personalized-needles-for-microinjections-in-the-rodent-brain

Personalized Needles for Microinjections in the Rodent Brain

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to ...

University of ferrara

The neuropeptide nociceptin/orphanin FQ (N/OFQ) has been shown to modulate neuronal excitability and neurotransmitter release. Previous studies indicate that the mRNA levels for the N/OFQ precursor (proN/OFQ) are increased after seizures. However, it is unclear whether N/OFQ plays a role in seizure expression. Therefore, (1) we analyzed proN/OFQ mRNA levels and NOP (the N/OFQ receptor) mRNA levels and receptor density in the kainate model of epilepsy, using Northern blot analysis, in situ hybridization, and receptor binding assay, and (2) we examined susceptibility to kainate seizure in mice treated with 1-[(3R, 4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1, 3-dihydro-benzimidazol-2-one (J-113397), a selective NOP receptor antagonist, and in proN/OFQ knock-out mice. After kainate administration, increased proN/OFQ gene expression was observed in the reticular nucleus of the thalamus and in the medial nucleus of the amygdala. In contrast, NOP mRNA levels and receptor density decreased in the amygdala, hippocampus, thalamus, and cortex. Mice treated with the NOP receptor antagonist J-113397 displayed reduced susceptibility to kainate-induced seizures (i.e., significant reduction of behavioral seizure scores). N/OFQ knock-out mice were less susceptible to kainate seizures compared with their wild-type littermates, in that lethality was reduced, latency to generalized seizure onset was prolonged, and behavioral seizure scores decreased. Intracerebroventricular administration of N/OFQ prevented reduced susceptibility to kainate seizures in N/OFQ knock-out mice. These data indicate that acute limbic seizures are associated with increased N/OFQ release in selected areas, causing downregulation of NOP receptors and activation of N/OFQ biosynthesis, and support the notion that the N/OFQ-NOP system plays a facilitatory role in kainate seizure expression.

Involvement of the neuropeptide nociceptin/orphanin FQ in kainate seizures.

The neuropeptide nociceptin/orphanin FQ (N/OFQ) is implicated in many biological functions, including nociception, locomotor activity, stress and anxiety, drinking and food-intake. N/OFQ has also been reported to play a facilitatory role in acute kainate-induced seizures. The aim of the present study was to investigate its involvement in a chronic model of temporal lobe epilepsy, kindling epileptogenesis, using N/OFQ knock-out mice and their wild-type littermates as controls. Kindling development was retarded in N/OFQ-deficient mice, in that (compared with controls) they required a significantly greater number of stimulations and a significantly longer time in electrical seizures to reach kindling criteria. These data indicate that N/OFQ is involved in the development of kindling and that it may play a pro-epileptogenic role.

Delayed epileptogenesis in nociceptin/orphanin FQ-deficient mice.

The role of the hippocampal somatostatin (somatotropin release-inhibiting factor, SRIF) system in the control of partial complex seizures is discussed in this review. The SRIF system plays a role in the inhibitory modulation of hippocampal circuitries under normal conditions: 1) SRIF neurons in the dentate gyrus are part of a negative feedback circuit modulating the firing rate of granule cells; 2) SRIF released in CA3 interacts both with presynaptic receptors located on associational/commissural terminals and with postsynaptic receptors located on pyramidal cell dendrites, reducing excitability of pyramidal neurons; 3) in CA1, SRIF exerts a feedback inhibition and reduces the excitatory drive on pyramidal neurons. Significant changes in the hippocampal SRIF system have been documented in experimental models of temporal lobe epilepsy (TLE), in particular in the kindling and in the kainate models. SRIF biosynthesis and release are increased in the kindled hippocampus, especially in the dentate gyrus. This hyper-function may be instrumental to control the latent hyperexcitability of the kindled brain, preventing excessive discharge of the principal neurons and the occurrence of spontaneous seizures. In contrast, the hippocampal SRIF system undergoes damage in the dentate gyrus following kainate-induced status epilepticus. Although surviving SRIF neurons appear to hyperfunction, the loss of hilar SRIF interneurons may compromise inhibitory mechanisms in the dentate gyrus, facilitating the occurrence of spontaneous seizures. In keeping with these data, pharmacological activation of SRIF1 (sst2) receptors, i.e. of the prominent receptor subtype on granule cells, exerts antiseizure effects. Taken together, the data presented suggest that the hippocampal SRIF system plays a role in the control of partial complex seizures and, therefore, that it may be proposed as a therapeutic target for TLE.

On the role of somatostatin in seizure control: clues from the hippocampus.

The neuropeptide nociceptin/orphanin FQ (N/OFQ) has been suggested to play a facilitatory role in kainate seizure expression. Furthermore, mRNA levels for the N/OFQ precursor are increased following kainate seizures, while its receptor (NOP) density is decreased. These data suggest increased N/OFQ release. To obtain direct evidence that this is the case, we have developed a microdialysis technique, coupled with a sensitive radioimmunoassay, that allows measurement of N/OFQ release from the hippocampus and thalamus of awake, freely moving animals. In both these brain areas, the spontaneous N/OFQ efflux decreased by approximately 50% and 65% when Ca2+ was omitted and when tetrodotoxin was added to the perfusion medium, respectively. Perfusion of the dialysis probe with high K+ increased N/OFQ release (approximately threefold) in a Ca2+-dependent and tetrodotoxin-sensitive manner. Kainate seizures caused a twofold increase in N/OFQ release followed, within 3 h, by a return to baseline levels. Approximately 5 h after kainate, a late increase in N/OFQ release was observed. On the following day, when animals were having only low grade seizures, N/OFQ release was not significantly different from normal. These phenomena were observed with similar patterns in the hippocampus and in the thalamus. The present data indicate that acute limbic seizures are associated with increased N/OFQ release, which may prime the molecular changes described above, i.e. cause down-regulation of NOP receptors and activation of N/OFQ biosynthesis.

Kainate seizures increase nociceptin/orphanin FQ release in the rat hippocampus and thalamus: a microdialysis study.

Fibroblast growth factor 2 (FGF-2) has multiple, pleiotropic effects on the nervous system that include neurogenesis, neuroprotection and neuroplasticity. Thus, alteration in FGF-2 expression patterns may have a profound impact in brain function, both in normal physiology and in pathology. Here, we used FGF-2 transgenic mice (TgFGF2) to study the effects of endogenous FGF-2 overexpression on susceptibility to seizures and to the pathological consequences of seizures. TgFGF2 mice display increased FGF-2 expression in hippocampal pyramidal neurons and dentate granule cells. Increased density of glutamatergic synaptic vesicles was observed in the hippocampus of TgFGF2 mice, and electrophysiological data (input/output curves and patch-clamp recordings in CA1) confirmed an increase in excitatory inputs in CA1, suggesting the presence of a latent hyperexcitability. Indeed, TgFGF2 mice displayed increased susceptibility to kainate-induced seizures compared with wild-type (WT) littermates, in that latency to generalized seizure onset was reduced, whereas behavioral seizure scores and lethality were increased. Finally, WT and TgFGF2 mice with similar seizure scores were used for examining seizure-induced cellular consequences. Neurogenesis and mossy fiber sprouting were not significantly different between the two groups. In contrast, cell damage (assessed with Fluoro-Jade B, silver impregnation and anti-caspase 3 immunohistochemistry) was significantly lower in TgFGF2 mice, especially in the areas of overexpression (CA1 and CA3), indicating reduction of seizure-induced necrosis and apoptosis. These data suggest that FGF-2 may be implicated in seizure susceptibility and in seizure-induced plasticity, exerting different, and apparently contrasting effects: favoring ictogenesis but reducing seizure-induced cell death.

Fgf-2 overexpression increases excitability and seizure susceptibility but decreases seizure-induced cell loss.

A loss of neurons is observed in the hippocampus of many patients with epilepsies of temporal lobe origin. It has been hypothesized that damage limitation or repair, for example using neurotrophic factors (NTFs), may prevent the transformation of a normal tissue into epileptic (epileptogenesis). Here, we used viral vectors to locally supplement two NTFs, fibroblast growth factor-2 (FGF-2) and brain-derived neurotrophic factor (BDNF), when epileptogenic damage was already in place. These vectors were first characterized in vitro, where they increased proliferation of neural progenitors and favored their differentiation into neurons, and they were then tested in a model of status epilepticus-induced neurodegeneration and epileptogenesis. When injected in a lesioned hippocampus, FGF-2/BDNF expressing vectors increased neuronogenesis, embanked neuronal damage, and reduced epileptogenesis. It is concluded that reduction of damage reduces epileptogenesis and that supplementing specific NTFs in lesion areas represents a new approach to the therapy of neuronal damage and of its consequences.

Localized delivery of fibroblast growth factor-2 and brain-derived neurotrophic factor reduces spontaneous seizures in an epilepsy model.

Neurotrophic factors (NTFs) are involved in the regulation of neuronal survival and function and, thus, may be used to treat neurological diseases associated with neuronal death. A major hurdle for their clinical application is the delivery mode. We describe here a new strategy based on the use of progenitor cells called mesoangioblasts (MABs). MABs can be isolated from postnatal mesoderm tissues and, because of a high adhesin-dependent migratory capacity, can reach perivascular targets especially in damaged areas. We generated genetically modified MABs producing nerve growth factor (MABs-NGF) or brain-derived neurotrophic factor (MABs-BDNF) and assessed their bystander effects in vitro using PC12 cells, primary cultures, and organotypic cultures of adult hippocampal slices. MABs-NGF-conditioned medium induced differentiation of PC12 cells, while MABs-BDNF-conditioned medium increased viability of cultured neurons and slices. Slices cultured with MABs-BDNF medium also better retained their morphology and functional connections, and all these effects were abolished by the TrkB kinase blocker K252a or the BDNF scavenger TrkB-IgG. Interestingly, the amount of BDNF released by MABs-BDNF produced greater effects than an identical amount of recombinant BDNF, suggesting that other NTFs produced by MABs synergize with BDNF. Thus, MABs can be an effective vehicle for NTF delivery, promoting differentiation, survival, and functionality of neurons. In summary, MABs hold distinct advantages over other currently evaluated approaches for NTF delivery in the CNS, including synergy of MAB-produced NTF with the neurotrophins. Since MABs may be capable of homing into damaged brain areas, they represent a conceptually novel, promising therapeutic approach to treat neurodegenerative diseases.

Bystander effect on brain tissue of mesoangioblasts producing neurotrophins.

The alterations in GABA release have not yet been systematically measured along the natural course of temporal lobe epilepsy. In this work, we analyzed GABA extracellular concentrations (using in vivo microdialysis under basal and high K(+)-evoked conditions) and loss of two GABA interneuron populations (parvalbumin and somatostatin neurons) in the ventral hippocampus at different time-points after pilocarpine-induced status epilepticus in the rat, i.e. during development and progression of epilepsy. We found that (i) during the latent period between the epileptogenic insult, status epilepticus, and the first spontaneous seizure, basal GABA outflow was reduced to about one third of control values while the number of parvalbumin-positive cells was reduced by about 50% and that of somatostatin-positive cells by about 25%; nonetheless, high K(+) stimulation increased extracellular GABA in a proportionally greater manner during latency than under control conditions; (ii) at the time of the first spontaneous seizure (i.e., when the diagnosis of epilepsy is made in humans) this increased responsiveness to stimulation disappeared, i.e. there was no longer any compensation for GABA cell loss; (iii) thereafter, this dysfunction remained constant until a late phase of the disease. These data suggest that a GABAergic hyper-responsiveness can compensate for GABA cell loss and protect from occurrence of seizures during latency, whereas impaired extracellular GABA levels can favor the occurrence of spontaneous recurrent seizures and the maintenance of an epileptic state.

Impairment of GABA release in the hippocampus at the time of the first spontaneous seizure in the pilocarpine model of temporal lobe epilepsy.

The identification of biomarkers of the transformation of normal to epileptic tissue would help to stratify patients at risk of epilepsy following brain injury, and inform new treatment strategies. MicroRNAs (miRNAs) are an attractive option in this direction. In this study, miRNA microarrays were performed on laser-microdissected hippocampal granule cell layer (GCL) and on plasma, at different time points in the development of pilocarpine-induced epilepsy in the rat: latency, first spontaneous seizure and chronic epileptic phase. Sixty-three miRNAs were differentially expressed in the GCL when considering all time points. Three main clusters were identified that separated the control and chronic phase groups from the latency group and from the first spontaneous seizure group. MiRNAs from rats in the chronic phase were compared to those obtained from the laser-microdissected GCL of epileptic patients, identifying several miRNAs (miR-21-5p, miR-23a-5p, miR-146a-5p and miR-181c-5p) that were up-regulated in both human and rat epileptic tissue. Analysis of plasma samples revealed different levels between control and pilocarpine-treated animals for 27 miRNAs. Two main clusters were identified that segregated controls from all other groups. Those miRNAs that are altered in plasma before the first spontaneous seizure, like miR-9a-3p, may be proposed as putative biomarkers of epileptogenesis.

MicroRNA profiles in hippocampal granule cells and plasma of rats with pilocarpine-induced epilepsy--comparison with human epileptic samples.

Brain-derived neurotrophic factor (BDNF) has been found to produce pro- but also anti-epileptic effects. Thus, its validity as a therapeutic target must be verified using advanced tools designed to block or to enhance its signal. The aim of this study was to develop tools to silence the BDNF signal. We generated Herpes simplex virus type 1 (HSV-1) derived amplicon vectors, i.e. viral particles containing a genome of 152 kb constituted of concatameric repetitions of an expression cassette, enabling the expression of the gene of interest in multiple copies. HSV-1 based amplicon vectors are non-pathogenic and have been successfully employed in the past for gene delivery into the brain of living animals. Therefore, amplicon vectors should represent a logical choice for expressing a silencing cassette, which, in multiple copies, is expected to lead to an efficient knock-down of the target gene expression. Here, we employed two amplicon-based BDNF silencing strategies. The first, antisense, has been chosen to target and degrade the cytoplasmic mRNA pool of BDNF, whereas the second, based on the convergent transcription technology, has been chosen to repress transcription at the BDNF gene. Both these amplicon vectors proved to be effective in down-regulating BDNF expression in vitro, in BDNF-expressing mesoangioblast cells. However, only the antisense strategy was effective in vivo, after inoculation in the hippocampus in a model of status epilepticus in which BDNF mRNA levels are strongly increased. Interestingly, the knocking down of BDNF levels induced with BDNF-antisense was sufficient to produce significant behavioral effects, in spite of the fact that it was produced only in a part of a single hippocampus. In conclusion, this study demonstrates a reliable effect of amplicon vectors in knocking down gene expression in vitro and in vivo. Therefore, this approach may find broad applications in neurobiological studies.

Article	Year
Involvement of the neuropeptide nociceptin/orphanin FQ in kainate seizures. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 12427860	2002
Delayed epileptogenesis in nociceptin/orphanin FQ-deficient mice. Neuroreport\| PubMed ID: 12858040	2003
On the role of somatostatin in seizure control: clues from the hippocampus. Reviews in the neurosciences\| PubMed ID: 14513869	2003
Kainate seizures increase nociceptin/orphanin FQ release in the rat hippocampus and thalamus: a microdialysis study. Journal of neurochemistry\| PubMed ID: 15379884	2004
Fgf-2 overexpression increases excitability and seizure susceptibility but decreases seizure-induced cell loss. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 19052202	2008
Localized delivery of fibroblast growth factor-2 and brain-derived neurotrophic factor reduces spontaneous seizures in an epilepsy model. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 19366663	2009
Bystander effect on brain tissue of mesoangioblasts producing neurotrophins. Cell transplantation\| PubMed ID: 22525962	2012
Impairment of GABA release in the hippocampus at the time of the first spontaneous seizure in the pilocarpine model of temporal lobe epilepsy. Experimental neurology\| PubMed ID: 24768627	2014
MicroRNA profiles in hippocampal granule cells and plasma of rats with pilocarpine-induced epilepsy--comparison with human epileptic samples. Scientific reports\| PubMed ID: 26382856	2015
Silencing Status Epilepticus-Induced BDNF Expression with Herpes Simplex Virus Type-1 Based Amplicon Vectors. PloS one\| PubMed ID: 26954758	2016

Anna Binaschi

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