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Synaptic vesicle glycoprotein 2A (SV2A) is a transmembrane protein that binds levetiracetam and is involved in neurotransmission via an unknown mechanism. SV2A-immunoreactivity is reduced in animal models of epilepsy, and in postmortem hippocampi from patients with temporal lobe epilepsy. It is not known if other regions outside the hippocampus are affected in epilepsy, and whether SV2A expression is permanently reduced or regulated over time. In this study, we induced a generalized status epilepticus (SE) by systemic administration of lithium-pilocarpine to adult female rats. The brains from all animals experiencing SE were collected at different time points after the treatment. The radiotracer, [11C]-UCB-J, binds to SV2A with high affinity, and has been used for in vivo imaging as an a-proxy marker for synaptic density. Here we determined the level of tritiated UCB-J binding by semiquantitative autoradiography in the cerebral cortex, hippocampus, thalamus, and hypothalamus, and in cortical subregions. A prominent and highly significant reduction in SV2A binding capacity was observed over the first days after SE in the cerebral cortex and the hippocampus, but not in the thalamus and hypothalamus. The magnitude in reduction was larger and occurred earlier in the hippocampus and the piriform cortex, than in other cortical subregions. Interestingly, in all areas examined, the binding capacity returned to control levels 12 weeks after the SE comparable to the chronic epileptic phase. These data indicate that lithium-pilocarpine-induced epileptogenesis involves both loss and gain of synapses in the in a time-dependent manner.

Synaptic Vesicle Glycoprotein 2A (SV2A) has been proposed as a presynaptic marker in several neurological disorders. Not only is SV2A the target for the antiepileptic drug levetiracetam, but also considered a marker of mature pre-synapses. In this study, we aimed to assess the binding of [3H]UCB-J as a selective radioligand for SV2A to visualize and determine changes during different stages of epileptogenesis by in-vitro autoradiography in rat models of temporal lobe epilepsy. Two different kainic acid (KA) injection routes were used to model temporal lobe epilepsy in the rat; a systemic (10 mg/kg KA injected intraperitoneally) and a local model (1.875 mM KA injected intrahippocampally). Brain tissue was sampled at different time points after the initial status epilepticus and semi-quantitative [3H]UCB-J autoradiography was performed to determine temporal and spatial changes under the progression of epileptogenesis. A decrease in [3H]UCB-J binding was observed in many brain areas in the acute phases after both types of kainic acid administration. Peak reductions occurred slightly before in systemic-treated animals (within 3–10 days) than after local-treated animals (within 5–15 days). Interestingly in the systemic model, we observed a full restoration in the binding level 30 days after the treatment in most areas probably reflecting neuronal reorganization. However, after the local injection in the hippocampus, the binding in the hippocampus, and in temporal and piriform cortices did not return to basal levels. The time-course profile displayed lateralization in the local model. These results demonstrate changes in the amount of a presynaptic SV2A binding site after seizures and suggest that SV2A may have importance in eliciting spontaneous seizures and/or be a biomarker for epileptogenesis. The present study shows that SV2A is a biomarker of acute phase epileptogenesis in specific brain regions.

Parkinson’s disease (PD) is caused by progressive neurodegeneration and characterised by motor dysfunction. Neurodegeneration of dopaminergic neurons also causes aberrations within the cortico-striato-thalamo-cortical (CSTC) circuit, which has been hypothesised to lead to non-motor symptoms such as depression. Individuals with PD have both lower synaptic density and changes in neuronal metabolic function in the basal ganglia, as measured using [11C]UCB-J and [18F]FDG positron emission tomography (PET), respectively. However, the two radioligands have not been directly compared in the same PD subject or in neurodegeneration animal models. Here, we investigate [11C]UCB-J binding and [18F]FDG uptake in the CSTC circuit following a unilateral dopaminergic lesion in rats and compare it to sham lesioned rats. Rats received either a unilateral injection of 6-hydroxydopamine (6-OHDA) or saline in the medial forebrain bundle and rostral substantia nigra (n = 4/group). After 3 weeks, all rats underwent two PET scans using [18F]FDG, followed by [11C]UCB-J on a separate day. [18F]FDG uptake and [11C]UCB-J binding were both lower in the ipsilateral striatal regions compared to the contralateral regions. Using [11C]UCB-J, we could detect an 8.7% decrease in the ipsilateral ventral midbrain, compared to a 2.9% decrease in ventral midbrain using [18F]FDG. Differential changes between hemispheres for [11C]UCB-J and [18F]FDG outcomes were also evident in the CSTC circuit’s cortical regions, especially in the orbitofrontal cortex and medial prefrontal cortex where higher synaptic density yet lower neuronal metabolic function was observed, following lesioning. In conclusion, [11C]UCB-J and [18F]FDG PET can detect divergent changes following a dopaminergic lesion in rats, especially in cortical regions that are not directly affected by the neurotoxin. These results suggest that combined [11C]UCB-J and [18F]FDG scans could yield a better picture of the heterogeneous cerebral changes in neurodegenerative disorders.

BackgroundThe gold standard for symptomatic relief of Parkinson’s disease is L-DOPA. However, long-term treatment often leads to motor complications such as L-DOPA-induced dyskinesia (LID). While amantadine (Gocovri™) is the only approved therapy for dyskinesia in people with Parkinson’s disease on the American market, it is associated with neurological side effects and limited efficacy. Thus, there remains a high unmet need for addressing LID in Parkinson’s disease patients worldwide.ObjectiveThe objective of this study was to evaluate the efficacy, safety and performance compared to approved treatments of the serotonin receptor 1A (5-HT1A) and 5-HT1B/D agonists buspirone and zolmitriptan in the 6-hydroxydopamine unilaterally lesioned rat model for Parkinson’s disease.MethodsThe hemi-parkinsonian 6-OHDA lesioned rats underwent chronic treatment with L-DOPA to induce dyskinesia and were subsequently used for efficacy testing of buspirone, zolmitriptan and comparison with amantadine, measured as abnormal involuntary movement (AIM) scores after L-DOPA challenge. Safety testing was performed in model and naive animals using forelimb adjusting, rotarod and open field tests.Results5-HT1A and 5-HT1B/D agonism effectively reduced AIM scores in a synergistic manner. The drug combination of buspirone and zolmitriptan was safe and did not lead to tolerance development following sub-chronic administration. Head-to-head comparison with amantadine showed superior performance of buspirone and zolmitriptan in the model.ConclusionsThe strong anti-dyskinetic effect found with combined 5-HT1A and 5-HT1B/D agonism renders buspirone and zolmitriptan a potential clinical candidate for LID in Parkinson’s disease.

Of the 18 sub-Saharan elephant-shrew species, only eastern rock elephant-shrews reproduce seasonally throughout their distribution, a process seemingly independent of photoperiod. The present study characterizes gonadal status and location/intensity of gonadotrophin-releasing hormone-1 (GnRH-1) and kisspeptin immunoreactivities in this polyovulating species in the breeding and nonbreeding seasons. GnRH-1-immunoreactive (ir) cell bodies are predominantly in the medial septum, diagonal band, and medial preoptic area; processes are generally sparse except in the external median eminence. Kisspeptin-ir cell bodies are detected only within the arcuate nucleus; the density of processes is generally low, except in the septohypothalamic nucleus, ventromedial bed nucleus of the stria terminalis, arcuate nucleus, and internal and external median eminence. Kisspeptin-ir processes are negligible at locations containing GnRH-1-ir cell bodies. The external median eminence is the only site with conspicuously overlapping distributions of the respective immunoreactivities and, accordingly, a putative site for kisspeptin's regulation of GnRH-1 release in this species. In the nonbreeding season in males, there is an increase in the rostral population of GnRH-1-ir cell bodies and density of GnRH-1-ir processes in the median eminence. In both sexes, the breeding season is associated with increased kisspeptin-ir process density in the rostral periventricular area of the third ventricle and arcuate nucleus; at the latter site, this is positively correlated with gonadal mass. Cross-species comparisons lead us to hypothesize differential mechanisms within these peptidergic systems: that increased GnRH-1 immunoreactivity during the nonbreeding season reflects increased accumulation with reduced release; that increased kisspeptin immunoreactivity during the breeding season reflects increased synthesis with increased release.

123I-CLINDE is a radiotracer developed for SPECT and targets the 18-kDa translocator protein (TSPO). TSPO is upregulated in glial cells and used as a measure of neuroinflammation in a variety of central nervous system diseases. The aim of this study was to examine the test-retest variability of 123I-CLINDE binding in healthy subjects. Methods: SPECT scans were acquired over 90 min in 16 healthy controls (9 women, 8 mixed-affinity binders [MABs] and 8 high-affinity binders [HABs] twice with an interval of 35 ± 15 d). Arterial input functions were based on individual blood measurements in 8 subjects and a population-based approach in combination with individual whole-blood time-activity curves in the other 8 subjects. Seven brain volumes of interest were extracted and quantified by SUVs and by 2-tissue-compartment modeling for calculation of distribution volumes (VT). Test-retest variability was measured by percentage difference (PD), the absolute PD, intraclass correlation coefficient (ICC), and coefficient of variation. Results: The absolute PD of brain SUV and the VT had similar values. The ICC values were higher for VTs than for brain SUVs, which were both moderate to high; however, lower ICC values were observed when calculated separately for HABs and MABs. Test-retest reproducibility was higher in subjects with immediate centrifugation of blood samples. The population-based method efficiently recovered data with delayed centrifugation. The VT of a 49-y-old male HAB was 7.5 ± 1.4 mL/cm3 compared with 4.6 ± 1.4 mL/cm3 of a sex- and age-matched MAB. The SUVs of a 49-y-old male HAB and MAB were 1.03 ± 0.14 and 0.88 ± 0.15 g/mL, respectively. Conclusion: The test-retest reproducibility of 123I-CLINDE is comparable or better than that reported for commonly used PET TSPO tracers. Because of the binding of 123I-CLINDE to blood cells and peripheral tissues, SUV is not a sufficient surrogate of VT from 2-tissue-compartment modeling. The population-adjusted method has the potential to reduce the complexity of blood analyses of TSPO tracers.

Dysfunction of N-Methyl-d-aspartate receptors (NMDARs) is believed to underlie some of the symptoms in schizophrenia, and non-competitive NMDAR antagonists (including phencyclidine (PCP)) are widely used as pharmacological schizophrenia models. Furthermore, mounting evidence suggests that impaired γ-aminobutyric acid (GABA) neurotransmission contributes to the cognitive deficits in schizophrenia. Thus alterations in GABAergic interneurons have been observed in schizophrenia patients and animal models. Acute systemic administration of PCP increases levels of c-Fos in several cortical and subcortical areas, but whether such induction occurs in specific populations of GABAergic interneuron subtypes still remains to be established. We performed an immunohistochemical analysis of the PCP-induced c-Fos-immunoreactivity (IR) in parvalbumin (PV) and calbindin (CB) interneuron subtypes in the cortex and thalamus of rats. A single dose of PCP (10mg/kg, s.c.) significantly increased total number of c-Fos-IR in: (1) the prelimbic, infralimbic, anterior cingulate, ventrolateral orbital, motor, somatosensory and retrosplenial cortices as well as the nucleus accumbens (NAc), field CA1 of the hippocampus (CA1) field of hippocampus and mediodorsal thalamus (MD); (2) PV-IR cells in the ventrolateral orbitofrontal and retrosplenial cortices and CA1 field of hippocampus; and (3) CB-IR cells in the motor cortex. Overall, our data indicate that PCP activates a wide range of cortical and subcortical brain regions and that a substantial part of this activation is present in GABAergic interneurons in certain regions. This suggests that the psychotomimetic effect of PCP may be mediated via GABAergic interneurons.

Central Kv7 (KCNQ) channels are voltage-dependent potassium channels composed of different combinations of four Kv7 subunits, being differently expressed in the brain. Notably, striatal dopaminergic neurotransmission is strongly suppressed by systemic administration of the pan-Kv7 channel opener retigabine. The effect of retigabine likely involves the inhibition of the activity in mesencephalic dopaminergic neurons projecting to the striatum, but whether Kv7 channels expressed in the striatum may also play a role is not resolved. We therefore assessed the effect of intrastriatal retigabine administration on striatal neuronal excitability in the rat determined by c-Fos immunoreactivity, a marker of neuronal activation. When retigabine was applied locally in the striatum, this resulted in a marked reduction in the number of c-Fos-positive neurons after a strong excitatory striatal stimulus induced by acute systemic haloperidol administration in the rat. The relative mRNA levels of Kv7 subunits in the rat striatum were found to be Kv7.2 = Kv7.3 = Kv7.5 > >Kv7.4. These data suggest that intrastriatal Kv7 channels play a direct role in regulating striatal excitability in vivo.

The KCa 3.1 channel (KCNN4) is an important modulator of microglia responses in rodents, but no information exists on functional expression on microglia from human adults. We isolated and cultured microglia (max 1% astrocytes, no neurons or oligodendrocytes) from neocortex surgically removed from epilepsy patients and employed electrophysiological whole-cell measurements and selective pharmacological tools to elucidate functional expression of KCa 3.1. The channel expression was demonstrated as a significant increase in the voltage-independent current by NS309, a KCa 3.1/KCa 2 activator, followed by full inhibition upon co-application with NS6180, a highly selective KCa 3.1 inhibitor. A major fraction (79%) of unstimulated human microglia expressed KCa 3.1, and the difference in current between full activation and inhibition (ΔKCa 3.1) was estimated at 292 ± 48 pA at -40 mV (n = 75), which equals at least 585 channels per cell. Serial KCa 3.1 activation/inhibition significantly hyperpolarized/depolarized the membrane potential. The isolated human microglia were potently activated by lipopolysaccharide (LPS) shown as a prominent increase in TNF-α production. However, incubation with LPS neither changed the KCa 3.1 current nor the fraction of KCa 3.1 expressing cells. In contrast, the anti-inflammatory cytokine IL-4 slightly increased the KCa 3.1 current per cell, but as the membrane area also increased, there was no significant change in channel density. A large fraction of the microglia also expressed a voltage-dependent current sensitive to the KCa 1.1 modulators NS1619 and Paxilline and an inward-rectifying current with the characteristics of a Kir channel. The high functional expression of KCa 3.1 in microglia from epilepsy patients accentuates the need for further investigations of its role in neuropathological processes. GLIA 2016;64:2065-2078.

The α7 nicotinic acetylcholine receptor (α7 nAChR) is highly expressed in the brain, where it maintains various neuronal functions including (but not limited to) learning and memory. In addition, the protein expression levels of α7 nAChRs are altered in various brain disorders. The classic rule governing α7 nAChR assembly in the mammalian brain was that it was assembled from five α7 subunits to form a homomeric receptor pentamer. However, emerging evidence demonstrates the presence of heteromeric α7 nAChRs in heterologously expressed systems and naturally in brain neurons, where α7 subunits are co-assembled with β2 subunits to form a novel type of α7β2 nAChR. Interestingly, the α7β2 nAChR exhibits distinctive function and pharmacology from traditional homomeric α7 nAChRs. We review recent advances in probing the distribution, function, pharmacology, pathophysiology, and stoichiometry of the heteromeric α7β2 nAChR, which have provided new insights into the understanding of a novel target of cholinergic signaling.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-020-19869-5.

Dopamine plays a key role in the cellular and behavioral responses to drugs of abuse, but the implication of metabotropic regulatory input to dopaminergic neurons on acute drug effects and subsequent drug-related behavior remains unclear. Here, we used chemogenetics [Designer Receptors Exclusively Activated by Designer Drugs (DREADDs)] to modulate dopamine signaling and activity before cocaine administration in mice. We show that chemogenetic inhibition of dopaminergic ventral tegmental area (VTA) neurons differentially affects locomotor and reward-related behavioral responses to cocaine. Stimulation of Gi-coupled DREADD (hM4Di) expressed in dopaminergic VTA neurons persistently reduced the locomotor response to repeated cocaine injections. An attenuated locomotor response was seen even when a dual-viral vector approach was used to restrict hM4Di expression to dopaminergic VTA neurons projecting to the nucleus accumbens. Surprisingly, despite the attenuated locomotor response, hM4Di-mediated inhibition of dopaminergic VTA neurons did not prevent cocaine sensitization, and the inhibitory effect of hM4Di-mediated inhibition was eliminated after withdrawal. In the conditioned place-preference paradigm, hM4Di-mediated inhibition did not affect cocaine-induced place preference; however, the extinction period was extended. Also, hM4Di-mediated inhibition had no effect on preference for a sugar-based reward over water but impaired motivation to work for the same reward in a touchscreen-based motivational assay. In addition, to support that VTA dopaminergic neurons operate as regulators of reward motivation toward both sugar and cocaine, our data suggest that repeated cocaine exposure leads to adaptations in the VTA that surmount the ability of Gi-signaling to suppress and regulate VTA dopaminergic neuronal activity.

Brain-derived neurotrophic factor (BDNF) has been implicated in multiple aspects of brain function including regulation of serotonin signaling. The BDNF val66met polymorphism (rs6265) has been linked to aspects of serotonin signaling in humans but its effects are not well understood. To address this, we evaluated whether BDNF val66met was predictive of a putative marker of brain serotonin levels, serotonin 4 receptor (5-HT4) binding assessed with [11C]SB207145 positron emission tomography, which has also been associated with the serotonin-transporter-linked polymorphic region (5-HTTLPR) polymorphism. We applied a linear latent variable model (LVM) using regional 5-HT4 binding values (neocortex, amygdala, caudate, hippocampus, and putamen) from 68 healthy humans, allowing us to explicitly model brain-wide and region-specific genotype effects on 5-HT4 binding. Our data supported an LVM wherein BDNF val66met significantly predicted a LV reflecting [11C]SB207145 binding across regions (P = 0.005). BDNF val66met met-carriers showed 2–9% higher binding relative to val/val homozygotes. In contrast, 5-HTTLPR did not predict the LV but S-carriers showed 7% lower neocortical binding relative to LL homozygotes (P = 7.3 × 10−6). We observed no evidence for genetic interaction. Our findings indicate that BDNF val66met significantly predicts a common regulator of brain [11C]SB207145 binding, which we hypothesize reflects brain serotonin levels. In contrast, our data indicate that 5-HTTLPR specifically affects 5-HT4 binding in the neocortex. These findings implicate serotonin signaling as an important molecular mediator underlying the effects of BDNF val66met and 5-HTTLPR on behavior and related risk for neuropsychiatric illness in humans. Hum Brain Mapp, 36:313–323, 2015. © 2014 Wiley Periodicals, Inc.

Both the serotonin and glutamate systems have been implicated in the pathophysiology of schizophrenia, as well as in the mechanism of action of antipsychotic drugs. Psychedelic drugs act through the serotonin 2A receptor (5-HT2AR), and elicit a head-twitch response (HTR) in mice, which directly correlates to 5-HT2AR activation and is absent in 5-HT2AR knockout mice. The precise mechanism of this response remains unclear, but both an intrinsic cortico-cortical pathway and a thalamo-cortical pathway involving glutamate release have been proposed. Here, we used a genetic model of NMDAR hypofunction, a serine racemase knockout (SRKO) mouse, to explore the role of glutamatergic transmission in regulating the 5-HT2AR-mediated cellular and behavioral responses. SRKO mice treated with the 5-HT2AR agonist (+/-)-2,5-dimethoxy-4-iodoamphetamine (DOI) showed a clearly diminished HTR and lower induction of c-fos mRNA. These altered functional responses in SRKO mice were not associated with changes in cortical or hippocampal 5-HT levels or in 5-HT2AR and metabotropic glutamate-2 receptor (mGluR2) mRNA and protein expression. Together, these findings suggest that D-serine-dependent NMDAR activity is involved in mediating the cellular and behavioral effects of 5-HT2AR activation.

The nicotinic acetylcholine receptor (nAChR) regulator chaperone (NACHO) was recently identified as an important regulator of nAChR maturation and surface expression. Here we show that NACHO levels decrease during early postnatal development in rats. This decrease occurs earlier and to a greater degree in the frontal cortex compared with the hippocampus. We further show that rats exposed to nicotine during pre- and postnatal development exhibit significantly higher NACHO levels in the frontal cortex at postnatal day (PND) 21, but not at PND60. Repeated exposure to nicotine selectively during early (PND8-14) or late (PND54-60) postnatal stages did not affect NACHO protein levels in the frontal cortex or hippocampus. Neither did exposure to high doses of the selective α7 nAChR agonists SSR180711, A-582941, or PNU-282987. However, we found significantly increased NACHO protein levels in the frontal cortex of PND36 rats after a single exposure to a combination of nicotine and the type II α7 nAChR positive allosteric modulator (PAM) PNU-120596, but not the type I PAM AVL-3288. These findings suggest that exposure to nAChR agonism affects NACHO protein levels, and that this effect is more pronounced during pre- or early postnatal development. The effect of PNU-120596 further suggests that the increase in NACHO expression is caused by activation rather than desensitization of nAChRs.

Nicotinic acetylcholine receptors (nAChRs) affect multiple physiological functions in the brain and their functions are modulated by regulatory proteins of the Lynx family. Here, we report for the first time a direct interaction of the Lynx protein LY6/PLAUR domain‐containing 6 (Lypd6) with nAChRs in human brain extracts, identifying Lypd6 as a novel regulator of nAChR function. Using protein cross‐linking and affinity purification from human temporal cortical extracts, we demonstrate that Lypd6 is a synaptically enriched membrane‐bound protein that binds to multiple nAChR subtypes in the human brain. Additionally, soluble recombinant Lypd6 protein attenuates nicotine‐induced hippocampal inward currents in rat brain slices and decreases nicotine‐induced extracellular signal‐regulated kinase phosphorylation in PC12 cells, suggesting that binding of Lypd6 is sufficient to inhibit nAChR‐mediated intracellular signaling. We further show that perinatal nicotine exposure in rats (4 mg/kg/day through minipumps to dams from embryonic day 7 to post‐natal day 21) significantly increases Lypd6 protein levels in the hippocampus in adulthood, which did not occur after exposure to nicotine in adulthood only. Our findings suggest that Lypd6 is a versatile inhibitor of cholinergic signaling in the brain, and that Lypd6 is dysregulated by nicotine exposure during early development. Regulatory proteins of the Lynx family modulate the function of nicotinic receptors (nAChRs). We report for the first time that the Lynx protein Lypd6 binds to nAChRs in human brain extracts, and that recombinant Lypd6 decreases nicotine‐induced ERK phosphorylation and attenuates nicotine‐induced hippocampal inward currents. Our findings suggest that Lypd6 is a versatile inhibitor of cholinergic signaling in the brain.

Long-term treatment with nicotine or selective α7 nicotinic acetylcholine receptor (nAChR) agonists increases the number of α7 nAChRs and this up-regulation may be involved in the mechanism underlying the sustained procognitive effect of these compounds. Here, we investigate the influence of type I and II α7 nAChR positive allosteric modulators (PAMs) on agonist-induced α7 nAChR up-regulation. We show that the type II PAMs, PNU-120596 (10 μM) or TQS (1 and 10 μM), inhibit up-regulation, as measured by protein levels, induced by the α7 nAChR agonist A-582941 (10 nM or 10 μM), in SH-EP1 cells stably expressing human α7 nAChR, whereas the type I PAMs AVL-3288 or NS1738 do not. Contrarily, neither type I nor II PAMs affect 10 μM nicotine-induced receptor up-regulation, suggesting that nicotine and A-582941 induce up-regulation through different mechanisms. We further show in vivo that 3 mg/kg PNU-120596 inhibits up-regulation of the α7 nAChR induced by 10 mg/kg A-582941, as measured by [125I]-bungarotoxin autoradiography, whereas 1 mg/kg AVL-3288 does not. Given that type II PAMs decrease desensitization of the receptor, whereas type I PAMs do not, these results suggest that receptor desensitization is involved in A-582941-induced up-regulation. Our results are the first to show an in vivo difference between type I and II α7 nAChR PAMs, and demonstrate an agonist-dependent effect of type II PAMs occurring on a much longer time scale than previously appreciated. Furthermore, our data suggest that nicotine and A-582941 induce up-regulation through different mechanisms, and that this confers differential sensitivity to the effects of α7 nAChR PAMs. These results may have implications for the clinical development of α7 nAChR PAMs.

Cognitive deficits represent a core symptom cluster in schizophrenia that are thought to reflect developmental dysregulations within a neural system involving the ventral hippocampus (VH), nucleus accumbens (NAC), and prefrontal cortex (PFC). The present experiments determined the cognitive effects of transiently inactivating VH in rats during a sensitive period of development. Neonatal (postnatal day 7, PD7) and adolescent (PD32) male rats received a single bilateral infusion of saline or tetrodotoxin (TTX) within the VH to transiently inactivate local circuitry and efferent outflow. Rats were tested as adults on an attentional set-shifting task. Performance in this task depends upon the integrity of the PFC and NAC. TTX infusions did not affect the initial acquisition or ability to learn an intra-dimensional shift. However, TTX rats required a greater number of trials than did controls to acquire the first reversal and extra-dimensional shift (ED) stages. These impairments were age and region-specific as rats infused with TTX into the VH at PD32, or into the dorsal hippocampus at PD7, exhibited performance in the task similar to that of controls. Finally, acute systemic administration of the partial α7 nicotinic acetylcholine receptor (nAChR) agonist SSR 180711 (3.0 mg/kg) eliminated the TTX-induced performance deficits. Given that patients with schizophrenia exhibit hippocampal pathophysiology and deficits in the ED stages of set-shifting tasks, our results support the significance of transient hippocampal inactivation as an animal model for studying the cognitive impairments in schizophrenia as well as the pro-cognitive therapeutic potential of α7 nAChR agonists.

In seasonal mammals, a distinct photoneuroendocrine circuit that involves the pineal hormone melatonin tightly synchronizes reproduction with seasons. In the Syrian hamster, a seasonal model in which sexual activity is inhibited by short days, we have previously shown that the potent GnRH stimulator, kisspeptin, is crucial to convey melatonin's message; however, the precise mechanisms through which melatonin affects kisspeptin remain unclear. Interestingly, rfrp gene expression in the neurons of the dorsomedial hypothalamic nucleus, a brain region in which melatonin receptors are present in the Syrian hamster, is strongly down-regulated by melatonin in short days. Because a large body of evidence now indicates that RFamide-related peptide (RFRP)-3, the product of the rfrp gene, is an inhibitor of gonadotropin secretion in various mammalian species, we sought to investigate its effect on the gonadotrophic axis in the Syrian hamster. We show that acute central injection of RFRP-3 induces c-Fos expression in GnRH neurons and increases LH, FSH, and testosterone secretion. Moreover, chronic central administration of RFRP-3 restores testicular activity and Kiss1 levels in the arcuate nucleus of hamsters despite persisting photoinhibitory conditions. By contrast RFRP-3 does not have a hypophysiotrophic effect. Overall, these findings demonstrate that, in the male Syrian hamster, RFRP-3 exerts a stimulatory effect on the reproductive axis, most likely via hypothalamic targets. This places RFRP-3 in a decisive position between the melatonergic message and Kiss1 seasonal regulation. Additionally, our data suggest for the first time that the function of this peptide depends on the species and the physiological status of the animal model.

Kiss1 neurons have recently emerged as a putative conduit for the metabolic gating of reproduction, with leptin being a regulator of hypothalamic Kiss1 expression. Early perturbations of the nutritional status are known to predispose to different metabolic disorders later in life and to alter the timing of puberty; however, the potential underlying mechanisms remain poorly defined. Here we report how changes in the pattern of postnatal feeding affect the onset of puberty and evaluate key hormonal and neuropeptide [Kiss1/kisspeptin (Kp)] alterations linked to these early nutritional manipulations. Female rats were raised in litters of different sizes: small (four pups per dam: overfeeding), normal (12 pups per dam), and large litters (20 pups per litter: underfeeding). Postnatal overfeeding resulted in persistently increased body weight and earlier age of vaginal opening, as an external sign of puberty, together with higher levels of leptin and hypothalamic Kiss1 mRNA. Conversely, postnatal underfeeding caused a persistent reduction in body weight, lower ovarian and uterus weights, and delayed vaginal opening, changes that were paralleled by a decrease in leptin and Kiss1 mRNA levels. Kisspeptin-52 immunoreactivity (Kp-IR) in the hypothalamus displayed similar patterns, with lower numbers of Kp-IR neurons in the arcuate nucleus of postnatally underfed animals, and a trend for increased Kp-positive fibers in the periventricular area of early overfed rats. Yet, gonadotropin responses to Kp at puberty were similar in all groups, except for enhanced responsiveness to low doses of Kp-10 in postnatally underfed rats. In conclusion, our data document that the timing of puberty is sensitive to both overfeeding and subnutrition during early (postnatal) periods and suggest that alterations in hypothalamic expression of Kiss1/kisspeptin may underlie at least part of such programming phenomenon.

In spontaneously ovulating rodents, the preovulatory LH surge is initiated on the day of proestrus by a timed, stimulatory signal originating from the circadian clock in the suprachiasmatic nucleus (SCN). The present studies explored whether kisspeptin is part of the essential neural circuit linking the SCN to the GnRH system to stimulate ovulation in Syrian hamsters (Mesocricetus auratus). Kisspeptin neurons exhibit an estrogen-dependent, daily pattern of cellular activity consistent with a role in the circadian control of the LH surge. The SCN targets kisspeptin neurons via vasopressinergic (AVP), but not vasoactive intestinal polypeptide-ergic, projections. Because AVP administration can only stimulate the LH surge during a restricted time of day, we examined the possibility that the response to AVP is gated at the level of kisspeptin and/or GnRH neurons. Kisspeptin and GnRH activation were assessed after the administration of AVP during the morning (when AVP is incapable of initiating the LH surge) and the afternoon (when AVP injections stimulate the LH surge). Kisspeptin, but not GnRH, cellular activity was up-regulated after morning injections of AVP, suggesting that time-dependent sensitivity to SCN signaling is gated within GnRH but not kisspeptin neurons. In support of this possibility, we found that the GnRH system exhibits pronounced daily changes in sensitivity to kisspeptin stimulation, with maximal sensitivity in the afternoon. Together these studies reveal a novel mechanism of ovulatory control with interactions among the circadian system, kisspeptin signaling, and a GnRH gating mechanism of control.

Severe inflammatory challenges are frequently coupled to decreased food intake and disruption of reproductive function, the latter via deregulation of different signaling pathways that impinge onto GnRH neurons. Recently, the hypothalamic Kiss1 system, a major gatekeeper of GnRH function, was suggested as potential target for transmitting immune-mediated repression of the gonadotropic axis during acute inflammation, and yet key facets of such a phenomenon remain ill defined. Using lipopolysaccharide S (LPS)-treated male rats as model of inflammation, we document herein the pattern of hypothalamic kisspeptin immunoreactivity (IR) and hormonal responses to kisspeptin during the acute inflammatory phase. LPS injections induced a dramatic but transient drop of serum LH and testosterone levels. Suppression of gonadotropic function was associated with a significant decrease in kisspeptin-IR in the arcuate nucleus (ARC) that was not observed under conditions of metabolic stress induced by 48-h fasting. In addition, absolute responses to kisspeptin-10 (Kp-10), in terms of LH and testosterone secretion, were significantly attenuated in LPS-treated males that also displayed a decrease in food intake and body weight. Yet pair-fed males did not show similar alterations in LH and testosterone secretory responses to Kp-10, whose magnitude was preserved, if not augmented, during food restriction. In summary, our data document the impact of acute inflammation on kisspeptin content at the ARC as key center for the neuroendocrine control of reproduction. Our results also suggest that suppressed gonadotropic function following inflammatory challenges might involve a reduction in absolute responsiveness to kisspeptin that is independent of the anorectic effects of inflammation.

alpha(7) nicotinic acetylcholine receptor (nAChR) agonists are candidates for the treatment of cognitive deficits in schizophrenia. Selective alpha(7) nAChR agonists, such as SSR180711, activate neurons in the medial prefrontal cortex (mPFC) and nucleus accumbens shell (ACCshell) in rats, regions important for cognitive function. However, the neural substrates involved in these effects remain elusive. Here we identify cortically projecting cholinergic neurons in the horizontal limb of the diagonal band of Broca (HDB) in the basal forebrain (BF) as important targets for alpha(7) nAChR activation, as measured by c-Fos immunoreactivity, a marker of neuronal activation. Selective depletion of these cholinergic neurons abolishes the SSR180711-induced activation of the mPFC but not the ACCshell, demonstrating their critical importance for alpha(7) nAChR-dependent activation of the mPFC. Contrarily, selective depletion of dopaminergic neurons in the ventral tegmental area abolishes the SSR180711-induced activation of the ACCshell but not the mPFC or HDB. These results demonstrate 2 distinct neural pathways activated by SSR180711. The BF and mPFC are important for attentional function and may subserve the procognitive effects of alpha(7) nAChR agonists, whereas activation of the ACCshell is implicated in the beneficial effect of antipsychotics on the positive symptoms of schizophrenia.

Serotonergic systems in the dorsal raphe nucleus are thought to play an important role in the regulation of anxiety states. To investigate responses of neurons in the dorsal raphe nucleus to a mild anxiety-related stimulus, we exposed rats to an open-field, under low-light or high-light conditions. Treatment effects on c-Fos expression in serotonergic and non-serotonergic cells in the midbrain raphe nuclei were determined 2h following open-field exposure or home cage control (CO) conditions. Rats tested under both light conditions responded with increases in c-Fos expression in serotonergic neurons within subdivisions of the midbrain raphe nuclei compared to CO rats. However, the total numbers of serotonergic neurons involved were small suggesting that exposure to the open-field may affect a subpopulation of serotonergic neurons. To determine if exposure to the open-field activates a subset of neurons in the midbrain raphe complex that projects to forebrain circuits regulating anxiety states, we used Cholera Toxin B subunit (CTb) as a retrograde tracer to identify neurons projecting to the basolateral amygdaloid complex (BL) in combination with c-Fos immunostaining to identify cells that responded to open-field exposure. Rats received a unilateral injection of CTb into the BL. Seven to eleven days following CTb injection rats were either, 1) exposed to an open-field in low-light conditions, 2) briefly handled or 3) left undisturbed in home cages. Dual immunostaining for c-Fos and CTb revealed an increase in the percentage of c-Fos-immunoreactive BL-projecting neurons in open-field-exposed rats compared with handled and control rats. Dual immunostaining for tryptophan hydroxylase and CTb revealed that a majority (65%) of BL-projecting neurons were serotonergic, leaving open the possibility that activated neurons were serotonergic, non-serotonergic, or both. These data are consistent with the hypothesis that exposure to anxiogenic stimuli activates a subset of neurons in the midbrain raphe complex projecting to amygdala anxiety circuits.

Anxiety states and anxiety-related behaviors appear to be regulated by a distributed and highly interconnected system of brain structures including the basolateral amygdala. Our previous studies demonstrate that exposure of rats to an open-field in high- and low-light conditions results in a marked increase in c-Fos expression in the anterior part of the basolateral amygdaloid nucleus (BLA) compared to controls. The neural mechanisms underlying the anatomically specific effects of open-field exposure on c-Fos expression in the BLA are not clear, however, it is likely that this reflects activation of specific afferent input to this region of the amygdala. In order to identify candidate brain regions mediating anxiety-induced activation of the basolateral amygdaloid complex in rats, we used Cholera Toxin B subunit (CTb) as a retrograde tracer to identify neurons with direct afferent projections to this region in combination with c-Fos immunostaining to identify cells responding to exposure to an open-field arena in low-light (8–13 lux) conditions (an anxiogenic stimulus in rats). Adult male Wistar rats received a unilateral microinjection of 4% CTb in phosphate-buffered saline into the basolateral amygdaloid complex. Rats were housed individually for 11 days after CTb injections and handled for 2 min each day. On the test day rats were either, 1) exposed to an open-field in low-light conditions (8–13 lux) for 15 minutes; 2) briefly handled or 3) left undisturbed (control). We report that dual immunohistochemical staining for c-Fos and CTb revealed an increase in the percentage of c-Fos-immunopositive basolateral amygdaloid complex-projecting neurons in open-field-exposed rats compared with handled and control rats in the ipsilateral CA1 region of the ventral hippocampus, subiculum and lateral entorhinal cortex. These data are consistent with the hypothesis that exposure to the open-field arena activates an anxiety-related neuronal system with convergent input to the basolateral amygdaloid complex.

SLURP-1 is a secreted toxin-like Ly-6/uPAR protein found in epithelium, sensory neurons and immune cells. Point mutations in the slurp-1 gene cause the autosomal inflammation skin disease Mal de Meleda. SLURP-1 is considered an autocrine/paracrine hormone that regulates growth and differentiation of keratinocytes and controls inflammation and malignant cell transformation. The majority of previous studies of SLURP-1 have been made using fusion constructs containing, in addition to the native protein, extra polypeptide sequences. Here we describe the activity and pharmacological profile of a recombinant analogue of human SLURP-1 (rSLURP-1) differing from the native protein only by one additional N-terminal Met residue. rSLURP-1 significantly inhibited proliferation (up to ~ 40%, EC50 ~ 4 nM) of human oral keratinocytes (Het-1A cells). Application of mecamylamine and atropine,--non-selective inhibitors of nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors, respectively, and anti-α7-nAChRs antibodies revealed α7 type nAChRs as an rSLURP-1 target in keratinocytes. Using affinity purification from human cortical extracts, we confirmed that rSLURP-1 binds selectively to the α7-nAChRs. Exposure of Xenopus oocytes expressing α7-nAChRs to rSLURP-1 caused a significant non-competitive inhibition of the response to acetylcholine (up to ~ 70%, IC50 ~ 1 μM). It was shown that rSLURP-1 binds to α7-nAChRs overexpressed in GH4Cl cells, but does not compete with 125I-α-bungarotoxin for binding to the receptor. These findings imply an allosteric antagonist-like mode of SLURP-1 interaction with α7-nAChRs outside the classical ligand-binding site. Contrary to rSLURP-1, other inhibitors of α7-nAChRs (mecamylamine, α-bungarotoxin and Lynx1) did not suppress the proliferation of keratinocytes. Moreover, the co-application of α-bungarotoxin with rSLURP-1 did not influence antiproliferative activity of the latter. This supports the hypothesis that the antiproliferative activity of SLURP-1 is related to 'metabotropic' signaling pathway through α7-nAChR, that activates intracellular signaling cascades without opening the receptor channel.

The neuropathology following traumatic brain injury (TBI) is poorly understood. From the primary biomechanical injury, secondary injuries develop, including neuro-inflammatory processes. These secondary injuries are regarded as a potential targets for treatment and diagnostics. The translocator protein 18 kDa (TSPO) is robustly upregulated in response to brain injury, making it a suitable biomarker for glia activation and the neuro-inflammatory response. Second-generation radioligands of TSPO, such as [123I]CLINDE, offer higher affinity and signal-to-noise ratio compared to PK11195, the prototypical ligand. Applicability of [123I]CLINDE was demonstrated in both neurodegenerative disease models and patient studies and therefore demonstrate the translational value of this tracer. We therefore investigated TSPO expression in a rat model of TBI with [123I]CLINDE , a selective and clinically relevant TSPO-radioligand. Adult Sprague-Dawley rats were subjected to moderate Controlled-Cortical-Impact injury (CCI, n=5 per group). Sham (n=5), craniotomy (n=3), and naïve animals (n=3) served as different control groups. Animals were sacrificed at 6h, 24h, 72h, and 28 days post-surgery, and TSPO expression was assessed in brain section employing [123I]CLINDE in vitro autoradiography. From 24 h to 28 d post-surgery, injured animals exhibited a marked and time-dependent increase of binding in the ipsilateral motor, somatosensory and parietal cortex, as well as in the hippocampus and thalamus. Furthermore, binding was significantly elevated in the contralateral motor cortex following TBI. Craniotomy also caused a significant increase in [123I]CLINDE binding per se. Radioligand binding was consistent with an increase in TSPO mRNA expression and OX-42 immunoreactivity at the contusion site. In conclusion, this study demonstrates the applicability of [123I]CLINDE for brain regional and quantitative assessment of neuro-inflammatory activity in experimental models of TBI.

The existence of α7β2 nicotinic acetylcholine receptors (nAChRs) has recently been demonstrated in both the rodent and human brain. Since α7-containing nAChRs are promising drug targets for schizophrenia and Alzheimer’s disease, it is critical to determine whether α7β2 nAChRs are present in the human brain, in which brain areas, and whether they differ functionally from α7 nAChR homomers. We used α-bungarotoxin to affinity purify α7-containing nAChRs from surgically excised human temporal cortex, and found that α7 subunits co-purify with β2 subunits, indicating the presence of α7β2 nAChRs in the human brain. We validated these results by demonstrating co-purification of β2 from wild-type, but not α7 or β2 knock-out mice. The pharmacology and kinetics of human α7β2 nAChRs differed significantly from that of α7 homomers in response to nAChR agonists when expressed in Xenopus oocytes and HEK293 cells. Notably, α7β2 heteromers expressed in HEK293 cells display markedly slower rise and decay phases. These results demonstrate that α7 subunits in the human brain form heteromeric complexes with β2 subunits, and that human α7β2 nAChR heteromers respond to nAChR agonists with a unique pharmacology and kinetic profile. α7β2 nAChRs thus represent an alternative mechanism for the reported clinical efficacy of α7 nAChR ligands.

Objective It is well known that reproductive capacity is lower in obese individuals, but what mediators and signals are involved is unclear. Kisspeptin is a potent stimulator of GnRH release, and it has been suggested that kisspeptin neurons located in the arcuate nucleus transmit metabolic signals to the GnRH neurons. Methods In this study, we measured body weight and plasma concentrations of leptin, insulin, testosterone, and triglycerides after high fat diet exposure and correlated these parameters with the number of kisspeptin-immunoreactive neurons in the arcuate nucleus of male rats. In this model, a high fat diet (45% or 60% energy from fat, respectively) or a control diet (10% energy from fat) was provided after weaning for three months. Results We find a significant increase in body weight and plasma leptin concentration, but no change in the number of kisspeptin-immunoreactive cells with increased fat in the diet. Kisspeptin-immunoreactive cells are not correlated with body weight, testosterone, leptin or insulin. However, we find that the number of kisspeptin-immunoreactive cells is strongly and negatively correlated with the level of plasma triglycerides (R2=0.49, p=0.004). Conclusion We find a strong negative correlation between plasma triglyceride concentrations and the number of kisspeptin neurons in the rat arcuate nucleus regardless of the percentage of fat in the diet. In line with the lipotoxicity hypothesis, our results suggest that it is the level of hypertriglyceridemia per se that is a detrimental factor for kisspeptin expression in the arcuate nucleus.

Here we compare translocator protein (TSPO) imaging using 6-chloro-2-(4′-123I-iodophenyl)-3-(N,N-diethyl)-imidazo[1,2-a]pyridine-3-acetamide SPECT (123I-CLINDE) and amino acid transport imaging using O-(2-18F-fluoroethyl)-l-tyrosine PET (18F-FET) and investigate whether 123I-CLINDE is superior to 18F-FET in predicting progression of glioblastoma multiforme (GBM) at follow-up. Methods: Three patients with World Health Organization grade IV GBM were scanned with 123I-CLINDE SPECT, 18F-FET PET, and gadolinium-enhanced MR imaging. Molecular imaging data were compared with follow-up gadolinium-enhanced MR images or contrast-enhanced CT scans. Results: The percentage overlap between volumes of interest (VOIs) of increased 18F-FET uptake and 123I-CLINDE binding was variable (12%–42%). The percentage overlap of MR imaging baseline VOIs was greater for 18F-FET (79%–93%) than 123I-CLINDE (15%–30%). In contrast, VOIs of increased contrast enhancement at follow-up compared with baseline overlapped to a greater extent with baseline 123I-CLINDE VOIs than 18F-FET VOIs (21% vs. 8% and 72% vs. 55%). Conclusion: Our preliminary results suggest that TSPO brain imaging in GBM may be a useful tool for predicting tumor progression at follow-up and may be less susceptible to changes in blood–brain barrier permeability than 18F-FET. Larger studies are warranted to test the clinical potential of TSPO imaging in GBM, including presurgical planning and radiotherapy.

Members of the inducible transcription factor Fos family, that are part of the AP-1 complex that binds to the corresponding promoter site, are implicated in the regulation of gene transcription after acute and chronic seizures. However, little is known about the temporal expression of the AP-1 transcription factors and if individual proteins composing this complex have distinct roles in development and maintenance of permanent epilepsy. In this study, the AP-1 binding capacity, its content of different Fos proteins, and the anatomical specificity, were analyzed 2 or 18 h after achieving full kindling in rats. The same analysis was performed in fully kindled animal receiving a new stimulus after a 3-week pause to determine the extent of stability of the AP-1 transcription factors. While both c-Fos and FosB were induced in all cortical areas after a single stimulus, only FosB-immunoreactivity remained after 18 h. A single stimulation to kindled animals left undisturbed for 3 weeks induced a long-lasting upregulation of AP-1 binding in the frontal cortex, but not in the hippocampus suggesting a permanent exposure of AP-1 heterocomplexes in the frontal cortex. Supershift assays showed that FosB is the dominant component of the long-term AP-1 complex. It is concluded that the AP-1 binding complex in fully kindled rats is composed of different proteins, and that FosB-containing AP-1 complexes mediate long-term effects in the frontal cortex.

Anxiety is a complex emotional state associated with sustained heightened autonomic and behavioral arousal and an increase in avoidance behavior. Anxiety-related behavior is a form of risk assessment behavior that is associated with a level of uncertainty or unpredictability regarding the outcome of emotionally salient events, often when both rewarding and aversive outcomes are possible. In this review, we highlight recent advances in our understanding of the neural circuits regulating anxiety states and anxiety-related behavior with an emphasis on the role of brainstem serotonergic systems in modulating anxiety-related circuits. In particular, we explore the possibility that the regulation of anxiety states and anxiety-related behavior by serotonergic systems is dependent on a specific, topographically organized mesolimbocortical serotonergic system that originates in the mid-rostrocaudal and caudal parts of the dorsal raphe nucleus.

Ovarian hormones have potent effects on key features of the serotonergic neurotransmission. This includes effects that may counterbalance each other, e.g. postsynaptic receptor changes and change in serotonin transporter (SERT) levels, which regulates synaptic serotonin. Such mechanisms may be implicated in the heightened risk for depressive episodes seen in perimenopause or postpartum. However, to what extent transition to hypogonadism in it-self drives such serotonin related risk mechanism remains elusive. Here we evaluate if ovarian hormone withdrawal affects postsynaptic serotonin receptor 2A (5-HT2A) and presynaptic SERT availability differently across the early withdrawal phase in ovariectomized (OVXed) relative to sham operated rats. Cortical (prefrontal cortex (PFC)) and subcortical (striatum) 5-HT2A receptor and SERT binding were quantified with autoradiography at 8 and 23 days after ovariectomy (OVX). We observed that 8 and 23 days after withdrawal the level of 5-HT2A receptor binding was decreased relative to sham, while SERT binding was unaltered, however, with a weak trend to decrease at day 23. The dataset available is small and the results should be viewed as preliminary. If replicated, these data highlight a potential phase-specific and predominantly early challenge of serotonergic neurotransmission when ovarian hormone levels decline abruptly, which may translate to the risk for psychopathology, e.g. depressive episodes postpartum or during menopausal transition in humans.

GnRH is a key player in the hypothalamic control of gonadotropin secretion from the anterior pituitary gland. It has been shown that the mammalian counterpart of the avian gonadotropin inhibitory hormone named RFamide-related peptide (RFRP) is expressed in hypothalamic neurons that innervate and inhibit GnRH neurons. The RFRP precursor is processed into 2 mature peptides, RFRP-1 and RFRP-3. These are characterized by a conserved C-terminal motif RF-NH2 but display highly different N termini. Even though the 2 peptides are equally potent in vitro, little is known about their relative distribution and their distinct roles in vivo. In this study, we raised an antiserum selective for RFRP-1 and defined the distribution of RFRP-1-immunoreactive (ir) neurons in the rat brain. Next, we analyzed the level of RFRP-1-ir during postnatal development in males and females and investigated changes in RFRP-1-ir during the estrous cycle. RFRP-1-ir neurons were distributed along the third ventricle from the caudal part of the medial anterior hypothalamus throughout the medial tuberal hypothalamus and were localized in, but mostly in between, the dorsomedial hypothalamic, ventromedial hypothalamic, and arcuate nuclei. The number of RFRP-1-ir neurons and the density of cellular immunoreactivity were unchanged from juvenile to adulthood in male rats during the postnatal development. However, both parameters were significantly increased in female rats from peripuberty to adulthood, demonstrating prominent gender difference in the developmental control of RFRP-1 expression. The percentage of c-Fos-positive RFRP-1-ir neurons was significantly higher in diestrus as compared with proestrus and estrus. In conclusion, we found that adult females, as compared with males, have significantly more RFRP-1-ir per cell, and these cells are regulated during the estrous cycle.

Kisspeptin, neurokinin B (NKB) and dynorphin A are coexpressed in a population of neurons in the arcuate nucleus (ARC), termed KNDy neurons, which were recently recognized as important elements for the generation of GnRH pulses. However, the topographic distribution of these peptides and their regulated expression by sex steroids are still not well understood. In this study, detailed examination of NKB and kisspeptin immunoreactivity in the rat ARC was carried out, including comparison between sexes, with and without sex steroid replacement. Neurons expressing kisspeptin and NKB were more prominent in the caudal ARC of females, whereas neurons expressing NKB, but not kisspeptin, were the most abundant in the male. Sex steroid manipulation revealed differential regulation of kisspeptin and NKB; although kisspeptin immunoreactive (ir) cells increased in response to gonadectomy, NKB remained unchanged. Furthermore, the number of NKB-ir cells increased upon sex steroid replacement compared with gonadectomy, whereas kisspeptin did not, suggesting that sex steroids differently regulate these peptides. In addition, only in females did the density of kisspeptin- and NKB-ir fibers in the ARC increase upon sex steroid replacement in relation to sham and ovariectomy, respectively, suggesting sex-specific regulation of release. In conclusion, our observations reveal sex differences in the number of kisspeptin- and NKB-ir cells, which are more prominent in the caudal ARC. The divergent regulation of kisspeptin and NKB peptide contents in the ARC as a function of sex and steroid milieu enlarge our understanding on how these neuropeptides are posttranscriptionally regulated in KNDy neurons.

Alzheimer's disease (AD) is a neurodegenerative disorder involving impaired cholinergic neurotransmission and dysregulation of nicotinic acetylcholine receptors (nAChRs). Ly-6/neurotoxin (Lynx) proteins have been shown to modulate cognition and neural plasticity by binding to nAChR subtypes and modulating their function. Hence, changes in nAChR regulatory proteins such as Lynx proteins could underlie the dysregulation of nAChRs in AD. Using Western blotting, we detected bands corresponding to the Lynx proteins prostate stem cell antigen (PSCA) and Lypd6 in human cortex indicating that both proteins are present in the human brain. We further showed that PSCA forms stable complexes with the α4 nAChR subunit and decreases nicotine-induced extracellular-signal regulated kinase phosphorylation in PC12 cells. In addition, we analyzed protein levels of PSCA and Lypd6 in postmortem tissue of medial frontal gyrus from AD patients and found significantly increased PSCA levels (approximately 70%). In contrast, no changes in Lypd6 levels were detected. In concordance with our findings in AD patients, PSCA levels were increased in the frontal cortex of triple transgenic mice with an AD-like pathology harboring human transgenes that cause both age-dependent β-amyloidosis and tauopathy, whereas Tg2576 mice, which display β-amyloidosis only, had unchanged PSCA levels compared to wild-type animals. These findings identify PSCA as a nAChR-binding protein in the human brain that is affected in AD, suggesting that PSCA-nAChR interactions may be involved in the cognitive dysfunction observed in AD. Alzheimer's disease (AD) is a neurodegenerative disorder involving impaired cholinergic neurotransmission and dysregulation of nicotinic acetylcholine receptors (nAChRs). Ly-6/neurotoxin (Lynx) proteins have been shown to modulate cognition and neural plasticity by binding to nAChR subtypes and modulating their function. Hence, changes in nAChR regulatory proteins such as Lynx proteins could underlie the dysregulation of nAChRs in AD. Using Western blotting, we detected bands corresponding to the Lynx proteins prostate stem cell antigen (PSCA) and Lypd6 in human cortex indicating that both proteins are present in the human brain. We further showed that PSCA forms stable complexes with the α4 nAChR subunit and decreases nicotine-induced extracellular-signal regulated kinase phosphorylation in PC12 cells. In addition, we analyzed protein levels of PSCA and Lypd6 in postmortem tissue of medial frontal gyrus from AD patients and found significantly increased PSCA levels (approximately 70%). In contrast, no changes in Lypd6 levels were detected. In concordance with our findings in AD patients, PSCA levels were increased in the frontal cortex of triple transgenic mice with an AD-like pathology harboring human transgenes that cause both age-dependent β-amyloidosis and tauopathy, whereas Tg2576 mice, which display β-amyloidosis only, had unchanged PSCA levels compared to wild-type animals. These findings identify PSCA as a nAChR-binding protein in the human brain that is affected in AD, suggesting that PSCA-nAChR interactions may be involved in the cognitive dysfunction observed in AD.

Brain‐derived neurotrophic factor (BDNF) has been implicated in multiple aspects of brain function including regulation of serotonin signaling. The BDNF val66met polymorphism (rs6265) has been linked to aspects of serotonin signaling in humans but its effects are not well understood. To address this, we evaluated whether BDNF val66met was predictive of a putative marker of brain serotonin levels, serotonin 4 receptor (5‐HT(4)) binding assessed with [(11)C]SB207145 positron emission tomography, which has also been associated with the serotonin‐transporter‐linked polymorphic region (5‐HTTLPR) polymorphism. We applied a linear latent variable model (LVM) using regional 5‐HT(4) binding values (neocortex, amygdala, caudate, hippocampus, and putamen) from 68 healthy humans, allowing us to explicitly model brain‐wide and region‐specific genotype effects on 5‐HT(4) binding. Our data supported an LVM wherein BDNF val66met significantly predicted a LV reflecting [(11)C]SB207145 binding across regions (P = 0.005). BDNF val66met met‐carriers showed 2–9% higher binding relative to val/val homozygotes. In contrast, 5‐HTTLPR did not predict the LV but S‐carriers showed 7% lower neocortical binding relative to LL homozygotes (P = 7.3 × 10(−6)). We observed no evidence for genetic interaction. Our findings indicate that BDNF val66met significantly predicts a common regulator of brain [(11)C]SB207145 binding, which we hypothesize reflects brain serotonin levels. In contrast, our data indicate that 5‐HTTLPR specifically affects 5‐HT(4) binding in the neocortex. These findings implicate serotonin signaling as an important molecular mediator underlying the effects of BDNF val66met and 5‐HTTLPR on behavior and related risk for neuropsychiatric illness in humans. Hum Brain Mapp, 36:313–323, 2015. © 2014 Wiley Periodicals, Inc.

Neuropeptide Y (NPY) is widely distributed in interneurons of the central nervous system (CNS), including the hippocampus and cerebral cortex, in concentrations exceeding those of any other known neuropeptides1,2. Sequence data comparing different species show that NPY is highly conserved3. This suggests a critical role in regulation of regional neuronal excitability. Kainic acid, a glutamate agonist at kainic acid receptors, causes severe limbic motor seizures culminating in status epilepticus4. We here report that NPY administered into the lateral ventricle is a powerful inhibitor of motor as well as electroencephalographic (EEG) seizures induced by kainic acid. This effect was mediated via receptors with a pharmacological profile similar to the recently cloned rat Y5 receptor5. The present study is the first to demonstrate that NPY possesses anticonvulsant activity. This is consistent with the concept that NPY is an endogenous anticonvulsant and suggests that agonists acting at Y5-like receptors may constitute a novel group of drugs in antiepileptic therapy.

The retinohypothalamic tract (RHT) relays photic information from the eyes to the suprachiasmatic nucleus (SCN). Activation of this pathway by light plays a role in adjusting circadian timing via a glutamatergic pathway at night. Here we report a new signaling pathway by which the RHT may regulate circadian timing in the daytime as well. We used dual immunocytochemistry for pituitary adenylate cyclase-activating peptide (PACAP) and thein vivotracer cholera toxin subunit B and observed intense PACAP-immunoreactivity (PACAP-IR) in retinal afferents in the rat SCN as well as in the intergeniculate leaflet (IGL) of the thalamus. This PACAP-IR in the SCN as well as in the IGL was nearly lost after bilateral eye enucleation. PACAP afferents originated from small ganglion cells distributed throughout the retina. The phase of circadian rhythm measured as SCN neuronal activityin vitrowas significantly advanced (3.5 ± 0.4 hr) by application of 1 × 10−6mPACAP-38 during the subjective day [circadian time (CT)-6] but not at night (CT14 and CT19). The phase-shifting effect is channeled to the clock via a PACAP-R1 receptor, because mRNA from this receptor was demonstrated in the ventral SCN byin situhybridization. Furthermore, vasoactive intestinal peptide was nearly 1000-fold less potent in stimulating a phase advance at CT6. The signaling mechanism was through a cAMP-dependent pathway, which could be blocked by a specific cAMP antagonist, Rp-cAMPS. Thus, in addition to its role in nocturnal regulation by glutamatergic neurotransmission, the RHT may adjust the biological clock by a PACAP/cAMP-dependent mechanism during the daytime.