cord, electrical stimulation caused increased axon length ipsilateral, but not contralateral, to stimulation. Thus, stimulation promoted outgrowth preferentially to the sparsely corticospinal-innervated and impaired side. Outgrowth resulted in greater axon density in the ipsilateral dorsal horn and intermediate zone, resembling the contralateral termination pattern. Importantly, as in spinal cord, increase in axon length in brain stem also was preferentially directed towards areas less densely innervated by the stimulated system. Thus, M1 electrical stimulation promotes increases in corticofugal axon length to multiple M1 targets. We propose the axon length change was driven by competition into an adaptive pattern resembling lost connections.
doi:10.1111/ejn.12119
PMCID: PMC3618589  PMID: 23360401
Corticospinal; electrical stimulation; injury; recovery; motor cortex; rat
4.  Gaze is driven by an internal goal trajectory in a visuomotor task 
The European journal of neuroscience  2012;37(7):1112-1119.
When we make hand movements to visual targets, gaze usually leads hand position by a series of saccades to task-relevant locations. Recent research suggests that the slow smooth pursuit eye movement system may interact with the saccadic system in complex tasks, suggesting that the smooth pursuit system can receive non-retinal input. We hypothesize that a combination of saccades and smooth pursuit guides the hand movements toward a goal in a complex environment, using an internal representation of future trajectories as input to the visuomotor system. This would imply that smooth pursuit leads hand position, which is remarkable since the general idea is that smooth pursuit is driven by retinal slip. To test this hypothesis, we designed a video-game task in which human subjects used their thumbs to move two cursors to a common goal position while avoiding stationary obstacles. We found that gaze led the cursors by a series of saccades interleaved with ocular fixation or pursuit. Smooth pursuit was neither correlated with cursor position nor cursor velocity. We conclude that a combination of fast and slow eye-movements, driven by an internal goal instead of a retinal goal, led the cursor movements and that both saccades and pursuit are driven by an internal representation of future trajectories of the hand. The lead distance of gaze relative to the hand may reflect a compromise between exploring future hand (cursor) paths and verifying that the cursors move along the desired paths.
doi:10.1111/ejn.12107
PMCID: PMC3618614  PMID: 23279153
eye-hand coordination; smooth pursuit; saccades; oculomotor neurophysiology; human
5.  GABAergic inhibition through synergistic astrocytic neuronal interaction transiently decreases vasopressin neuronal activity during hypoosmotic challenge 
The European journal of neuroscience  2013;37(8):1260-1269.
The neuropeptide vasopressin is crucial to mammalian osmotic regulation. Local hypoosmotic challenge transiently decreases and then increases vasopressin secretion. To investigate mechanisms underlying this transient response, we examined effects of hypoosmotic challenge on the electrical activity of rat hypothalamic supraoptic nucleus (SON) vasopressin neurones using patch-clamp recordings. We found that 5 min exposure of hypothalamic slices to hypoosmotic solution transiently increased IPSC frequency and reduced firing rate of vasopressin neurones. Recovery occurred by 10 min exposure, even though the osmolality remained low. The GABAA receptor blocker, gabazine, blocked the IPSCs and the hypoosmotic suppression of firing. The gliotoxin l-aminoadipic acid blocked the increase in IPSC frequency at 5 min and the recovery of firing at 10 min, indicating astrocytic involvement in hypoosmotic modulation of vasopressin neuronal activity. Moreover, β-alanine, an osmolyte of astrocytes and GABA transporter inhibitor, blocked the increase in IPSC frequency at 5 min of hypoosmotic challenge. Confocal microscopy of immunostained SON sections revealed that astrocytes and magnocellular neurones both showed positive staining of vesicular GABA transporters (VGAT). Hypoosmotic stimulation in vivo reduced the number of VGAT-expressing neurones and increased co-localisation and molecular association of VGAT with glial fibrillary acidic protein that increased significantly by 10 min. By 30 min, neuronal VGAT labelling was partially restored and astrocytic VGAT was relocated to ventral portion while decreased in the somatic zone of the SON. Thus, synergistic astrocytic and neuronal GABAergic inhibition could ensure that vasopressin neurone firing is only transiently suppressed under hypoosmotic conditions.
doi:10.1111/ejn.12137
PMCID: PMC3627741  PMID: 23406012
glia; hypothalamo-neurohypophyseal system; in vitro brainstem slice; neuroendocrinology; transporters
6.  Dopamine suppresses persistent network activity via D1-like dopamine receptors in rat medial entorhinal cortex 
The European journal of neuroscience  2013;37(8):1242-1247.
Cortical networks display persistent activity in the form of periods of sustained synchronous depolarisations (‘UP states’) punctuated by periods of relative hyperpolarisation (‘DOWN states’), which together form the slow oscillation. UP states are known to be synaptically generated and are sustained by a dynamic balance of excitation and inhibition, with fast ionotropic glutamatergic excitatory and GABAergic inhibitory conductances increasing during the UP state. Previously, work from our group demonstrated that slow metabotropic GABA receptors also play an important role in terminating the UP state, but the effects of other neuromodulators on this network phenomenon received little attention. Given that persistent activity is a neural correlate of working memory and that signalling through dopamine receptors has been shown to be critical for working memory tasks, we examined whether dopaminergic neurotransmission affected the slow oscillation. Here, using an in vitro model of the slow oscillation in rat medial entorhinal cortex, we show that dopamine strongly and reversibly suppresses cortical UP states. We show that this effect is mediated through D1-like and not D2-like dopamine receptors, and we found no evidence that tonic dopaminergic transmission affected UP states in our model.
doi:10.1111/ejn.12125
PMCID: PMC3628042  PMID: 23336973
slow oscillation; UP states; cortex
7.  Viral transduction of the neonatal brain delivers controllable genetic mosaicism for visualizing and manipulating neuronal circuits in vivo 
The European journal of neuroscience  2013;37(8):1203-1220.
Neonatal intraventricular injection of adeno-associated virus has been shown to transduce neurons widely throughout the brain, but its full potential for experimental neuroscience has not been adequately explored. We report a detailed analysis of the method’s versatility with an emphasis on experimental applications where tools for genetic manipulation are currently lacking. Viral injection into the neonatal mouse brain is fast, easy, and accesses regions of the brain including cerebellum and brain stem that have been difficult to target with other techniques such as electroporation. We show that viral transduction produces an inherently mosaic expression pattern that can be exploited by varying the titer to transduce isolated neurons or densely-packed populations. We demonstrate that expression of virally-encoded proteins is active much sooner than previously believed, allowing genetic perturbation during critical periods of neuronal plasticity, but is also long-lasting and stable, allowing chronic studies of aging. We harness these features to visualize and manipulate neurons in the hindbrain that have been recalcitrant to approaches commonly applied in the cortex. We show that viral labeling aids the analysis of postnatal dendritic maturation in cerebellar Purkinje neurons by allowing individual cells to be readily distinguished, and then demonstrate that the same sparse labeling allows live in vivo imaging of mature Purkinje neurons at resolution sufficient for complete analytical reconstruction. Given the rising availability of viral constructs, packaging services, and genetically modified animals, these techniques should facilitate a wide range of experiments into brain development, function, and degeneration.
doi:10.1111/ejn.12126
PMCID: PMC3628093  PMID: 23347239
Adeno-associated virus; AAV; neonatal viral transgenesis; 2-photon in vivo imaging; mouse
8.  Musicians Show General Enhancement of Complex Sound Encoding and Better Inhibition of Irrelevant Auditory Change in Music: An ERP Study 
The European journal of neuroscience  2013;37(8):1295-1307.
Using electrophysiology, we have examined two questions in relation to musical training – namely, whether it enhances sensory encoding of the human voice and whether it improves the ability to ignore irrelevant auditory change. Participants performed an auditory distraction task, in which they identified each sound as either short (350 ms) or long (550 ms) and ignored a change in sounds’ timbre. Sounds consisted of a male and a female voice saying a neutral sound [a], and of a cello and a French Horn playing an F3 note. In some blocks, musical sounds occurred on 80% of trials, while voice sounds on 20% of trials. In other blocks, the reverse was true. Participants heard naturally recorded sounds in half of experimental blocks and their spectrally-rotated versions in the other half. Regarding voice perception, we found that musicians had a larger N1 ERP component not only to vocal sounds but also to their never before heard spectrally-rotated versions. We, therefore, conclude that musical training is associated with a general improvement in the early neural encoding of complex sounds. Regarding the ability to ignore irrelevant auditory change, musicians’ accuracy tended to suffer less from the change in sounds’ timbre, especially when deviants were musical notes. This behavioral finding was accompanied by a marginally larger re-orienting negativity in musicians, suggesting that their advantage may lie in a more efficient disengagement of attention from the distracting auditory dimension.
doi:10.1111/ejn.12110
PMCID: PMC3628406  PMID: 23301775
musical training; auditory perception in musicians; timbre perception; auditory change; auditory distraction
9.  Yohimbine Anxiogenesis in the Elevated Plus Maze Requires Hindbrain Noradrenergic Neurons that Target the Anterior Ventrolateral Bed Nucleus of the Stria Terminalis 
The European journal of neuroscience  2013;37(8):1340-1349.
The α2 adrenergic receptor antagonist yohimbine (YO) increases transmitter release from noradrenergic (NA) terminals in cortical and subcortical brain regions, including the bed nucleus of the stria terminalis (BST). YO activates the HPA stress axis and is potently anxiogenic in rats and humans. We previously reported that hindbrain NA neurons within the caudal nucleus of the solitary tract (NST-A2/C2) and ventrolateral medulla (VLM-A1/C1) that innervate the anterior ventrolateral (vl)BST contribute to the ability of YO to activate the HPA stress axis in rats. To determine whether the same NA pathway also contributes to YO-induced anxiogenesis in the elevated plus maze (EPMZ), a selective saporin ribotoxin conjugate (DSAP) was microinjected bilaterally into the anterior vlBST to destroy its NA inputs. Sham-lesioned controls were microinjected with vehicle. Two experiments were conducted to determine DSAP lesion effects on EPMZ behavior. DSAP lesions did not alter maze behavior in rats after i.p. saline, and did not alter the significant effect of prior maze experience to reduce exploratory and open arm maze activities. However, in maze-naïve rats, DSAP lesions abolished YO anxiogenesis in the EPMZ. Postmortem immunocytochemical analyses confirmed that DSAP consistently ablated caudal NST-A2/C2 and VLM-A1/C1 neurons that innervate the anterior vlBST. DSAP lesions did not destroy non-NA inputs to the anterior vlBST, and produced inconsistent cell loss within the pontine locus coeruleus (A6 cell group) that was unrelated to YO anxiogenesis. Thus, the ability of YO to increase anxiety-like behavior in the EPMZ depends on hindbrain NA neurons that target the anterior vlBST.
doi:10.1111/ejn.12123
PMCID: PMC3637934  PMID: 23368289
norepinephrine; anxiety; rat; nucleus of the solitary tract; ventrolateral medulla; locus coeruleus
10.  High-fat diet acutely affects circadian organization and eating behavior 
The European journal of neuroscience  2013;37(8):1350-1356.
The organization of timing in mammalian circadian clocks optimally coordinates behavior and physiology with daily environmental cycles. Chronic consumption of a high-fat diet alters circadian rhythms, but the acute effects on circadian organization are unknown. To investigate the proximate effects of a high-fat diet on circadian physiology, we examined the phase relationship between central and peripheral clocks in mice fed a high-fat diet for 1 week. By 7 days, the phase of the liver rhythm was markedly advanced (by 5 h), whereas rhythms in other tissues were not affected. In addition, immediately upon consumption of a high-fat diet, the daily rhythm of eating behavior was altered. As the tissue rhythm of the suprachiasmatic nucleus was not affected by 1 week of high-fat diet consumption, the brain nuclei mediating the effect of a high-fat diet on eating behavior are likely to be downstream of the suprachiasmatic nucleus.
doi:10.1111/ejn.12133
PMCID: PMC3645495  PMID: 23331763
C57BL/6J; hypothalamus; liver; luciferase reporter; mouse; obesity
11.  Cognitive deficits in a mouse model of pre-manifest Parkinson's disease 
Early cognitive deficits are increasingly recognized in patients with Parkinson's disease (PD), and represent an unmet need for the treatment of PD. These early deficits have been difficult to model in mice, and their mechanisms are poorly understood. α-Synuclein is linked to both familial and sporadic forms of PD, and is believed to accumulate in brains of patients with PD before cell loss. Mice expressing human wild-type a-synuclein under the Thy1 promoter (Thy1-aSyn mice) exhibit broad overexpression of α-synuclein throughout the brain and dynamic alterations in dopamine release several months before striatal dopamine loss. We now show that these mice exhibit deficits in cholinergic systems involved in cognition, and cognitive deficits in domains affected in early PD. Together with an increase in extracellular dopamine and a decrease in cortical acetylcholine at 4–6 months of age, Thy1-aSyn mice made fewer spontaneous alternations in the Y-maze and showed deficits in tests of novel object recognition (NOR), object–place recognition, and operant reversal learning, as compared with age-matched wild-type littermates. These data indicate that cognitive impairments that resemble early PD manifestations are reproduced by α-synuclein overexpression in a murine genetic model of PD. With high power to detect drug effects, these anomalies provide a novel platform for testing improved treatments for these pervasive cognitive deficits.
doi:10.1111/j.1460-9568.2012.08012.x
PMCID: PMC3967873  PMID: 22356593
α-synuclein; novel object recognition; novel place recognition; operant-reversal learning; Y-maze
12.  CXCR4 receptors in the dorsal medulla: implications for autonomic dysfunction 
The chemokine receptor, CXCR4, plays an essential role in guiding neural development of the CNS. Its natural agonist, CXCL12 [or stromal cell-derived factor-1 (SDF-1)], normally is derived from stromal cells, but is also produced by damaged and virus-infected neurons and glia. Pathologically, this receptor is critical to the proliferation of the HIV virus and initiation of metastatic cell growth in the brain. Anorexia, nausea and failed autonomic regulation of gastrointestinal (GI) function cause morbidity and contribute to the mortality associated with these disease states. Our previous work on the peripheral cytokine, tumor necrosis factor-alpha, demonstrated that similar morbidity factors involving GI dysfunction are attributable to agonist action on neural circuit elements of the dorsal vagal complex (DVC) of the hindbrain. The DVC includes vagal afferent terminations in the solitary nucleus, neurons in the solitary nucleus (NST) and area postrema, and visceral efferent motor neurons in the dorsal motor nucleus (DMN) that are responsible for the neural regulation of digestive functions from the oral cavity to the transverse colon. Immunohistochemical techniques demonstrate a dense concentration of CXCR4 receptors on neurons throughout the DVC and the hypoglossal nucleus. CXCR4-immunoreactivity is also intense on microglia within the DVC, though not on the astrocytes. Physiological studies show that nanoinjection of SDF-1 into the DVC produces a significant reduction in gastric motility in parallel with an elevation in the numbers of cFOS-activated neurons in the NST and DMN. These results suggest that this chemokine receptor may contribute to autonomically mediated pathophysiological events associated with CNS metastasis and infection.
doi:10.1111/j.1460-9568.2008.06058.x
PMCID: PMC3951345  PMID: 18333961
chemokines; gastric; hindbrain; vagus
13.  Noise-rearing disrupts the maturation of multisensory integration 
It is commonly believed that the ability to integrate information from different senses develops according to associative learning principles as neurons acquire experience with co-active cross-modal inputs. However, previous studies have not distinguished between requirements for co-activation versus co-variation. To determine whether cross-modal co-activation is sufficient for this purpose in visual–auditory superior colliculus (SC) neurons, animals were reared in constant omnidirectional noise. By masking most spatiotemporally discrete auditory experiences, the noise created a sensory landscape that decoupled stimulus co-activation and co-variance. Although a near-normal complement of visual–auditory SC neurons developed, the vast majority could not engage in multisensory integration, revealing that visual–auditory co-activation was insufficient for this purpose. That experience with co-varying stimuli is required for multisensory maturation is consistent with the role of the SC in detecting and locating biologically significant events, but it also seems likely that this is a general requirement for multisensory maturation throughout the brain.
doi:10.1111/ejn.12423
PMCID: PMC3944832  PMID: 24251451
cat; cross-modal; hearing; vision
14.  Enduring increases in anxiety-like behavior and rapid nucleus accumbens dopamine signaling in socially isolated rats 
The European journal of neuroscience  2013;37(6):10.1111/ejn.12113.
Social isolation (SI) rearing, a model of early life stress, results in profound behavioral alterations, including increased anxiety-like behavior, impaired sensorimotor gating and increased self-administration of addictive substances. These changes are accompanied by alterations in mesolimbic dopamine function, such as increased dopamine and metabolite tissue content, increased dopamine responses to cues and psychostimulants, and increased dopamine neuron burst firing. Using voltammetric techniques, we examined the effects of SI rearing on dopamine transporter activity, vesicular release and dopamine D2-type autoreceptor activity in the nucleus accumbens core. Long–Evans rats were housed in group (GH; 4/cage) or SI (1/cage) conditions from weaning into early adulthood [postnatal day (PD) 28–77]. After this initial housing period, rats were assessed on the elevated plus-maze for an anxiety-like phenotype, and then slice voltammetry experiments were performed. To study the enduring effects of SI rearing on anxiety-like behavior and dopamine terminal function, another cohort of similarly reared rats was isolated for an additional 4 months (until PD 174) and then tested. Our findings demonstrate that SI rearing results in lasting increases in anxiety-like behavior, dopamine release and dopamine transporter activity, but not D2 activity. Interestingly, GH-reared rats that were isolated as adults did not develop the anxiety-like behavior or dopamine changes seen in SI-reared rats. Together, our data suggest that early life stress results in an anxiety-like phenotype, with lasting increases in dopamine terminal function.
doi:10.1111/ejn.12113
PMCID: PMC3746484  PMID: 23294165
fast-scan cyclic voltammetry; release; social; stress; uptake
15.  Distinct neuroendocrine mechanisms control neural activity underlying sex differences in sexual motivation and performance 
Sexual behavior can be usefully parsed into an appetitive and a consummatory component. Both appetitive and consummatory male-typical sexual behaviors (respectively ASB and CSB) are activated in male Japanese quail by testosterone (T) acting in the medial preoptic nucleus (POM) but never observed in females. This sex difference is based on a demasculinization (= organizational effect) by estradiol during embryonic life for CSB, but a differential activation by T in adulthood for ASB. Males expressing rhythmic cloacal sphincter movements (RCSM, a form of ASB) or allowed to copulate display increased Fos expression in POM. We investigated Fos brain responses in females exposed to behavioral tests after various endocrine treatments. T-treated females displayed RCSM but never copulated when exposed to another female. Accordingly they showed an increased Fos expression in POM after ASB but not CSB tests. Females treated with the aromatase inhibitor Vorozole in ovo and T in adulthood displayed both male-typical ASB and CSB and Fos expression in POM was increased after both types of tests. Thus the neural circuit mediating ASB is present or can develop in both sexes but is inactive in females unless they are exposed to exogenous T. In contrast, the neural mechanism mediating CSB is not normally present in females but can be preserved by blocking the embryonic production of estrogens. Overall these data confirm the difference in endocrine controls and probably neural mechanisms supporting ASB and CSB in quail and highlight the complexity of mechanisms underlying sexual differentiation of behavior.
doi:10.1111/ejn.12102
PMCID: PMC3594409  PMID: 23282041
Appetitive sexual behavior; Consumatory sexual behavior; Fos expression; Sexual differentiation; Japanese quail
16.  Knockdown of orexin type 2 receptor in the lateral pontomesencephalic tegmentum of rats increases REM sleep 
Dysfunction of the orexin/hypocretin neurotransmitter system causes the sleep disorder narcolepsy, characterized by intrusion of rapid-eye-movement (REM) sleep-like events into normal wakefulness. The sites where orexins act to suppress REM sleep are incompletely understood. Previous studies suggested that the lateral pontomesencephalic tegmentum (lPMT) contains an important REM sleep inhibitory area, and proposed that orexins inhibit REM sleep via orexin type 2 receptors (OxR2) in this region. However, this hypothesis has heretofore not been tested. We thus performed bilateral injection of small interfering RNAs (siRNAs) targeting Ox2R into the lPMT on two consecutive days. This led to a ~30 % increase of time spent in REM sleep in both the dark and light periods for the first two days after injection, with a return to baseline over the next two post-injection days. This increase was mainly due to more longer (>120 s) REM episodes. Cataplexy-like episodes were not observed. The percentage of time spent in wakefulness and NREM sleep, as well as the power spectral profile of NREM and REM sleep, were unaffected. Control animals injected with scrambled siRNA had no sleep changes post-injection. Quantification of the knockdown revealed that unilateral microinjection of siRNAs targeting OxR2 into the lPMT induced a ~40% reduction of OxR2 mRNA two days following the injections when compared to the contralateral side receiving control (scrambled) siRNA. Orexin type 1 receptor (OxR1) mRNA level was unaffected. Our results indicate that removal of OxR2 neurotransmission in the lPMT enhances REM sleep by increasing the duration of REM episodes.
doi:10.1111/ejn.12101
PMCID: PMC3604039  PMID: 23282008
Hypocretin; Narcolepsy; RNA interference; Diurnal; siRNA
17.  Putative Excitatory and Putative Inhibitory Inputs Localize to Different Dendritic Domains in a Drosophila Flight Motoneuron 
Input-output computations of individual neurons may be affected by the three-dimensional structure of their dendrites and by the targeting of input synapses to specific parts of their dendrites. However, only few examples exist where dendritic architecture can be related to behaviorally relevant computations of a neuron. By combining genetic, immunohistochemical, and confocal laser scanning methods this study estimates the location of the spike initiating zone and the dendritic distribution patterns of putative synaptic inputs on an individually identified Drosophila flight motorneuron, MN5. MN5 is a monopolar neuron with more than 4000 dendritic branches. The site of spike initiation was estimated by mapping sodium channel immunolabel onto geometric reconstructions of MN5. Maps of putative excitatory cholinergic and of putative inhibitory GABAergic inputs on MN5 dendrites were created by charting tagged Dα7 nicotinic acetylcholine receptors and Rdl GABAA receptors onto MN5 dendritic surface reconstructions. Although these methods provided only an estimate of putative input synapse distributions, the data indicated that inhibitory and excitatory synapses were targeted preferentially to different dendritic domains of MN5, and thus, computed mostly separately. Most putative inhibitory inputs were close to spike initiation, which was consistent with sharp inhibition, as predicted previously based on recordings of motoneuron firing patterns during flight. By contrast, highest densities of putative excitatory inputs at more distant dendritic regions were consistent with the prediction that in response to different power demands during flight, tonic excitatory drive to flight motoneuron dendrites must be smoothly translated into different tonic firing frequencies.
doi:10.1111/ejn.12104
PMCID: PMC3604049  PMID: 23279094
motoneuron; dendrite; flight; insect
18.  Intermittent nicotine exposure upregulates nAChRs in VTA dopamine neurons and sensitizes locomotor responding to the drug 
The European journal of neuroscience  2013;37(6):1004-1011.
Dopaminergic projections from the ventral tegmental area (VTA) to the nucleus accumbens (NAcc) mediate the behavioral and motivational effects of many drugs of abuse, including nicotine. Repeated intermittent administration of these drugs, a pattern often associated with initial drug exposure, sensitizes the reactivity of dopamine (DA) neurons in this pathway, enhances the locomotor behaviors the drugs emit, and promotes their pursuit and self-administration. Here we show that activation of nicotinic acetylcholine receptors (nAChRs) in the VTA, but not the NAcc, is essential for the induction of locomotor sensitization by nicotine. Repeated intermittent nicotine exposure (4 × 0.4 mg/kg, base, i.p., administered over 7 days), a regimen leading to long-lasting locomotor sensitization, also produced upregulation of nAChRs in the VTA, but not the NAcc, in the hours following the last exposure injection. Functional nAChR upregulation was observed selectively in DA but not GABA neurons in the VTA. These effects were followed by long-term potentiation of excitatory inputs to these cells and increased nicotine-evoked DA overflow in the NAcc. Withdrawal symptoms were not observed following this exposure regimen. Thus, intermittent activation and upregulation by nicotine of nAChRs in DA neurons in the VTA may contribute to the development of behavioral sensitization and increased liability for nicotine addiction.
doi:10.1111/ejn.12114
PMCID: PMC3604051  PMID: 23331514
dopaminergic neurons; GABAergic neurons; LTP; nAChR upregulation; nucleus accumbens; sensitization; ventral tegmental area
19.  Multisensory and unisensory neurons in ferret parietal cortex exhibit distinct functional properties 
Despite the fact that unisensory and multisensory neurons are comingled in every neural structure in which they have been identified, no systematic comparison of their response features has been conducted. Towards that goal, the present study was designed to examine and compare measures of response magnitude, latency, duration and spontaneous activity in unisensory and bimodal neurons from the ferret parietal cortex. Using multichannel single-unit recording, bimodal neurons were observed to demonstrate significantly higher response levels and spontaneous discharge rates than did their unisensory counterparts. These results suggest that, rather than merely reflect different connectional arrangements, unisensory and multisensory neurons are likely to differ at the cellular level. Thus, it can no longer be assumed that the different populations of bimodal and unisensory neurons within a neural region respond similarly to a given external stimulus.
doi:10.1111/ejn.12085
PMCID: PMC3604143  PMID: 23279600
Multisensory Integration; Parietal Cortex; Somatosensation; Vision; Neurophysiology
20.  Striatum-dependent habits are insensitive to both increases and decreases in reinforcer value in mice 
The European journal of neuroscience  2013;37(6):1012-1021.
The mouse has emerged as an advantageous species for studying the brain circuitry that underlies complex behavior and for modeling neuropsychiatric disease. The transition from flexible, goal-directed actions to inflexible, habitual responses is argued to be a valid and reliable behavioral model for studying a core aspect of corticostriatal systems that is implicated in certain forms of psychopathology. This transition is thought to correspond to a progression of behavioral control from associative to sensorimotor cortico-basal ganglia networks. Habits form following extensive training and are characterized by reduced sensitivity of instrumental responding to reinforcer revaluation; few studies have examined this form of behavioral control in mice. Here we examine the involvement of the dorsolateral and dorsomedial striatum in this transition in the C57BL/6 inbred mouse strain. We provide evidence that damage to the dorsolateral striatum disrupts habitual responding – that is, it preserves sensitivity to changes in outcome value following either outcome devaluation or, for the first time in mice, outcome inflation. Together, these data show that instrumental responding in lesioned mice tracks the current value of a reinforcer and provide evidence that neuroanatomical mechanisms underlying habit learning in rats are preserved in mouse. This will allow for genetic and molecular dissection of neural factors involved in decision-making and mechanisms of aberrant habit formation.
doi:10.1111/ejn.12106
PMCID: PMC3604187  PMID: 23298231
instrumental; learning; goal-directed; habitual; devaluation
21.  Brain-derived neurotrophic factor rapidly increases AMPA receptor surface expression in rat nucleus accumbens 
In the rodent nucleus accumbens (NAc), cocaine elevates levels of brain-derived neurotrophic factor (BDNF). Conversely, BDNF can augment cocaine-related behavioral responses. The latter could reflect enhancement of AMPA receptor (AMPAR) transmission, because AMPARs in the NAc mediate some cocaine-induced behaviors. Furthermore, in vitro studies in other cell types show that BDNF can promote AMPAR synaptic delivery. In this study, we investigated whether BDNF similarly promotes AMPAR trafficking in the adult rat NAc. After unilateral intracranial injection of BDNF into NAc core or shell, rats were killed at post-injection times ranging from 30 min to 3 days. NAc core or shell tissue from both injected and non-injected hemispheres was analyzed by Western blotting. A protein crosslinking assay was used to measure AMPAR surface expression. Assessment of tropomyosin receptor kinase B (TrkB) signaling demonstrated that injected BDNF was biologically active. BDNF injection into NAc core, but not NAc shell, led to a protein synthesis and extracellular signal-regulated kinase (ERK) dependent increase in cell surface GluA1 and a trend towards increased total GluA1. This was detected 30 min post-injection but not at longer time-points. GluA2 and GluA3 were unaffected, suggesting an effect of BDNF on homomeric GluA1 Ca2+-permeable AMPARs. These results demonstrate that exogenous BDNF rapidly increases AMPAR surface expression in the rat NAc core, raising the possibility of a relationship between increases in endogenous BDNF levels and alterations in AMPAR transmission observed in the NAc of cocaine-experienced rats.
doi:10.1111/j.1460-9568.2011.07754.x
PMCID: PMC3936351  PMID: 21692887
Ca2+ permeable AMPA receptor; cocaine; ERK; TrkB
22.  Developmental regulation of G protein-gated inwardly-rectifying K+ (GIRK/KIR3) channel subunits in the brain 
The European journal of neuroscience  2011;34(11):1724-1736.
G protein-gated inwardly-rectifying K+ (GIRK/family 3 of inwardly-rectifying K+) channels are coupled to neurotransmitter action and can play important roles in modulating neuronal excitability. We investigated the temporal and spatial expression of GIRK1, GIRK2 and GIRK3 subunits in the developing and adult rodent brain using biochemical, immunohistochemical and immunoelectron microscopic techniques. At all ages analysed, the overall distribution patterns of GIRK1-3 were very similar, with high expression levels in the neocortex, cerebellum, hippocampus and thalamus. Focusing on the hippocampus, histoblotting and immunohistochemistry showed that GIRK1-3 protein levels increased with age, and this was accompanied by a shift in the subcellular localization of the subunits. Early in development (postnatal day 5), GIRK subunits were predominantly localized to the endoplasmic reticulum in the pyramidal cells, but by postnatal day 60 they were mostly found along the plasma membrane. During development, GIRK1 and GIRK2 were found primarily at postsynaptic sites, whereas GIRK3 was predominantly detected at presynaptic sites. In addition, GIRK1 and GIRK2 expression on the spine plasma membrane showed identical proximal-to-distal gradients that differed from GIRK3 distribution. Furthermore, although GIRK1 was never found within the postsynaptic density (PSD), the level of GIRK2 in the PSD progressively increased and GIRK3 did not change in the PSD during development. Together, these findings shed new light on the developmental regulation and subcellular diversity of neuronal GIRK channels, and support the contention that distinct subpopulations of GIRK channels exert separable influences on neuronal excitability. The ability to selectively target specific subpopulations of GIRK channels may prove effective in the treatment of disorders of excitability.
doi:10.1111/j.1460-9568.2011.07886.x
PMCID: PMC3936682  PMID: 22098295
electron microscopy; family 3 of inwardly-rectifying K+ channels; G protein-gated inwardly-rectifying K+ channel; histoblot; immunohistochemistry
23.  Diminished trkA receptor signaling reveals cholinergic-attentional vulnerability of aging 
The cellular mechanisms underlying the exceptional vulnerability of the basal forebrain (BF) cholinergic neurons during pathological aging have remained elusive. Here we employed an adeno-associated viral vector-based RNA interference (AAV-RNAi) strategy to suppress the expression of trkA receptors by cholinergic neurons in the nucleus basalis of Meynert/ substantia innominata (nMB/SI) of adult and aged rats. Suppression of trkA receptor expression impaired attentional performance selectively in aged rats. Performance correlated with trkA levels in the nMB/SI. TrkA knockdown neither affected nMB/SI cholinergic cell counts nor the decrease in cholinergic cell size observed in aged rats. However, trkA suppression augmented an age-related decrease in the density of cortical cholinergic processes and attenuated the capacity of cholinergic neurons to release ACh. The capacity of cortical synapses to release acetylcholine (ACh) in vivo was also lower in aged/trkA-AAV-infused rats than in aged or young controls, and it correlated with their attentional performance. Furthermore, age-related increases in cortical proNGF and p75 receptor levels interacted with the vector-induced loss of trkA receptors to shift NGF signaling toward p75-mediated suppression of the cholinergic phenotype, thereby attenuating cholinergic function and impairing attentional performance. These effects model the abnormal trophic regulation of cholinergic neurons and cognitive impairments in patients with early Alzheimer's disease. This rat model is useful for identifying the mechanisms rendering aging cholinergic neurons vulnerable as well as for studying the neuropathological mechanisms that are triggered by disrupted trophic signaling.
doi:10.1111/ejn.12090
PMCID: PMC3932048  PMID: 23228124
acetylcholine; trophic; cognition; RNAi; rats
24.  Basal ganglia control of sleep–wake behavior and cortical activation 
The basal ganglia (BG) are involved in numerous neurobiological processes that operate on the basis of wakefulness, including motor function, learning, emotion and addictive behaviors. We hypothesized that the BG might play an important role in the regulation of wakefulness. To test this prediction, we made cell body-specific lesions in the striatum and globus pallidus (GP) using ibotenic acid. We found that rats with striatal (caudoputamen) lesions exhibited a 14.95% reduction in wakefulness and robust fragmentation of sleep–wake behavior, i.e. an increased number of state transitions and loss of ultra-long wake bouts (> 120 min). These lesions also resulted in a reduction in the diurnal variation of sleep–wakefulness. On the other hand, lesions of the accumbens core resulted in a 26.72% increase in wakefulness and a reduction in non-rapid eye movement (NREM) sleep bout duration. In addition, rats with accumbens core lesions exhibited excessive digging and scratching. GP lesions also produced a robust increase in wakefulness (45.52%), and frequent sleep–wake transitions and a concomitant decrease in NREM sleep bout duration. Lesions of the subthalamic nucleus or the substantia nigra reticular nucleus produced only minor changes in the amount of sleep–wakefulness and did not alter sleep architecture. Finally, power spectral analysis revealed that lesions of the striatum, accumbens and GP all resulted in a shifting of fast theta power to slow delta power, i.e. a slowing of the cortical electroencephalogram. Collectively, our results suggest that the BG, via a cortico-striato-pallidal loop, are important neural circuitry regulating sleep–wake behaviors and cortical activation.
doi:10.1111/j.1460-9568.2009.07062.x
PMCID: PMC3928571  PMID: 20105243
globus pallidus; ibotenic acid; sleep–wake cycle; striatum; ultradian oscillation
25.  Stereological methods reveal the robust size and stability of ectopic hilar granule cells after pilocarpine-induced status epilepticus in the adult rat 
The European journal of neuroscience  2006;24(8):2203-2210.
Following status epilepticus in the rat, dentate granule cell neurogenesis increases greatly, and many of the new neurons appear to develop ectopically, in the hilar region of the hippocampal formation. It has been suggested that the ectopic hilar granule cells could contribute to the spontaneous seizures that ultimately develop after status epilepticus. However, the population has never been quantified, so it is unclear whether it is substantial enough to have a strong influence on epileptogenesis. To quantify this population, the total number of ectopic hilar granule cells was estimated using unbiased stereology at different times after pilocarpine-induced status epilepticus. The number of hilar neurons immunoreactive for Prox-1, a granule-cell-specific marker, was estimated using the optical fractionator method. The results indicate that the size of the hilar ectopic granule cell population after status epilepticus is substantial, and stable over time. Interestingly, the size of the population appears to be correlated with the frequency of behavioral seizures, because animals with more ectopic granule cells in the hilus have more frequent behavioral seizures. The hilar ectopic granule cell population does not appear to vary systematically across the septotemporal axis, although it is associated with an increase in volume of the hilus. The results provide new insight into the potential role of ectopic hilar granule cells in the pilocarpine model of temporal lobe epilepsy.
doi:10.1111/j.1460-9568.2006.05101.x
PMCID: PMC3924324  PMID: 17042797
dentate gyrus; epilepsy; hippocampus; neurogenesis; seizures

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