Interleukin 1ß triggers synaptic and memory deficits in Herpes simplex virus type-1-infected mice by downregulating the expression of synaptic plasticity-related genes via the epigenetic MeCP2/HDAC4 complex.

Extensive research provides evidence that neuroinflammation underlies numerous brain disorders. However, the molecular mechanisms by which inflammatory mediators determine synaptic and cognitive dysfunction occurring in neurodegenerative diseases (e.g., Alzheimer's disease) are far from being fully understood. Here we investigated the role of interleukin 1ß (IL-1ß), and the molecular cascade downstream the activation of its receptor, to the synaptic dysfunction occurring in the mouse model of multiple Herpes simplex virus type-1 (HSV-1) reactivations within the brain. These mice are characterized by neuroinflammation and memory deficits associated with a progressive accumulation of neurodegenerative hallmarks (e.g., amyloid-ß protein and tau hyperphosphorylation). Here we show that mice undergone two HSV-1 reactivations in the brain exhibited increased levels of IL-1ß along with significant alterations of: (1) cognitive performances; (2) hippocampal long-term potentiation; (3) expression synaptic-related genes and pre- and post-synaptic proteins; (4) dendritic spine density and morphology. These effects correlated with activation of the epigenetic repressor MeCP2 that, in association with HDAC4, affected the expression of synaptic plasticity-related genes. Specifically, in response to HSV-1 infection, HDAC4 accumulated in the nucleus and promoted MeCP2 SUMOylation that is a post-translational modification critically affecting the repressive activity of MeCP2. The blockade of IL-1 receptors by the specific antagonist Anakinra prevented the MeCP2 increase and the consequent downregulation of gene expression along with rescuing structural and functional indices of neurodegeneration. Collectively, our findings provide novel mechanistic evidence on the role played by HSV-1-activated IL-1ß signaling pathways in synaptic deficits leading to cognitive impairment.

Noise-induced auditory damage affects hippocampus causing memory deficits in a model of early age-related hearing loss.

Several studies identified noise-induced hearing loss (NIHL) as a risk factor for sensory aging and cognitive decline processes, including neurodegenerative diseases, such as dementia and age-related hearing loss (ARHL). Although the association between noise- and age-induced hearing impairment has been widely documented by epidemiological and experimental studies, the molecular mechanisms underlying this association are not fully understood as it is not known how these risk factors (aging and noise) can interact, affecting memory processes. We recently found that early noise exposure in an established animal model of ARHL (C57BL/6 mice) accelerates the onset of age-related cochlear dysfunctions. Here, we extended our previous data by investigating what happens in central brain structures (auditory cortex and hippocampus), to assess the relationship between hearing and memory impairment and the possible combined effect of noise and sensory aging on the cognitive domain. To this aim, we exposed juvenile C57BL/6 mice of 2 months of age to repeated noise sessions (60 min/day, pure tone of 100 dB SPL, 10 kHz, 10 consecutive days) and we monitored auditory threshold by measuring auditory brainstem responses (ABR), spatial working memory, by using the Y-maze test, and basal synaptic transmission by using ex vivo electrophysiological recordings, at different time points (1, 4 and 7 months after the onset of noise exposure, corresponding to 3, 6 and 9 months of age). We found that hearing loss, along with accelerated presbycusis onset, can induce persistent synaptic alterations in the auditory cortex. This was associated with decreased memory performance and oxidative-inflammatory injury in the hippocampus, the extra-auditory structure involved in memory processes. Collectively, our data confirm the critical relationship between auditory and memory circuits, suggesting that the combined detrimental effect of noise and sensory aging on hearing function can be considered a high-risk factor for both sensory and cognitive degenerative processes, given that early noise exposure accelerates presbycusis phenotype and induces hippocampal-dependent memory dysfunctions.

Maternal High Fat Diet Anticipates the AD-like Phenotype in 3xTg-AD Mice by Epigenetic Dysregulation of Aß Metabolism.

Maternal overnutrition has been reported to affect brain plasticity of the offspring by altering gene expression, regulating both synaptic plasticity and adult neurogenesis. However, whether perinatal metabolic stress may influence the accumulation of misfolded proteins and the development of neurodegeneration remains to be clarified. We investigated the impact of maternal high fat diet (HFD) in an experimental model of Alzheimer's disease (AD). The 3xTg-AD mice born to overfed mothers showed an impairment of synaptic plasticity and cognitive deficits earlier than controls. Maternal HFD also altered the expression of genes regulating amyloid-ß-protein (Aß) metabolism (i.e., Bace1, Ern1, Ide and Nicastrin) and enhanced Aß deposition in the hippocampus. Finally, we found an epigenetic derangement and an aberrant recruitment of transcription factors NF-kB and STAT3 and chromatin remodeler HDAC2 on the regulatory sequences of the same genes. Collectively, our data indicate that early life metabolic stress worsens the AD phenotype via epigenetic alteration of genes regulating Aß synthesis and clearance.

Auditory sensory deprivation induced by noise exposure exacerbates cognitive decline in a mouse model of Alzheimer's disease.

Although association between hearing impairment and dementia has been widely documented by epidemiological studies, the role of auditory sensory deprivation in cognitive decline remains to be fully understood. To address this issue we investigated the impact of hearing loss on the onset and time-course of cognitive decline in an animal model of Alzheimer's disease (AD), that is the 3×Tg-AD mice and the underlying mechanisms. We found that hearing loss induced by noise exposure in the 3×Tg-AD mice before the phenotype is manifested caused persistent synaptic and morphological alterations in the auditory cortex. This was associated with earlier hippocampal dysfunction, increased tau phosphorylation, neuroinflammation, and redox imbalance, along with anticipated memory deficits compared to the expected time-course of the neurodegenerative phenotype. Our data suggest that a mouse model of AD is more vulnerable to central damage induced by hearing loss and shows reduced ability to counteract noise-induced detrimental effects, which accelerates the neurodegenerative disease onset.

Whole Blood Transcriptome Characterization of 3xTg-AD Mouse and Its Modulation by Transcranial Direct Current Stimulation (tDCS).

The 3xTg-AD mouse is a widely used model in the study of Alzheimer's Disease (AD). It has been extensively characterized from both the anatomical and behavioral point of view, but poorly studied at the transcriptomic level. For the first time, we characterize the whole blood transcriptome of the 3xTg-AD mouse at three and six months of age and evaluate how its gene expression is modulated by transcranial direct current stimulation (tDCS). RNA-seq analysis revealed 183 differentially expressed genes (DEGs) that represent a direct signature of the genetic background of the mouse. Moreover, in the 6-month-old 3xTg-AD mice, we observed a high number of DEGs that could represent good peripheral biomarkers of AD symptomatology onset. Finally, tDCS was associated with gene expression changes in the 3xTg-AD, but not in the control mice. In conclusion, this study provides an in-depth molecular characterization of the 3xTg-AD mouse and suggests that blood gene expression can be used to identify new biomarkers of AD progression and treatment effects.

Passive immunotherapy for N-truncated tau ameliorates the cognitive deficits in two mouse Alzheimer's disease models.

Clinical and neuropathological studies have shown that tau pathology better correlates with the severity of dementia than amyloid plaque burden, making tau an attractive target for the cure of Alzheimer's disease. We have explored whether passive immunization with the 12A12 monoclonal antibody (26-36aa of tau protein) could improve the Alzheimer's disease phenotype of two well-established mouse models, Tg2576 and 3xTg mice. 12A12 is a cleavage-specific monoclonal antibody which selectively binds the pathologically relevant neurotoxic NH226-230 fragment (i.e. NH2htau) of tau protein without cross-reacting with its full-length physiological form(s). We found out that intravenous administration of 12A12 monoclonal antibody into symptomatic (6 months old) animals: (i) reaches the hippocampus in its biologically active (antigen-binding competent) form and successfully neutralizes its target; (ii) reduces both pathological tau and amyloid precursor protein/amyloidß metabolisms involved in early disease-associated synaptic deterioration; (iii) improves episodic-like type of learning/memory skills in hippocampal-based novel object recognition and object place recognition behavioural tasks; (iv) restores the specific up-regulation of the activity-regulated cytoskeleton-associated protein involved in consolidation of experience-dependent synaptic plasticity; (v) relieves the loss of dendritic spine connectivity in pyramidal hippocampal CA1 neurons; (vi) rescues the Alzheimer's disease-related electrophysiological deficits in hippocampal long-term potentiation at the CA3-CA1 synapses; and (vii) mitigates the neuroinflammatory response (reactive gliosis). These findings indicate that the 20-22 kDa NH2-terminal tau fragment is crucial target for Alzheimer's disease therapy and prospect immunotherapy with 12A12 monoclonal antibody as safe (normal tau-preserving), beneficial approach in contrasting the early Amyloidß-dependent and independent neuropathological and cognitive alterations in affected subjects.

Plasma BDNF Levels Following Transcranial Direct Current Stimulation Allow Prediction of Synaptic Plasticity and Memory Deficits in 3×Tg-AD Mice.

Early diagnosis of Alzheimer's disease (AD) supposedly increases the effectiveness of therapeutic interventions. However, presently available diagnostic procedures are either invasive or require complex and expensive technologies, which cannot be applied at a larger scale to screen populations at risk of AD. We were looking for a biomarker allowing to unveil a dysfunction of molecular mechanisms, which underly synaptic plasticity and memory, before the AD phenotype is manifested and investigated the effects of transcranial direct current stimulation (tDCS) in 3×Tg-AD mice, an experimental model of AD which does not exhibit any long-term potentiation (LTP) and memory deficits at the age of 3 months (3×Tg-AD-3M). Our results demonstrated that tDCS differentially affected 3×Tg-AD-3M and age-matched wild-type (WT) mice. While tDCS increased LTP at CA3-CA1 synapses and memory in WT mice, it failed to elicit these effects in 3×Tg-AD-3M mice. Remarkably, 3×Tg-AD-3M mice did not show the tDCS-dependent increases in pCREB Ser133 and pCaMKII Thr286 , which were found in WT mice. Of relevance, tDCS induced a significant increase of plasma BDNF levels in WT mice, which was not found in 3×Tg-AD-3M mice. Collectively, our results showed that plasticity mechanisms are resistant to tDCS effects in the pre-AD stage. In particular, the lack of BDNF responsiveness to tDCS in 3×Tg-AD-3M mice suggests that combining tDCS with dosages of plasma BDNF levels may provide an easy-to-detect and low-cost biomarker of covert impairment of synaptic plasticity mechanisms underlying memory, which could be clinically applicable. Testing proposed here might be useful to identify AD in its preclinical stage, allowing timely and, hopefully, more effective disease-modifying interventions.

Enhancing Plasticity Mechanisms in the Mouse Motor Cortex by Anodal Transcranial Direct-Current Stimulation: The Contribution of Nitric Oxide Signaling.

Consistent body of evidence shows that transcranial direct-current stimulation (tDCS) over the primary motor cortex (M1) facilitates motor learning and promotes recovery after stroke. However, the knowledge of molecular mechanisms behind tDCS effects needs to be deepened for a more rational use of this technique in clinical settings. Here we characterized the effects of anodal tDCS of M1, focusing on its impact on glutamatergic synaptic transmission and plasticity. Mice subjected to tDCS displayed increased long-term potentiation (LTP) and enhanced basal synaptic transmission at layer II/III horizontal connections. They performed better than sham-stimulated mice in the single-pellet reaching task and exhibited increased forelimb strength. Dendritic spine density of layer II/III pyramidal neurons was also increased by tDCS. At molecular level, tDCS enhanced: 1) BDNF expression, 2) phosphorylation of CREB, CaMKII, and GluA1, and 3) S-nitrosylation of GluA1 and HDAC2. Blockade of nitric oxide synthesis by L-NAME prevented the tDCS-induced enhancement of GluA1 phosphorylation at Ser831 and BDNF levels, as well as of miniature excitatory postsynaptic current (mEPSC) frequency, LTP and reaching performance. Collectively, these findings demonstrate that anodal tDCS engages plasticity mechanisms in the M1 and highlight a role for nitric oxide (NO) as a novel mediator of tDCS effects.

Maternal insulin resistance multigenerationally impairs synaptic plasticity and memory via gametic mechanisms.

Metabolic diseases harm brain health and cognitive functions, but whether maternal metabolic unbalance may affect brain plasticity of next generations is still unclear. Here, we demonstrate that maternal high fat diet (HFD)-dependent insulin resistance multigenerationally impairs synaptic plasticity, learning and memory. HFD downregulates BDNF and insulin signaling in maternal tissues and epigenetically inhibits BDNF expression in both germline and hippocampus of progeny. Notably, exposure of the HFD offspring to novel enriched environment restores Bdnf epigenetic activation in the male germline and counteracts the transmission of cognitive impairment to the next generations. BDNF administration to HFD-fed mothers or preserved insulin sensitivity in HFD-fed p66Shc KO mice also prevents the intergenerational transmission of brain damage to the progeny. Collectively, our data suggest that maternal diet multigenerationally impacts on descendants' brain health via gametic mechanisms susceptible to lifestyle.

Glutamate/GABA co-release selectively influences postsynaptic glutamate receptors in mouse cortical neurons.

Cultured rat cortical neurons co-expressing VGLUT1 and VGAT (mixed synapses) co-release Glu and GABA. Here, mixed synapses were studied in cultured mouse cortical neurons to verify whether in mice mixed synapses co-release Glu and GABA, and to gain insight into how they may influence excitation/inhibition balance. Results showed the existence of synapses and autapses that co-release Glu and GABA in cultured mouse cortical neurons, and the ability of both neurotransmitters to evoke postsynaptic responses mediated by ionotropic receptors. We studied the short-term plasticity of glutamatergic, GABAergic, and mixed responses and we found that the kinetics of mixPSC amplitude depression was similar to that observed in EPSCs, but it was different from that of IPSCs. We found similar presynaptic release characteristics in glutamatergic and mixed synapses. Analysis of postsynaptic features, obtained by measuring AMPAR- and NMDAR-mediated currents, showed that AMPAR-mediated currents were significantly higher in pure glutamatergic than in mixed synapses, whereas NMDAR-mediated currents were not significantly different from those measured in mixed synapses. Overall, our findings demonstrate that glutamatergic and mixed synapses share similar electrophysiological properties. However, co-release of GABA and Glu influences postsynaptic ionotropic glutamatergic receptor subtypes, thus selectively influencing AMPAR-mediated currents. These findings strengthen the view that mixed neurons can play a key role in CNS development and in maintaining the excitation-inhibition balance.

Neuromodulatory Action of Picomolar Extracellular Aß42 Oligomers on Presynaptic and Postsynaptic Mechanisms Underlying Synaptic Function and Memory.

Failure of anti-amyloid-ß peptide (Aß) therapies against Alzheimer's disease (AD), a neurodegenerative disorder characterized by high amounts of the peptide in the brain, raised the question of the physiological role of Aß released at low concentrations in the healthy brain. To address this question, we studied the presynaptic and postsynaptic mechanisms underlying the neuromodulatory action of picomolar amounts of oligomeric Aß42 (oAß42) on synaptic glutamatergic function in male and female mice. We found that 200 pm oAß42 induces an increase of frequency of miniature EPSCs and a decrease of paired pulse facilitation, associated with an increase in docked vesicle number, indicating that it augments neurotransmitter release at presynaptic level. oAß42 also produced postsynaptic changes as shown by an increased length of postsynaptic density, accompanied by an increased expression of plasticity-related proteins such as cAMP-responsive element binding protein phosphorylated at Ser133, calcium-calmodulin-dependent kinase II phosphorylated at Thr286, and brain-derived neurotrophic factor, suggesting a role for Aß in synaptic tagging. These changes resulted in the conversion of early into late long-term potentiation through the nitric oxide/cGMP/protein kinase G intracellular cascade consistent with a cGMP-dependent switch from short- to long-term memory observed in vivo after intrahippocampal administration of picomolar amounts of oAß42 These effects were present upon extracellular but not intracellular application of the peptide and involved α7 nicotinic acetylcholine receptors. These observations clarified the physiological role of oAß42 in synaptic function and memory formation providing solid fundamentals for investigating the pathological effects of high Aß levels in the AD brains.SIGNIFICANCE STATEMENT High levels of oligomeric amyloid-ß42 (oAß42) induce synaptic dysfunction leading to memory impairment in Alzheimer's disease (AD). However, at picomolar concentrations, the peptide is needed to ensure long-term potentiation (LTP) and memory. Here, we show that extracellular 200 pm oAß42 concentrations increase neurotransmitter release, number of docked vesicles, postsynaptic density length, and expression of plasticity-related proteins leading to the conversion of early LTP into late LTP and of short-term memory into long-term memory. These effects require α7 nicotinic acetylcholine receptors and are mediated through the nitric oxide/cGMP/protein kinase G pathway. The knowledge of Aß function in the healthy brain might be useful to understand the causes leading to its increase and detrimental effect in AD.

Role of BDNF Signaling in Memory Enhancement Induced by Transcranial Direct Current Stimulation.

In the recent years numerous studies have provided encouraging results supporting the use of transcranial direct current stimulation (tDCS) as non-invasive brain stimulation technique to improve motor and cognitive functions in patients suffering from neurological and neuropsychiatric disorders as well as in healthy subjects. Among the multiple effects elicited by tDCS on cognitive functions, experimental evidence and clinical findings have highlighted the beneficial impact on long-term memory. Memory deficits occur during physiological aging as well as in neurological and neurodegenerative disorders, including Alzheimer's disease (AD). In this scenario, non-invasive techniques for memory enhancement, such as tDCS, are receiving increasing attention. The knowledge of molecular mechanisms subtending tDCS effects is of pivotal importance for a more rationale use of this technique in clinical settings. Although we are still far from having a clear picture, recent literature on human and animal studies has pointed to the involvement of synaptic plasticity mechanisms in mediating tDCS effects on long-term memory. Here we review these studies focusing on the neurotrophin "brain-derived neurotrophic factor" (BDNF) as critical tDCS effector.

Anodal transcranial direct current stimulation affects auditory cortex plasticity in normal-hearing and noise-exposed rats.

BACKGROUND: Transcranial direct current stimulation (tDCS) is a non-invasive tool capable to modulate cortical functions by affecting neuronal excitability and synaptic plasticity. OBJECTIVE: Here we investigated the effects of anodal tDCS on auditory cortex (ACx) in normal-hearing rats and following a paradigm of noise-induced hearing loss (NIHL), that causes morphological alterations in ACx pyramidal neurons. METHODS: Male rats exposed to intense pure tone (10 kHz) were subsequently subjected to unilateral anodal tDCS of ACx and changes in dendritic morphology and spines were assessed by Golgi-Cox staining 30 days after the onset of the acoustic trauma. Molecular and functional changes were investigated by Western immunoblotting, immunofluorescence experiments and electrophysiological recordings in brain slices. RESULTS: We found that NIHL altered dendritic morphology by decreasing spine density, mostly in layer 2/3 pyramidal neurons. Interestingly, tDCS increased ACx spine density, targeting apical dendrites of layer 2/3 and 5/6 pyramidal neurons in rats with normal auditory function and both apical and basal arborizations in layer 2/3 of NIHL rats. Twenty-four hours after tDCS, Bdnf and synaptophysin levels in ACx increased both in normal-hearing and noise-exposed rats. Field recordings showed that basal synaptic transmission at layer 2/3 horizontal connections was significantly reduced in noise-exposed rats compared to normal-hearing animals and, notably, input-output curves of noise-exposed animals subjected to tDCS were similar to those of normal-hearing rats. CONCLUSIONS: Our findings provide novel evidence that anodal tDCS affects structural plasticity in the ACx suggesting that it might be beneficial in treating cortical alterations due to cochlear damage.

LTP and memory impairment caused by extracellular Aß and Tau oligomers is APP-dependent.

The concurrent application of subtoxic doses of soluble oligomeric forms of human amyloid-beta (oAß) and Tau (oTau) proteins impairs memory and its electrophysiological surrogate long-term potentiation (LTP), effects that may be mediated by intra-neuronal oligomers uptake. Intrigued by these findings, we investigated whether oAß and oTau share a common mechanism when they impair memory and LTP in mice. We found that as already shown for oAß, also oTau can bind to amyloid precursor protein (APP). Moreover, efficient intra-neuronal uptake of oAß and oTau requires expression of APP. Finally, the toxic effect of both extracellular oAß and oTau on memory and LTP is dependent upon APP since APP-KO mice were resistant to oAß- and oTau-induced defects in spatial/associative memory and LTP. Thus, APP might serve as a common therapeutic target against Alzheimer's Disease (AD) and a host of other neurodegenerative diseases characterized by abnormal levels of Aß and/or Tau.

Anodal transcranial direct current stimulation boosts synaptic plasticity and memory in mice via epigenetic regulation of Bdnf expression.

The effects of transcranial direct current stimulation (tDCS) on brain functions and the underlying molecular mechanisms are yet largely unknown. Here we report that mice subjected to 20-min anodal tDCS exhibited one-week lasting increases in hippocampal LTP, learning and memory. These effects were associated with enhanced: i) acetylation of brain-derived neurotrophic factor (Bdnf) promoter I; ii) expression of Bdnf exons I and IX; iii) Bdnf protein levels. The hippocampi of stimulated mice also exhibited enhanced CREB phosphorylation, pCREB binding to Bdnf promoter I and recruitment of CBP on the same regulatory sequence. Inhibition of acetylation and blockade of TrkB receptors hindered tDCS effects at molecular, electrophysiological and behavioral levels. Collectively, our findings suggest that anodal tDCS increases hippocampal LTP and memory via chromatin remodeling of Bdnf regulatory sequences leading to increased expression of this gene, and support the therapeutic potential of tDCS for brain diseases associated with impaired neuroplasticity.

NO-donor thiacarbocyanines as multifunctional agents for Alzheimer's disease.

Some symmetrical and unsymmetrical thiacarbocyanines bearing NO-donor nitrooxy and furoxan moieties were synthesized and studied as candidate anti-Alzheimer's drugs. All products activated soluble guanylate cyclase (sGC) in a dose-dependent manner, depending on the presence in their structures of NO-donor groups. None displayed toxicity when tested at concentrations below 10 µM on human brain microvascular endothelial cells (hCMEC/D3). Some products were capable of inhibiting amyloid ß-protein (Aß) aggregation, with a potency in the low µM concentration range, and of inhibiting aggregation of human recombinant tau protein in amyloid fibrils when incubated with the protein at 1 µM concentration. Nitrooxy derivative 21 and furoxan derivative 22 were selected to investigate synaptic plasticity. Both products, tested at 2 µM concentration, counteracted the inhibition of long-term potentiation (LTP) induced by Aß42 in hippocampal brain slices.

Intracellular accumulation of amyloid-ß (Aß) protein plays a major role in Aß-induced alterations of glutamatergic synaptic transmission and plasticity.

Intracellular accumulation of amyloid-ß (Aß) protein has been proposed as an early event in AD pathogenesis. In patients with mild cognitive impairment, intraneuronal Aß immunoreactivity was found especially in brain regions critically involved in the cognitive deficits of AD. Although a large body of evidence demonstrates that Aß42 accumulates intraneuronally ((in)Aß), the action and the role of Aß42 buildup on synaptic function have been poorly investigated. Here, we demonstrate that basal synaptic transmission and LTP were markedly depressed following Aß42 injection into the neuron through the patch pipette. Control experiments performed with the reverse peptide (Aß42-1) allowed us to exclude that the effects of (in)Aß depended on changes in oncotic pressure. To further investigate (in)Aß synaptotoxicity we used an Aß variant harboring oxidized methionine in position 35 that does not cross the neuronal plasma membrane and is not uploaded from the extracellular space. This Aß42 variant had no effects on synaptic transmission and plasticity when applied extracellularly, but induced synaptic depression and LTP inhibition after patch-pipette dialysis. Finally, the injection of an antibody raised against human Aß42 (6E10) in CA1 pyramidal neurons of mouse hippocampal brain slices and autaptic microcultures did not, per se, significantly affect LTP and basal synaptic transmission, but it protected against the toxic effects of extracellular Aß42. Collectively, these findings suggest that Aß42-induced impairment of glutamatergic synaptic function depends on its internalization and intracellular accumulation thus paving the way to a systemic proteomic analysis of intracellular targets/partners of Aß42.

The neurogenic effects of exogenous neuropeptide Y: early molecular events and long-lasting effects in the hippocampus of trimethyltin-treated rats.

Modulation of endogenous neurogenesis is regarded as a promising challenge in neuroprotection. In the rat model of hippocampal neurodegeneration obtained by Trimethyltin (TMT) administration (8 mg/kg), characterised by selective pyramidal cell loss, enhanced neurogenesis, seizures and cognitive impairment, we previously demonstrated a proliferative role of exogenous neuropeptide Y (NPY), on dentate progenitors in the early phases of neurodegeneration. To investigate the functional integration of newly-born neurons, here we studied in adult rats the long-term effects of intracerebroventricular administration of NPY (2 µg/2 µl, 4 days after TMT-treatment), which plays an adjuvant role in neurodegeneration and epilepsy. Our results indicate that 30 days after NPY administration the number of new neurons was still higher in TMT+NPY-treated rats than in control+saline group. As a functional correlate of the integration of new neurons into the hippocampal network, long-term potentiation recorded in Dentate Gyrus (DG) in the absence of GABAA receptor blockade was higher in the TMT+NPY-treated group than in all other groups. Furthermore, qPCR analysis of Kruppel-like factor 9, a transcription factor essential for late-phase maturation of neurons in the DG, and of the cyclin-dependent kinase 5, critically involved in the maturation and dendrite extension of newly-born neurons, revealed a significant up-regulation of both genes in TMT+NPY-treated rats compared with all other groups. To explore the early molecular events activated by NPY administration, the Sonic Hedgehog (Shh) signalling pathway, which participates in the maintenance of the neurogenic hippocampal niche, was evaluated by qPCR 1, 3 and 5 days after NPY-treatment. An early significant up-regulation of Shh expression was detected in TMT+NPY-treated rats compared with all other groups, associated with a modulation of downstream genes. Our data indicate that the neurogenic effect of NPY administration during TMT-induced neurodegeneration involves early Shh pathway activation and results in a functional integration of newly-generated neurons into the local circuit.

Expression of olfactory-type cyclic nucleotide-gated channels in rat cortical astrocytes.

Cyclic nucleotide-gated (CNG) channels are nonselective cation channels activated by cyclic AMP (cAMP) or cyclic GMP (cGMP). They were originally identified in retinal and olfactory receptors, but evidence has also emerged for their expression in several mammalian brain areas. Because cGMP and cAMP control important aspects of glial cell physiology, we wondered whether CNG channels are expressed in astrocytes, the most functionally relevant glial cells in the CNS. Immunoblot and immunofluorescence experiments demonstrated expression of the CNG channel olfactory-type A subunit, CNGA2, in cultured rat cortical astrocytes. In patch-clamp experiments, currents elicited in these cells by voltage ramps from -100 to +100 mV in the presence of the cGMP analogue, dB-cGMP, were significantly reduced by the CNG channel blockers, L-cis-diltiazem (LCD) and Cd(2+) . The reversal potentials of the LCD- and Cd(2+) -sensitive currents were more positive than that of K(+) , as expected for a mixed cation current. Noninactivating, voltage-independent currents were also elicited by extracellular application of the membrane permeant cGMP analogue, 8-Br-cGMP. These effects were blocked by LCD and were mimicked by natriuretic peptide receptor activation and inhibition of phosphodiesterase activity. Voltage-independent, LCD-sensitive currents were also elicited by 8-Br-cGMP in astrocytes of hippocampal and neocortical brain slices. Immunohistochemistry confirmed a broad distribution of CNG channels in astrocytes of the rat forebrain, midbrain, and hindbrain. These findings suggest that CNG channels are downstream targets of cyclic nucleotides in astrocytes, and they may be involved in the glial-mediated regulation of CNS functions under physiological and pathological conditions.