Functional protection in J20/VLW mice: a model of non-demented with Alzheimer's disease neuropathology.

Alzheimer's disease comprises amyloid-ß and hyperphosphorylated Tau accumulation, imbalanced neuronal activity, aberrant oscillatory rhythms and cognitive deficits. Non-demented with Alzheimer's disease neuropathology defines a novel clinical entity with amyloid-ß and Tau pathologies but preserved cognition. The mechanisms underlying such neuroprotection remain undetermined and animal models of non-demented with Alzheimer's disease neuropathology are currently unavailable. We demonstrate that J20/VLW mice (accumulating amyloid-ß and hyperphosphorylated Tau) exhibit preserved hippocampal rhythmic activity and cognition, as opposed to J20 and VLW animals, which show significant alterations. Furthermore, we show that the overexpression of mutant human Tau in coexistence with amyloid-ß accumulation renders a particular hyperphosphorylated Tau signature in hippocampal interneurons. The GABAergic septohippocampal pathway, responsible for hippocampal rhythmic activity, is preserved in J20/VLW mice, in contrast to single mutants. Our data highlight J20/VLW mice as a suitable animal model in which to explore the mechanisms driving cognitive preservation in non-demented with Alzheimer's disease neuropathology. Moreover, they suggest that a differential Tau phosphorylation pattern in hippocampal interneurons prevents the loss of GABAergic septohippocampal innervation and alterations in local field potentials, thereby avoiding cognitive deficits.

Amyloid ß-Peptide Causes the Permanent Activation of CaMKIIα through Its Oxidation.

Alzheimer's disease (AD) is characterised by the presence of extracellular amyloid plaques in the brain. They are composed of aggregated amyloid beta-peptide (Aß) misfolded into beta-sheets which are the cause of the AD memory impairment and dementia. Memory depends on the hippocampal formation and maintenance of synapses by long-term potentiation (LTP), whose main steps are the activation of NMDA receptors, the phosphorylation of CaMKIIα and the nuclear translocation of the transcription factor CREB. It is known that Aß oligomers (oAß) induce synaptic loss and impair the formation of new synapses. Here, we have studied the effects of oAß on CaMKIIα. We found that oAß produce reactive oxygen species (ROS), that induce CaMKIIα oxidation in human neuroblastoma cells as we assayed by western blot and immunofluorescence. Moreover, this oxidized isoform is significantly present in brain samples from AD patients. We found that the oxidized CaMKIIα is active independently of the binding to calcium/calmodulin, and that CaMKIIα phosphorylation is mutually exclusive with CaMKIIα oxidation as revealed by immunoprecipitation and western blot. An in silico modelling of the enzyme was also performed to demonstrate that oxidation induces an activated state of CaMKIIα. In brains from AD transgenic models of mice and in primary cultures of murine hippocampal neurons, we demonstrated that the oxidation of CaMKIIα induces the phosphorylation of CREB and its translocation to the nucleus to promote the transcription of ARC and BDNF. Our data suggests that CaMKIIα oxidation would be a pro-survival mechanism that is triggered when a noxious stimulus challenges neurons as do oAß.

Cannabinoid type-1 receptor blockade restores neurological phenotypes in two models for Down syndrome.

Intellectual disability is the most limiting hallmark of Down syndrome, for which there is no gold-standard clinical treatment yet. The endocannabinoid system is a widespread neuromodulatory system involved in multiple functions including learning and memory processes. Alterations of this system contribute to the pathogenesis of several neurological and neurodevelopmental disorders. However, the involvement of the endocannabinoid system in the pathogenesis of Down syndrome has not been explored before. We used the best-characterized preclinical model of Down syndrome, the segmentally trisomic Ts65Dn model. In male Ts65Dn mice, cannabinoid type-1 receptor (CB1R) expression was enhanced and its function increased in hippocampal excitatory terminals. Knockdown of CB1R in the hippocampus of male Ts65Dn mice restored hippocampal-dependent memory. Concomitant with this result, pharmacological inhibition of CB1R restored memory deficits, hippocampal synaptic plasticity and adult neurogenesis in the subgranular zone of the dentate gyrus. Notably, the blockade of CB1R also normalized hippocampal-dependent memory in female Ts65Dn mice. To further investigate the mechanisms involved, we used a second transgenic mouse model overexpressing a single gene candidate for Down syndrome cognitive phenotypes, the dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A). CB1R pharmacological blockade similarly improved cognitive performance, synaptic plasticity and neurogenesis in transgenic male Dyrk1A mice. Our results identify CB1R as a novel druggable target potentially relevant for the improvement of cognitive deficits associated with Down syndrome.

Monoacylglycerol lipase blockade impairs fine motor coordination and triggers cerebellar neuroinflammation through cyclooxygenase-2.

Monoacylglycerol lipase (MAGL) is the main enzyme implicated in the degradation of the most abundant endocannabinoid in the brain, 2-arachidonoylglycerol (2-AG), producing arachidonic acid (AA) and glycerol. MAGL pharmacological inhibition with JZL184 or genetic deletion results in an exacerbated 2-AG signaling and reduced synthesis of prostaglandins (PGs), due to the reduced AA precursor levels. We found that acute JZL184 administration, previously described to exert anti-inflammatory effects, and MAGL knockout (KO) mice display cerebellar, but not hippocampal, microglial reactivity, accompanied with increased expression of the mRNA levels of neuroinflammatory markers, such as cyclooxygenase-2 (COX-2). Notably, this neuroinflammatory phenotype correlated with relevant motor coordination impairment in the beam-walking and the footprint tests. Treatment with the COX-2 inhibitor NS398 during 5 days prevented the deficits in cerebellar function and the cerebellar microglia reactivity in MAGL KO, without affecting hippocampal reactivity. Altogether, this study reveals the brain region-specific response to MAGL inhibition, with an important role of COX-2 in the cerebellar deficits associated, which should be taken into account for the use of MAGL inhibitors as anti-inflammatory drugs.

Peripheral and central CB1 cannabinoid receptors control stress-induced impairment of memory consolidation.

Stressful events can generate emotional memories linked to the traumatic incident, but they also can impair the formation of nonemotional memories. Although the impact of stress on emotional memories is well studied, much less is known about the influence of the emotional state on the formation of nonemotional memories. We used the novel object-recognition task as a model of nonemotional memory in mice to investigate the underlying mechanism of the deleterious effect of stress on memory consolidation. Systemic, hippocampal, and peripheral blockade of cannabinoid type-1 (CB1) receptors abolished the stress-induced memory impairment. Genetic deletion and rescue of CB1 receptors in specific cell types revealed that the CB1 receptor population specifically in dopamine ß-hydroxylase (DBH)-expressing cells is both necessary and sufficient for stress-induced impairment of memory consolidation, but CB1 receptors present in other neuronal populations are not involved. Strikingly, pharmacological manipulations in mice expressing CB1 receptors exclusively in DBH(+) cells revealed that both hippocampal and peripheral receptors mediate the impact of stress on memory consolidation. Thus, CB1 receptors on adrenergic and noradrenergic cells provide previously unrecognized cross-talk between central and peripheral mechanisms in the stress-dependent regulation of nonemotional memory consolidation, suggesting new potential avenues for the treatment of cognitive aspects on stress-related disorders.

Peripheral CB1 receptor blockade acts as a memory enhancer through a noradrenergic mechanism.

Peripheral inputs continuously shape brain function and can influence memory acquisition, but the underlying mechanisms have not been fully understood. Cannabinoid type-1 receptor (CB1R) is a well-recognized player in memory performance, and its systemic modulation significantly influences memory function. By assessing low arousal/non-emotional recognition memory in mice, we found a relevant role of peripheral CB1R in memory persistence. Indeed, the peripherally-restricted CB1R specific antagonist AM6545 showed significant mnemonic effects that were occluded in adrenalectomized mice, and after peripheral adrenergic blockade. AM6545 also transiently impaired contextual fear memory extinction. Vagus nerve chemogenetic inhibition reduced AM6545-induced mnemonic effect. Genetic CB1R deletion in dopamine ß-hydroxylase-expressing cells enhanced recognition memory persistence. These observations support a role of peripheral CB1R modulating adrenergic tone relevant for cognition. Furthermore, AM6545 acutely improved brain connectivity and enhanced extracellular hippocampal norepinephrine. In agreement, intra-hippocampal ß-adrenergic blockade prevented AM6545 mnemonic effects. Altogether, we disclose a novel CB1R-dependent peripheral mechanism with implications relevant for lengthening the duration of non-emotional memory.

Auricular Transcutaneous Vagus Nerve Stimulation Acutely Modulates Brain Connectivity in Mice.

Brain electrical stimulation techniques take advantage of the intrinsic plasticity of the nervous system, opening a wide range of therapeutic applications. Vagus nerve stimulation (VNS) is an approved adjuvant for drug-resistant epilepsy and depression. Its non-invasive form, auricular transcutaneous VNS (atVNS), is under investigation for applications, including cognitive improvement. We aimed to study the effects of atVNS on brain connectivity, under conditions that improved memory persistence in CD-1 male mice. Acute atVNS in the cymba conchae of the left ear was performed using a standard stimulation protocol under light isoflurane anesthesia, immediately or 3 h after the training/familiarization phase of the novel object-recognition memory test (NORT). Another cohort of mice was used for bilateral c-Fos analysis after atVNS administration. Spearman correlation of c-Fos density between each pair of the thirty brain regions analyzed allowed obtaining the network of significant functional connections in stimulated and non-stimulated control brains. NORT performance was enhanced when atVNS was delivered just after, but not 3 h after, the familiarization phase of the task. No alterations in c-Fos density were associated with electrostimulation, but a significant effect of atVNS was observed on c-Fos-based functional connectivity. atVNS induced a clear reorganization of the network, increasing the inter-hemisphere connections and the connectivity of locus coeruleus. Our results provide new insights into the effects of atVNS on memory performance and brain connectivity extending our knowledge of the biological mechanisms of bioelectronics in medicine.

Cannabinoid signaling modulation through JZL184 restores key phenotypes of a mouse model for Williams-Beuren syndrome.

Williams-Beuren syndrome (WBS) is a rare genetic multisystemic disorder characterized by mild-to-moderate intellectual disability and hypersocial phenotype, while the most life-threatening features are cardiovascular abnormalities. Nowadays, there are no pharmacological treatments to directly ameliorate the main traits of WBS. The endocannabinoid system (ECS), given its relevance for both cognitive and cardiovascular function, could be a potential druggable target in this syndrome. We analyzed the components of the ECS in the complete deletion (CD) mouse model of WBS and assessed the impact of its pharmacological modulation in key phenotypes relevant for WBS. CD mice showed the characteristic hypersociable phenotype with no preference for social novelty and poor short-term object-recognition performance. Brain cannabinoid type-1 receptor (CB1R) in CD male mice showed alterations in density and coupling with no detectable change in main endocannabinoids. Endocannabinoid signaling modulation with subchronic (10 days) JZL184, a selective inhibitor of monoacylglycerol lipase, specifically normalized the social and cognitive phenotype of CD mice. Notably, JZL184 treatment improved cardiovascular function and restored gene expression patterns in cardiac tissue. These results reveal the modulation of the ECS as a promising novel therapeutic approach to improve key phenotypic alterations in WBS.

Williams-Beuren syndrome (WBS) is a rare disorder that causes hyper-social behavior, intellectual disability, memory problems, and life-threatening overgrowth of the heart. Behavioral therapies can help improve the cognitive and social aspects of the syndrome and surgery is sometimes used to treat the effects on the heart, although often with limited success. However, there are currently no medications available to treat WBS. The endocannabinoid system ­ which consists of cannabis-like chemical messengers that bind to specific cannabinoid receptor proteins ­ has been shown to influence cognitive and social behaviors, as well as certain functions of the heart. This has led scientists to suspect that the endocannabinoid system may play a role in WBS, and drugs modifying this network of chemical messengers could help treat the rare condition. To investigate, Navarro-Romero, Galera-López et al. studied mice which had the same genetic deletion found in patients with WBS. Similar to humans, the male mice displayed hyper-social behaviors, had memory deficits and enlarged hearts. Navarro-Romero, Galera-López et al. found that these mutant mice also had differences in the function of the receptor protein cannabinoid type-1 (CB1). The genetically modified mice were then treated with an experimental drug called JZL184 that blocks the breakdown of endocannabinoids which bind to the CB1 receptor. This normalized the number and function of receptors in the brains of the WBS mice, and reduced their social and memory symptoms. The treatment also restored the animals' heart cells to a more normal size, improved the function of their heart tissue, and led to lower blood pressure. Further experiments revealed that the drug caused the mutant mice to activate many genes in their heart muscle cells to the same level as normal, healthy mice. These findings suggest that JZL184 or other drugs targeting the endocannabinoid system may help ease the symptoms associated with WBS. More studies are needed to test the drug's effectiveness in humans with this syndrome. Furthermore, the dramatic effect JZL184 has on the heart suggests that it might also help treat high blood pressure or conditions that cause the overgrowth of heart cells.

Role of the endocannabinoid system in a mouse model of Fragile X undergoing neuropathic pain.

BACKGROUND: Neuropathic pain is a complex condition characterized by sensory, cognitive and affective symptoms that magnify the perception of pain. The underlying pathogenic mechanisms are largely unknown and there is an urgent need for the development of novel medications. The endocannabinoid system modulates pain perception and drugs targeting the cannabinoid receptor type 2 (CB2) devoid of psychoactive side effects could emerge as novel analgesics. An interesting model to evaluate the mechanisms underlying resistance to pain is the fragile X mental retardation protein knockout mouse (Fmr1KO), a model of fragile X syndrome that exhibits nociceptive deficits and fails to develop neuropathic pain. METHODS: A partial sciatic nerve ligation was performed to wild-type (WT) and Fmr1KO mice having (HzCB2 and Fmr1KO-HzCB2, respectively) or not (WT and Fmr1KO mice) a partial deletion of CB2 to investigate the participation of the endocannabinoid system on the pain-resistant phenotype of Fmr1KO mice. RESULTS: Nerve injury induced canonical hypersensitivity in WT and HzCB2 mice, whereas this increased pain sensitivity was absent in Fmr1KO mice. Interestingly, Fmr1KO mice partially lacking CB2 lost this protection against neuropathic pain. Similarly, pain-induced depressive-like behaviour was observed in WT, HzCB2 and Fmr1KO-HzCB2 mice, but not in Fmr1KO littermates. Nerve injury evoked different alterations in WT and Fmr1KO mice at spinal and supra-spinal levels that correlated with these nociceptive and emotional alterations. CONCLUSIONS: This work shows that CB2 is necessary for the protection against neuropathic pain observed in Fmr1KO mice, raising the interest in targeting this receptor for the treatment of neuropathic pain. SIGNIFICANCE: Neuropathic pain is a complex chronic pain condition and current treatments are limited by the lack of efficacy and the incidence of important side effects. Our findings show that the pain-resistant phenotype of Fmr1KO mice against nociceptive and emotional manifestations triggered by persistent nerve damage requires the participation of the cannabinoid receptor CB2, raising the interest in targeting this receptor for neuropathic pain treatment. Additional multidisciplinary studies more closely related to human pain experience should be conducted to explore the potential use of cannabinoids as adequate analgesic tools.

Protein Kinase C-Gamma Knockout Mice Show Impaired Hippocampal Short-Term Memory While Preserved Long-Term Memory.

The brain encodes, stores, and retrieves relevant information in the form of memories that are classified as short-term (STM) and long-term memories (LTM) depending on the interval between acquisition and retrieval. It is classically accepted that STM undergo a consolidation process to form LTM, but the molecular determinants involved are not well understood. Among the molecular components relevant for memory formation, we focused our attention on the protein kinase C (PKC) family of enzymes since they control key aspects of the synaptic plasticity and memory. Within the different PKC isoforms, PKC-gamma has been specifically associated with learning and memory since mice lacking this isoform (PKC-gamma KO mice) showed mild cognitive impairment and deficits in hippocampal synaptic plasticity. We now reveal that PKC-gamma KO mice present a severe impairment in hippocampal-dependent STM using different memory tests including the novel object-recognition and novel place-recognition, context fear conditioning and trace fear conditioning. In contrast, no differences between genotypes were observed in an amygdala-dependent test, the delay fear conditioning. Strikingly, all LTM tasks that could be assessed 24 h after acquisition were not perturbed in the KO mice. The analysis of c-Fos expression in several brain areas after trace fear conditioning acquisition showed a blunted response in the dentate gyrus of PKC-gamma KO mice compared with WT mice, but such differences between genotypes were absent when the amygdala or the prefrontal cortex were examined. In the hippocampus, PKC-gamma was found to translocate to the membrane after auditory trace, but not after delay fear conditioning. Together, these results indicate that PKC-gamma dysfunction affects specifically hippocampal-dependent STM performance and disclose PKC-gamma as a molecular player differentially involved in STM and LTM processes.

An Alternative Maze to Assess Novel Object Recognition in Mice.

The novel object recognition (NOR) task is a behavioral test commonly used to evaluate episodic-like declarative memory and it relies on the innate tendency of rodents to explore novelty. Here we present a maze used to evaluate NOR memory in mice that reduces the time of the assay while improving reliability of the measurements by increasing the exploratory behavior. This memory test, being performed in a two-arms maze, is suitable for several strains of mice (including inbreed and outbreed) and does not require extended training sessions allowing an accurate temporal assessment of memory formation. This particular maze increases the mouse exploration time and reduces variability compared to other arenas used before to assess NOR. As both long- and short-term NOR memory can be easily and accurately quantified using this paradigm, this improved methodology can be easily applied to study pharmacological, genetic or age-related modulation of cognitive function.

Auricular transcutaneous vagus nerve stimulation improves memory persistence in naïve mice and in an intellectual disability mouse model.

BACKGROUND: Vagus nerve stimulation (VNS) using non-invasive approaches have attracted great attention due to their anti-epileptic, anti-depressive and pro-cognitive effects. It has been proposed that auricular transcutaneous VNS (atVNS) could benefit intellectual disability disorders, but preclinical data supporting this idea is limited. OBJECTIVE: To develop an atVNS device for mice and to test its efficacy on memory performance in naïve mice and in a mouse model for intellectual disability. METHODS: Naïve outbreed CD-1 mice and a model for fragile X syndrome, the Fmr1 knockout (Fmr1KO), were used to assess the effect of atVNS in the novel object-recognition memory performance. RESULTS: We found that atVNS significantly improves memory persistence in naïve mice. Notably, atVNS was efficacious in normalizing the object-recognition memory deficit in the Fmr1KO model. CONCLUSION: Our data show that atVNS improves memory persistence in naïve mice and in a model of intellectual disability and support further studies taking advantage of preclinical mouse models of cognitive disorders.

A phase 1, randomized double-blind, placebo controlled trial to evaluate safety and efficacy of epigallocatechin-3-gallate and cognitive training in adults with Fragile X syndrome.

BACKGROUND & AIMS: Despite the wide spectrum of experimental compounds tested in clinical trials, there is still no proven pharmacological treatment available for Fragile-X syndrome (FXS), since several targeted clinical trials with high expectations of success have failed to demonstrate significant improvements. Here we tested epigallocatechin-3-gallate (EGCG) as a treatment option for ameliorating core cognitive and behavioral features in FXS. METHODS: We conducted preclinical studies in Fmr1 knockout mice (Fmr1-/y) using novel object-recognition memory paradigm upon acute EGCG (10 mg/kg) administration. Furthermore we conducted a double-blind placebo-controlled phase I clinical trial (TESXF; NCT01855971). Twenty-seven subjects with FXS (18-55 years) were administered of EGCG (5-7 mg/kg/day) combined with cognitive training (CT) during 3 months with 3 months of follow-up after treatment discontinuation. RESULTS: Preclinical studies showed an improvement in memory using the Novel Object Recognition paradigm. We found that FXS patients receiving EGCG + CT significantly improved cognition (visual episodic memory) and functional competence (ABAS II-Home Living skills) in everyday life compared to subjects receiving Placebo + CT. CONCLUSIONS: Phase 2 clinical trials in larger groups of subjects are necessary to establish the therapeutic potential of EGCG for the improvement of cognition and daily life competences in FXS.

Use of the Vsoc-maze to Study Sociability and Preference for Social Novelty in Rodents.

Studying social behavior in mouse models empowers the understanding of the neurobiological mechanisms involved, which are affected in neuropsychiatric disorders, allowing the evaluation of therapeutic strategies. Behavioral methods available are time-consuming and reducing the length of behavioral sessions may render more manageable experiments and reduce animal stress. We validated a new reliable and sensitive method to study two features of social behavior (sociability and preference for social novelty) in two strains of male mice, the C57BL/6J inbreed strain and the CD1 (ICR) outbreed strain, using a modified version of the V-shaped maze (Vsoc-maze). The Vsoc-maze for sociability and preference for social novelty improves time performance by shortening the length of the sessions, and reduces variability compared to the classical approach performed in the three-chamber apparatus. Altogether, the Vsoc-maze allows evaluating the specific alterations of social behavior in mice in a time-efficient and reproducible manner.

The CD26-related dipeptidyl aminopeptidase-like protein DPPX is a critical component of neuronal A-type K+ channels.

Subthreshold-activating somatodendritic A-type potassium channels have fundamental roles in neuronal signaling and plasticity which depend on their unique cellular localization, voltage dependence, and kinetic properties. Some of the components of A-type K(+) channels have been identified; however, these do not reproduce the properties of the native channels, indicating that key molecular factors have yet to be unveiled. We purified A-type K(+) channel complexes from rat brain membranes and found that DPPX, a protein of unknown function that is structurally related to the dipeptidyl aminopeptidase and cell adhesion protein CD26, is a novel component of A-type K(+) channels. DPPX associates with the channels' pore-forming subunits, facilitates their trafficking and membrane targeting, reconstitutes the properties of the native channels in heterologous expression systems, and is coexpressed with the pore-forming subunits in the somatodendritic compartment of CNS neurons.

BDNF impairment in the hippocampus is related to enhanced despair behavior in CB1 knockout mice.

Stress can cause damage and atrophy of neurons in the hippocampus by deregulating the expression of neurotrophic factors that promote neuronal plasticity. The endocannabinoid system represents a physiological substrate involved in neuroprotection at both cellular and emotional levels. The lack of CB1 receptor alters neuronal plasticity and originates an anxiety-like phenotype in mice. In the present study, CB1 knockout mice exhibited an augmented response to stress revealed by the increased despair behavior and corticosterone levels showed in the tail suspension test and decreased brain derived neurotrophic factor (BDNF) levels in the hippocampus. Interestingly, local administration of BDNF in the hippocampus reversed the increased despair behavior of CB1 knockout mice, confirming the crucial role played by BDNF on the emotional impairment of these mutants. The neurotrophic deficiency seems to be specific for BDNF as no differences were found in the levels of nerve growth factor and NT-3, two additional neurotrophic factors. Moreover, BDNF impairment is not related to the activity of its specific tyrosine kinase receptor or the activity of the transcription factor cAMP responsive element binding. These results suggest that the lack of CB1 receptor originates an enhanced response to stress and deficiency in neuronal plasticity by decreasing BDNF levels in the hippocampus that lead to impairment in the responses to emotional disturbances.

Advances in the field of cannabinoid--opioid cross-talk.

A remarkable amount of literature has been generated demonstrating the functional similarities between the endogenous opioid and cannabinoid systems. Anatomical, biochemical and molecular data support the existence of reciprocal interactions between these two systems related to several pharmacological responses including reward, cognitive effects, and the development of tolerance and dependence. However, the assessment of the bidirectionality of these effects has been difficult due to their variety and complexity. Reciprocal interactions have been well established for the development of physical dependence. Cross-tolerance and cross-sensitization, although not always bidirectional, are also supported by a number of evidence, while less data have been gathered regarding the relationship of these systems in cognition and emotion. Nevertheless, the most recent advances in cannabinoid-opioid cross-modulation have been made in the area of drug craving and relapse processes. The present review is focused on the latest developments in the cannabinoid-opioid cross-modulation of their behavioural effects and the possible neurobiological substrates involved.

Serotonergic mechanisms involved in antidepressant-like responses evoked by GLT-1 blockade in rat infralimbic cortex.

Novel fast-acting antidepressant strategies, such as ketamine and deep brain stimulation, enhance glutamatergic neurotransmission in medial prefrontal cortex (mPFC) regions via AMPA receptor (AMPA-R) activation. We recently reported that the regionally-selective blockade of the glial glutamate transporter-1 (GLT-1) by dihydrokainic acid (DHK) microinfusion in rat infralimbic cortex (IL), the most ventral part of the mPFC, evoked immediate (10 min) antidepressant-like responses, which involved AMPA-R activation and were associated to increased serotonin (5-hydroxytryptamine, 5-HT) release. Given the reciprocal connectivity between the mPFC and the serotonergic dorsal raphe nucleus (DR), here we examined the serotoninergic mechanisms involved in the reported antidepressant-like responses of DHK microinfusion. First, we show that antidepressant-like responses evoked by IL application of DHK and citalopram are mediated by local 5-HT1A receptors (5-HT1A-R), since they are cancelled by previous IL WAY100635 microinfusion. Second, IL DHK microinfusion increases excitatory inputs onto DR, as shown by an increased glutamate and 5-HT release in DR and by a selective increase of c-Fos expression in DR 5-HT neurons, not occurring in putative GABAergic neurons. This view is also supported by an increased 5-HT release in ventral hippocampus following IL DHK microinfusion. Interestingly, antidepressant-like responses evoked by IL DHK lasted for 2 h and could be prolonged for up to 24 h by attenuating self-inhibitory effects via 5-HT1A autoreceptors. In contrast, the antidepressant-like effects of S-AMPA microinfusion in IL were short-lasting. Together, our results further support a prominent role of the IL-DR pathway and of ascending 5-HT pathways in mediating antidepressant-like responses evoked by glutamatergic mechanisms.

Hippocampal Protein Kinase C Signaling Mediates the Short-Term Memory Impairment Induced by Delta9-Tetrahydrocannabinol.

Cannabis affects cognitive performance through the activation of the endocannabinoid system, and the molecular mechanisms involved in this process are poorly understood. Using the novel object-recognition memory test in mice, we found that the main psychoactive component of cannabis, delta9-tetrahydrocannabinol (THC), alters short-term object-recognition memory specifically involving protein kinase C (PKC)-dependent signaling. Indeed, the systemic or intra-hippocampal pre-treatment with the PKC inhibitors prevented the short-term, but not the long-term, memory impairment induced by THC. In contrast, systemic pre-treatment with mammalian target of rapamycin complex 1 inhibitors, known to block the amnesic-like effects of THC on long-term memory, did not modify such a short-term cognitive deficit. Immunoblot analysis revealed a transient increase in PKC signaling activity in the hippocampus after THC treatment. Thus, THC administration induced the phosphorylation of a specific Ser residue in the hydrophobic-motif at the C-terminal tail of several PKC isoforms. This significant immunoreactive band that paralleled cognitive performance did not match in size with the major PKC isoforms expressed in the hippocampus except for PKCθ. Moreover, THC transiently enhanced the phosphorylation of the postsynaptic calmodulin-binding protein neurogranin in a PKC dependent manner. These data demonstrate that THC alters short-term object-recognition memory through hippocampal PKC/neurogranin signaling.

Distribution of Kv3.3 potassium channel subunits in distinct neuronal populations of mouse brain.

Kv3.3 proteins are pore-forming subunits of voltage-dependent potassium channels, and mutations in the gene encoding for Kv3.3 have recently been linked to human disease, spinocerebellar ataxia 13, with cerebellar and extracerebellar symptoms. To understand better the functions of Kv3.3 subunits in brain, we developed highly specific antibodies to Kv3.3 and analyzed immunoreactivity throughout mouse brain. We found that Kv3.3 subunits are widely expressed, present in important forebrain structures but particularly prominent in brainstem and cerebellum. In forebrain and midbrain, Kv3.3 expression was often found colocalized with parvalbumin and other Kv3 subunits in inhibitory neurons. In brainstem, Kv3.3 was strongly expressed in auditory and other sensory nuclei. In cerebellar cortex, Kv3.3 expression was found in Purkinje and granule cells. Kv3.3 proteins were observed in axons, terminals, somas, and, unlike other Kv3 proteins, also in distal dendrites, although precise subcellular localization depended on cell type. For example, hippocampal dentate granule cells expressed Kv3.3 subunits specifically in their mossy fiber axons, whereas Purkinje cells of the cerebellar cortex strongly expressed Kv3.3 subunits in axons, somas, and proximal and distal, but not second- and third-order, dendrites. Expression in Purkinje cell dendrites was confirmed by immunoelectron microscopy. Kv3 channels have been demonstrated to rapidly repolarize action potentials and support high-frequency firing in various neuronal populations. In this study, we identified additional populations and subcellular compartments that are likely to sustain high-frequency firing because of the expression of Kv3.3 and other Kv3 subunits.