Orexin‐A increases the firing activity of hippocampal CA1 neurons through orexin‐1 receptors

Orexins including two peptides, orexin‐A and orexin‐B, are produced in the posterior lateral hypothalamus. Much evidence has indicated that central orexinergic systems play numerous functions including energy metabolism, feeding behavior, sleep/wakefulness, and neuroendocrine and sympathetic activation. Morphological studies have shown that the hippocampal CA1 regions receive orexinergic innervation originating from the hypothalamus. Positive orexin‐1 (OX₁) receptors are detected in the CA1 regions. Previous behavioral studies have shown that microinjection of OX₁ receptor antagonist into the hippocampus impairs acquisition and consolidation of spatial memory. However, up to now, little has been known about the direct electrophysiological effects of orexin‐A on hippocampal CA1 neurons. Employing multibarrel single‐unit extracellular recordings, the present study showed that micropressure administration of orexin‐A significantly increased the spontaneous firing rate from 2.96 ± 0.85 to 8.45 ± 1.86 Hz (P < 0.001) in 15 out of the 23 hippocampal CA1 neurons in male rats. Furthermore, application of the specific OX₁ receptor antagonist SB‐334867 alone significantly decreased the firing rate from 4.02 ± 1.08 to 2.11 ± 0.58 Hz in 7 out of the 17 neurons (P < 0.05), suggesting that endogenous orexinergic systems modulate the firing activity of CA1 neurons. Coapplication of SB‐334867 completely blocked orexin‐A–induced excitation of hippocampal CA1 neurons. The PLC pathway may be involved in activation of OX₁ receptor–induced excitation of CA1 neurons. Taken together, the present study's results suggest that orexin‐A produces excitatory effects on hippocampal neurons via OX₁ receptors. © 2016 Wiley Periodicals, Inc.     

Subregion‐specific decreases in hippocampal serotonin transporter protein expression and function associated with endophenotypes of depression

Stress influences the development of depression, and depression is associated with structural and functional changes in the hippocampus. The current study sought to determine whether chronic corticosteroid (CORT) treatment influences serotonin transporter (5‐HTT) protein expression and function in the CA1, CA3, and dentate gyrus (DG) subregions of the hippocampus. Male CD‐1 mice were subcutaneously injected with CORT at a dose of 20 mg/kg once daily for 3 weeks. Behavioral state was assessed using sucrose preference, physical state of the coat, forced swimming test, and tail suspension test. We then determine 5‐HTT protein expression and synaptosomal 5‐HT uptake in the CA1, CA3 and DG subregions. CORT treatment induced anhedonia and behavioral despair, two core endophenotypes of clinical depression; 5‐HTT protein expression levels and synaptosomal 5‐HT uptake were both decreased in a subregion‐specific manner, with the greatest decrease observed in the DG, a moderate decrease in the CA3, and the CA1 showed no apparent change. In addition, a reduction in tissue mass was detected in the DG following the CORT treatment. These data indicate that subregion‐specific decreases in hippocampal 5‐HTT protein expression and function are associated with endophenotypes of depression. © 2014 Wiley Periodicals, Inc.     

Behavior‐driven arc expression is reduced in all ventral hippocampal subfields compared to CA1, CA3, and dentate gyrus in rat dorsal hippocampus

Anatomical connectivity and lesion studies reveal distinct functional heterogeneity along the dorsal–ventral axis of the hippocampus. The immediate early gene Arc is known to be involved in neural plasticity and memory and can be used as a marker for cell activity that occurs, for example, when hippocampal place cells fire. We report here, that Arc is expressed in a greater proportion of cells in dorsal CA1, CA3, and dentate gyrus (DG), following spatial behavioral experiences compared to ventral hippocampal subregions (dorsal CA1 = 33%; ventral CA1 = 13%; dorsal CA3 = 23%; ventral CA3 = 8%; and dorsal DG = 2.5%; ventral DG = 1.2%). The technique used here to obtain estimates of numbers of behavior‐driven cells across the dorsal–ventral axis, however, corresponds quite well with samples from available single unit recording studies. Several explanations for the two‐ to‐threefold reduction in spatial behavior‐driven cell activity in the ventral hippocampus can be offered. These include anatomical connectivity differences, differential gain of the self‐motion signals that appear to alter the scale of place fields and the proportion of active cells, and possibly variations in the neuronal responses to non‐spatial information within the hippocampus along its dorso‐ventral axis.     

The 5‐hydroxytryptamine4 receptor enables differentiation of informational content and encoding in the hippocampus

Long‐term synaptic plasticity, represented by long‐term depression (LTD) and long‐term potentiation (LTP) comprise cellular processes that enable memory. Neuromodulators such as serotonin regulate hippocampal function, and the 5‐HT₄‐receptor contributes to processes underlying cognition. It was previously shown that in the CA1‐region, 5‐HT₄‐receptors regulate the frequency‐response relationship of synaptic plasticity: patterned afferent stimulation that has no effect on synaptic strength (i.e., a θm‐frequency), will result in LTP or LTD, when given in the presence of a 5‐HT₄‐agonist, or antagonist, respectively. Here, we show that in the dentate gyrus (DG) and CA3 regions of freely behaving rats, pharmacological manipulations of 5‐HT₄‐receptors do not influence responses generated at θm‐frequencies, but activation of 5‐HT₄‐receptors prevents persistent LTD in mossy fiber (mf)‐CA3, or perforant path‐DG synapses. Furthermore, the regulation by 5‐HT₄‐receptors of LTP is subfield‐specific: 5‐HT₄‐receptor‐activation prevents mf‐CA3‐LTP, but does not strongly affect DG‐potentiation. These data suggest that 5‐HT₄‐receptor activation prioritises information encoding by means of LTP in the DG and CA1 regions, and suppresses persistent information storage in mf‐CA3 synapses. Thus, 5‐HT₄‐receptors serve to shape information storage across the hippocampal circuitry and specify the nature of experience‐dependent encoding. © 2016 The Authors Hippocampus Published by Wiley Periodicals, Inc.     

Identification of neuropeptide receptors expressed by melanin‐concentrating hormone neurons

Melanin‐concentrating hormone (MCH) is a 19‐amino‐acid cyclic neuropeptide that acts in rodents via the MCH receptor 1 (MCHR1) to regulate a wide variety of physiological functions. MCH is produced by a distinct population of neurons located in the lateral hypothalamus (LH) and zona incerta (ZI), but MCHR1 mRNA is widely expressed throughout the brain. The physiological responses and behaviors regulated by the MCH system have been investigated, but less is known about how MCH neurons are regulated. The effects of most classical neurotransmitters on MCH neurons have been studied, but those of most neuropeptides are poorly understood. To gain insight into how neuropeptides regulate the MCH system, we investigated which neuropeptide receptors are expressed by MCH neurons by using double in situ hybridization. In all, 20 receptors, selected based on either a suspected interaction with the MCH system or demonstrated high expression levels in the LH and ZI, were tested to determine whether they are expressed by MCH neurons. Overall, 11 neuropeptide receptors were found to exhibit significant colocalization with MCH neurons: nociceptin/orphanin FQ opioid receptor (NOP), MCHR1, both orexin receptors (ORX), somatostatin receptors 1 and 2 (SSTR1, SSTR2), kisspeptin recepotor (KissR1), neurotensin receptor 1 (NTSR1), neuropeptide S receptor (NPSR), cholecystokinin receptor A (CCKAR), and the κ‐opioid receptor (KOR). Among these receptors, six have never before been linked to the MCH system. Surprisingly, several receptors thought to regulate MCH neurons displayed minimal colocalization with MCH, suggesting that they may not directly regulate the MCH system. J. Comp. Neurol., 522:3817–3833, 2014. © 2014 Wiley Periodicals, Inc.     

GET73 increases rat extracellular hippocampal CA1 GABA levels through a possible involvement of local mGlu5 receptor

N‐[(4‐trifluoromethyl) benzyl] 4‐methoxybutyramide (GET73) is a newly synthesized compound displaying anti‐alcohol and anxiolytic properties. In light of the importance of the hippocampal CA1 subregion in alcohol addiction and anxiety‐like behaviors—this in vivo microdialysis study characterized the effect of GET73 on extracellular GABA levels in the hippocampal CA1 region of the freely moving rat—including a possible role for mGlu5 receptor in mediating this effect. Both intraperitoneal administration (2–10 mg/kg) and local intra‐hippocampal CA1 perfusion with GET73 (50–1000 nM) were associated with a transient, step‐wise increase in dialysate hippocampal CA1 GABA levels. The GET73 (10 mg/kg)‐induced increase in GABA levels was not affected by intra‐CA1 perfusion with either the GABA reuptake inhibitor SKF89976A (0.5 mM) or by local GABA_A (bicuculline; 1μM) and GABA_B (CGP35348; 500 μM) receptor antagonists. On the contrary, the GET73‐induced increase in GABA levels was partially counteracted by the intra‐CA1 perfusion with the mGlu5 receptor negative allosteric modulator MPEP (300 µM). Interestingly, GET73 at the lowest (2 mg/kg) dose tested, by itself ineffective, fully counteracted the increase in GABA levels induced by the mGlu5 receptor agonist CHPG (1000 µM). Taken together, these findings suggest that the GET73‐induced increase in hippocampal CA1 GABA levels operates independently of local GABA reuptake and/or GABA_A or GABA_B receptors. Furthermore, the present data lead to hypothesize a possible interaction between GET73 and mGluR5‐mediated regulation of hippocampal CA1 GABA transmission, an effect which may be relevant to the ability of GET73 to reduce alcohol intake in an alcohol‐preferring rat strain. Synapse 67:678–691, 2013 . © 2013 Wiley Periodicals, Inc.     

Anatomical characterization of the cannabinoid CB1 receptor in cell‐type–specific mutant mouse rescue models

Type 1 cannabinoid (CB₁) receptors are widely distributed in the brain. Their physiological roles depend on their distribution pattern, which differs remarkably among cell types. Hence, subcellular compartments with little but functionally relevant CB₁ receptors can be overlooked, fostering an incomplete mapping. To overcome this, knockin mice with cell‐type–specific rescue of CB₁ receptors have emerged as excellent tools for investigating CB₁ receptors’ cell‐type–specific localization and sufficient functional role with no bias. However, to know whether these rescue mice maintain endogenous CB₁ receptor expression level, detailed anatomical studies are necessary. The subcellular distribution of hippocampal CB₁ receptors of rescue mice that express the gene exclusively in dorsal telencephalic glutamatergic neurons (Glu‐CB₁‐RS) or GABAergic neurons (GABA‐CB₁‐RS) was studied by immunoelectron microscopy. Results were compared with conditional CB₁ receptor knockout lines. As expected, CB₁ immunoparticles appeared at presynaptic plasmalemma, making asymmetric and symmetric synapses. In the hippocampal CA1 stratum radiatum, the values of the CB₁ receptor‐immunopositive excitatory and inhibitory synapses were Glu‐CB₁‐RS, 21.89% (glutamatergic terminals); 2.38% (GABAergic terminals); GABA‐CB₁‐RS, 1.92% (glutamatergic terminals); 77.92% (GABAergic terminals). The proportion of CB₁ receptor‐immunopositive excitatory and inhibitory synapses in the inner one‐third of the dentate molecular layer was Glu‐CB₁‐RS, 53.19% (glutamatergic terminals); 2.30% (GABAergic terminals); GABA‐CB₁‐RS, 3.19% (glutamatergic terminals); 85.07% (GABAergic terminals). Taken together, Glu‐CB₁‐RS and GABA‐CB₁‐RS mice show the usual CB₁ receptor distribution and expression in hippocampal cell types with specific rescue of the receptor, thus being ideal for in‐depth anatomical and functional investigations of the endocannabinoid system. J. Comp. Neurol. 525:302–318, 2017. © 2016 Wiley Periodicals, Inc.     

Impaired maze performance in aged rats is accompanied by increased density of NMDA, 5-HT1A, and alpha-adrenoceptor binding in hippocampus

Using quantitative receptor autoradiography, we assessed binding site densities and distribution patterns of glutamate, GABA(A), acetylcholine (ACh), and monoamine receptors in the hippocampus of 32-month-old Fischer 344/Brown Norway rats. Prior to autoradiography, the rats were divided into two groups according to their retention performance in a water maze reference memory task, which was assessed 1 week after 8 days of daily maze training. The animals of the inferior group showed less long-term retention of the hidden-platform task but did not differ from superior rats in their navigation performance during place training and cued trials. The decreased retention performance in the group of inferior learners was primarily accompanied by increased alpha(1)-adrenoceptors in all hippocampal subregions under inspection (CA1-CA4 and dentate gyrus), while elevated alpha(2)-adrenoceptor binding was observed in the CA1 region and DG. Furthermore, inferior learners had higher NMDA binding in the CA2 and CA4 and increased 5-HT(1A) binding sites in the CA2, CA3, and CA4 region. No significant differences between inferior and superior learners were evident with regard to AMPA, kainate, GABA(A), muscarinergic M(1), dopamine D(1), and 5-HT(2) binding densities in any hippocampal region analyzed. These results show that increased NMDA, 5-HT(1A), and alpha-adrenoceptor binding in the hippocampus is associated with a decline in spatial memory. The increased receptor binding observed in the group of old rats with inferior maze performance might be the result of neural adaptation triggered by age-related changes in synaptic connectivity and/or synaptic activity.     

Depressive mood modulates the anterior lateral CA1 and DG/CA3 during a pattern separation task in cognitively intact individuals: A functional MRI study

Although patients with major depressive disorder typically have a reduced hippocampal volume, particularly in the cornu ammonis 1 (CA1), animal studies suggest that depressive mood is related to the dentate gyrus (DG). In this study, our objective was to clarify which hippocampal subregions are functionally associated with depressive mood in humans. We conducted a functional MRI (fMRI) study on 27 cognitively intact volunteers. Subjects performed a modified version of a delayed matching‐to‐sample task in an MRI scanner to investigate pattern separation‐related activity during each phase of encoding, delay, and retrieval. In each trial, subjects learned a pair of sample cues. Functional MR images were acquired at a high spatial resolution, focusing on the hippocampus. Subjects also completed the Beck Depression Inventory (BDI), a questionnaire about depressive mood. Depending on the similarity between sample cues, activity in the DG/CA3 and medial CA1 in the anterior hippocampus changed only during encoding. Furthermore, the DG/CA3 region was more active during successful encoding trials compared to false trials. Activity in the DG/CA3 and lateral CA1 was negatively correlated with BDI scores. These results suggest that the DG/CA3 is the core region for pattern separation during the encoding phase and interacts with the medial CA1, depending on the similarity of the stimuli, to achieve effective encoding. Impaired activity in the DG/CA3, as well as in the lateral CA1, was found to be associated with depressive symptoms, even at a subclinical level. © 2013 Wiley Periodicals, Inc.     

Protective role of neuropeptide Y Y2 receptors in cell death and microglial response following methamphetamine injury

It has been reported that the hippocampus is very susceptible to methamphetamine (METH) and that neuropeptide Y (NPY) is an important neuroprotective agent against hippocampal excitotoxicity. However, there is very little information regarding the role of the NPYergic system in this brain region under conditions of METH toxicity. To clarify this issue, we investigated the role of NPY and its receptors against METH‐induced neuronal cell death in hippocampal organotypic slice cultures. Our data show that NPY (1 μm ) is neuroprotective in DG, CA3 and CA1 subregions via Y₂ receptors. Moreover, the selective activation of Y₁ receptors (1 μm [Leu³¹,Pro³⁴]NPY) partially prevented the toxicity induced by METH in DG and CA3 subfields, but completely blocked its toxicity in the CA1 pyramidal cell layer. Regarding Y₂ receptors, its activation (300 nm NPY13–36) completely prevented METH‐induced toxicity in all subregions analysed, which involved changes in levels of pro‐ and anti‐apoptotic proteins Bcl‐2 and Bax, respectively. Besides neuronal cell death, we also showed that METH triggers a microglial response in the mouse hippocampus which was attenuated by Y₂ receptor activation. To better clarify the effect of METH and the NPY system on microglial cells, we further used the N9 microglial cell line. We found that both NPY and the Y₂ receptor agonist were able to protect microglia against METH‐induced cell death. Overall, our data demonstrate that METH is toxic to both neurons and microglial cells, and that NPY, mainly via Y₂ receptors, has an important protective role against METH‐induced cell death and microgliosis.     

Prepubertal castration‐associated developmental changes in sigma‐1 receptor gene expression levels regulate hippocampus area CA1 activity during adolescence

The functional relevance of sigma‐1 (σ₁) receptor expression in the rat hippocampal CA1 during adolescence (i.e., 35–60 days old) was explored. A selective antagonist for the σ₁ receptor subtype, BD‐1047, was applied to study hippocampal long‐term potentiation (LTP) and spatial learning performance. Changes in the expression of the σ₁ receptor subtype and its function were compared between castrated and sham‐castrated rats. Castration reduced the magnitude of both field excitatory postsynaptic potential (fEPSP)‐LTP and population spike (PS)‐LTP at 35 days (d). BD‐1047 decreased PS‐LTP in sham‐castrated rats, whereas BD‐1047 reversed the effect of castration on fEPSP‐LTP at 35 d. In addition, BD1047 impaired spatial learning and augmented σ₁ receptor mRNA levels in castrated rats at 35 d. Surprisingly, neither castration nor BD1047 had an effect on fEPSP‐LTP and PS‐LTP, spatial learning ability or gene expression levels at 45 d. Castration had no effect on fEPSP‐LTP but reduced PS‐LTP at 60 d. BD1047 increased the magnitude of fEPSP‐LTP, but had no effect on PS‐LTP in castrated rats at 60 d. However, BD1047 reduced spatial learning ability, and σ₁ receptor mRNA levels were decreased in castrated rats at 60 d. This study shows that σ₁ receptors play a role in the regulation of both CA1 synaptic efficacy and spatial learning performance. The regulatory role of σ₁ receptors in activity‐dependent CA1‐LTP is locality‐ and age‐dependent, whereas its role in spatial learning ability is only age‐dependent. Prepubertal castration‐associated changes in the expression and function of the σ₁ receptor during adolescence may play a developmental role in the regulation of hippocampal area CA1 activity and plasticity. © 2016 Wiley Periodicals, Inc.     

Inhibition of GABA release by presynaptic ionotropic GABA receptors in hippocampal CA3

Vesicular transmitter release can be regulated by transmitter-gated ion channels at presynaptic axon terminals. The central inhibitory transmitter GABA acts on such presynaptic ionotropic receptors in various cells, including inhibitory interneurons. Here we report that GABA-mediated postsynaptic inhibitory currents in CA3 pyramidal cells of rat hippocampal slices are suppressed by agonists of GABAA receptors. The effect is present for both stimulus-induced and miniature IPSCs, indicating a reduction in the probability of vesicular release by presynaptic, action-potential-independent mechanisms. We conclude that the release of GABA from hippocampal CA3 interneurons is regulated by a negative feedback via presynaptic ionotropic GABA autoreceptors.     

New boundaries and dissociation of the mouse hippocampus along the dorsal‐ventral axis based on glutamatergic,GABAergic and catecholaminergic receptor densities

In rodents, gene‐expression, neuronal tuning, connectivity and neurogenesis studies have postulated that the dorsal, the intermediate and the ventral hippocampal formation (HF) are distinct entities. These findings are underpinned by behavioral studies showing a dissociable role of dorsal and ventral HF in learning, memory, stress and emotional processing. However, up to now, the molecular basis of such differences in relation to discrete boundaries is largely unknown. Therefore, we analyzed binding site densities for glutamatergic AMPA, NMDA, kainate and mGluR_2/3, GABAergic GABA_A (including benzodiazepine binding sites), GABA_B, dopaminergic D_1/5 and noradrenergic α₁ and α₂ receptors as key modulators for signal transmission in hippocampal functions, using quantitative in vitro receptor autoradiography along the dorsal‐ventral axis of the mouse HF. Beside general different receptor profiles of the dentate gyrus (DG) and Cornu Ammonis fields (CA1, CA2, CA3, CA4/hilus), we detected substantial differences between dorsal, intermediate and ventral subdivisions and individual layers for all investigated receptor types, except GABA_B. For example, striking higher densities of α₂ receptors were detected in the ventral DG, while the dorsal DG possesses higher numbers of kainate, NMDA, GABA_A and D_1/5 receptors. CA1 dorsal and intermediate subdivisions showed higher AMPA, NMDA, mGluR_2/3, GABA_A, D_1/5 receptors, while kainate receptors are higher expressed in ventral CA1, and noradrenergic α₁ and α₂ receptors in the intermediate region of CA1. CA2 dorsal was distinguished by higher kainate, α₁ and α₂ receptors in the intermediate region, while CA3 showed a more complex dissociation. Our findings resulted not only in a clear segmentation of the mouse hippocampus along the dorsal‐ventral axis, but also provides insights into the neurochemical basis and likely associated physiological processes in hippocampal functions. Therein, the presented data has a high impact for future studies modeling and investigating dorsal, intermediate and ventral hippocampal dysfunction in relation to neurodegenerative diseases or psychiatric disorders.     

Differential effects of the group II mGluR agonist, DCG-IV, on depolarization-induced suppression of inhibition in hippocampal CA1 and CA3 neurons

We investigated the role of metabotropic glutamate receptors in the mediation of depolarization-induced suppression of inhibition (DSI), using whole-cell electrophysiological techniques in rat hippocampal slice preparation. In a previous work, we showed that a retrograde signal travels from CA1 pyramidal cells to GABA interneurons and prevents them from releasing GABA for tens of seconds at 30 degrees C. The resulting suppression of inhibition is DSI. The retrograde signal appeared to be glutamate, or a glutamate analog, which acted on group I metabotropic receptors on the interneurons. It is not known if DSI occurs in hippocampal subregions besides CA1. If DSI does occur in other regions, it will be important to know if the role of metabotropic glutamate receptors (mGluRs) in mediating DSI is the same everywhere. The distribution of mGluR subtypes varies among hippocampal subregions. In the CA3 region, unlike CA1, group II mGluRs are prevalent. It was possible, therefore, that in CA3, the group II mGluRs would mediate DSI. We have begun to investigate these issues. We now report that: 1) DSI does occur in CA3. 2) Carbachol induces IPSC activity that can be recorded in CA1 and CA3a. This carbachol-induced activity can be reduced by the selective group II mGluR agonist, DCG-IV, and by DSI. 3) Evoked IPSCs in CA3a, but not in CA1, can be reduced by DCG-IV; hence the interneurons activated by carbachol may reside in CA3a. 4) Despite the group II mGluR agonist sensitivity of CA3a interneurons, DSI in this region is not affected by a group II mGluR antagonist, CPPG, and therefore does not appear to be mediated by group II mGluRs.     

Distribution of corticotropin‐releasing factor (CRF) receptor binding in the mouse brain using a new,high‐affinity radioligand, [125I]‐PD‐Sauvagine

The corticotropin‐releasing factor (CRF) family of peptides includes CRF and three urocortins, which signal through two distinct G‐protein coupled receptors, CRF₁ and CRF₂. Although the cellular distribution of CRF receptor expression has been well characterized at the mRNA level, the localization of receptor protein, and, by inference, of functional receptors, has been limited by a lack of reliable immunohistochemical evidence. Recently, a CRF‐related peptide, termed PD‐sauvagine, was isolated from the skin of the frog, Pachymedusa dacnicolor, and validated as a high‐affinity ligand for CRF receptor studies. A radiolabeled analog, [¹²⁵I]‐PD‐sauvagine, with high signal‐to‐noise ratio, was used in autoradiographic studies to map the distribution of CRF receptor binding sites in the mouse brain. Through the use of receptor‐deficient mice and subtype‐specific antagonists, CRF₁ and CRF₂ binding sites were isolated, and found to be readily reconcilable with regional patterns of mRNA expression. Binding site distributions within a given structure sometimes differed from mRNA patterns, however, particularly in laminated structures of the isocortex, hippocampus, and cerebellum, presumably reflecting the trafficking of receptors to their operational homes on neuronal (mostly dendritic) processes. Binding patterns of [¹²⁵I]‐PD‐sauvagine provided independent assessments of controversial receptor localizations, failing to provide support for CRF₁ expression in central autonomic components of the limbic forebrain, the locus coeruleus and cerebellar Purkinje cells, or for CRF₂ in any aspect of the cerebellar cortex. Though lacking in ideal resolution, in vitro binding of the PD‐sauvagine radioligand currently provides the most sensitive and accurate available tool for localizing CRF receptors in rodent brain.     

Rapamycin has age‐, treatment paradigm‐, and model‐specific anticonvulsant effects and modulates neuropeptide Y expression in rats

Purpose: Rapamycin (RAP) has certain antiepileptogenic features. However, it is unclear whether these effects can be explained by the anticonvulsant action of RAP, which has not been studied. To address this question, we tested potential anticonvulsant effects of RAP in immature and adult rats using different seizure models and treatment paradigms. In addition, we studied changes in the expression of neuropeptide Y (NPY) induced by RAP, which may serve as an indirect target of the RAP action. Methods: A complex approach was adopted to evaluate the anticonvulsant potential of RAP: We used flurothyl‐, pentylenetetrazole (PTZ)–, N‐methyl‐d ‐aspartate (NMDA)–, and kainic acid (KA)–induced seizures to test the effects of RAP using different pretreatment protocols in immature and adult rats. We also evaluated expression of NPY within the primary motor cortex, hippocampal CA1, and dentate gyrus (DG) after different pretreatments with RAP in immature rats. Key Findings: We found the following: (1) RAP administered with short‐term pretreatment paradigms has a weak anticonvulsant potential in the seizure models with compromised inhibition. (2) Lack of RAP efficacy correlates with decreased NPY expression in the cortex, CA1, and DG. Specifically in immature rats, a single dose of RAP (3 mg/kg) 4 or 24 h before seizure testing had anticonvulsant effects against PTZ‐induced seizures. In the flurothyl seizure model only the 4‐h pretreatment with RAP was anticonvulsant in the both age groups. Short‐term pretreatments with RAP had no effects against NMDA‐ and KA‐induced seizures tested in immature rats. Long‐term pretreatments with RAP over 8 days did not show beneficial effect in all tested seizure models in developing rats. Moreover, the long‐term pretreatment with RAP had a slight proconvulsant effect on KA‐induced seizures. In immature rats, any lack of anticonvulsant effect (including proconvulsant effect of multiple doses of RAP) was associated with downregulation of NPY expression in the cortex and DG. In immature animals, after a single dose of RAP with 24 h delay, we found a decrease of NPY expression in DG, and CA1 as well. Significance: Our data show weak age‐, treatment paradigm‐, and model‐specific anticonvulsant effects of RAP as well as loss of those effects after long‐term RAP pretreatment associated with downregulation of NPY expression. These findings suggest that RAP is a poor anticonvulsant and may have beneficial effects only against epileptogenesis. In addition, our data present new insights into mechanisms of RAP action on seizures indicating a possible connection between mammalian target of rapamycin (mTOR) signaling and NPY system.     

Autism‐associated Shank3 mutations alter mGluR expression and mGluR‐dependent but not NMDA receptor‐dependent long‐term depression

SHANK3 is a postsynaptic structural protein localized at excitatory glutamatergic synapses in which deletions and mutations have been implicated in patients with autism spectrum disorders (ASD). The expression of Shank3 ASD mutations causes impairments in ionotropic glutamate receptor‐mediated synaptic responses in neurons, which is thought to underlie ASD‐related behaviors, thereby indicating glutamatergic synaptopathy as one of the major pathogenic mechanisms. However, little is known about the functional consequences of ASD‐associated mutations in Shank3 on another important set of glutamate receptors, group I metabotropic glutamate receptors (mGluRs). Here, we further assessed how Shank3 mutations identified in patients with ASD (one de novo InsG mutation and two inherited point mutations, R87C and R375C) disrupt group I mGluR (mGluR1 and mGluR5) expression and function. To identify potential isoform‐specific deficits induced by ASD‐associated Shank3 mutations on group I mGluRs, we surface immunolabeled mGluR1 and mGluR5 independently. We also induced mGluR‐dependent synaptic plasticity (R,S‐3,5‐dihydroxyphenylglycine [DHPG]‐induced long‐term depression [LTD]) as well as N‐methyl‐D‐aspartate receptor (NMDAR)‐dependent LTD. ASD‐associated mutations in Shank3 differentially interfered with the ability of cultured hippocampal neurons to express mGluR5 and mGluR1 at synapses. Intriguingly, all ASD Shank3 mutations impaired mGluR‐dependent LTD without altering NMDAR‐dependent LTD. Our data show that the specific perturbation in mGluR‐dependent synaptic plasticity occurs in neurons expressing ASD‐associated Shank3 mutations, which may underpin synaptic dysfunction and subsequent behavioral deficits in ASD.