Rodrigo N. Romcy-Pereira

Distinct modulatory effects of sleep on the maintenance of hippocampal and medial prefrontal cortex LTP

Both human and animal studies support the idea that memory consolidation of waking experiences occurs during sleep. In experimental models, rapid‐eye‐movement (REM) sleep has been shown to be necessary for cortical synaptic plasticity and for the acquisition of spatial and nonspatial memory. Because the hippocampus and medial prefrontal cortex (mPFC) play distinct and important roles in memory processing, we sought to determine the role of sleep in the maintenance of long‐term potentiation (LTP) in the dentate gyrus (DG) and mPFC of freely behaving rats. Animals were implanted with stimulating and recording electrodes, either in the medial perforant path and DG or CA1 and mPFC, for the recording of field potentials. Following baseline recordings, LTP was induced and the animals were assigned to three different groups: REM sleep‐deprived (REMD), total sleep‐deprived (TSD) and control which were allowed to sleep (SLEEP). The deprivation protocol lasted for 4 h and the recordings were made during the first hour and at 5, 24 and 48 h following LTP induction. Our results show that REMD impaired the maintenance of late‐phase (48‐h) LTP in the DG, whereas it enhanced it in the mPFC. Sleep, therefore, could have distinct effects on the consolidation of different forms of memory.

Hippocampal cell proliferation and epileptogenesis after audiogenic kindling are not accompanied by mossy fiber sprouting or Fluoro-Jade staining.

Repetitive sound-induced seizures, known as audiogenic kindling (AK), gradually induce the transference of epileptic activity from brainstem to forebrain structures along with behavioral changes. The aim of our work was to correlate the behavioral changes observed during the AK with possible alterations in neuronal proliferation, cell death, hippocampal mossy fiber sprouting and in the EEG pattern of Wistar audiogenic rats, a genetically susceptible strain from our laboratory. Susceptible and non-susceptible animals were submitted to repeated sound stimulations for 14-16 days and hippocampal mitotic activity was studied through the incorporation of bromodeoxyuridine (BrdU). Cell death and mossy fiber sprouting were assessed, respectively, by using Fluoro-Jade and Timm staining, 2 and 32 days after the last kindling stimulation. In addition, we used immunofluorescent double labeling for a glial and a mitotic marker to evaluate newly born cell identity. Some animals had hippocampus and amygdala electrodes for EEG recordings. Our results show that kindled animals with 6-11 generalized limbic seizures (class IV-V) had increased cell proliferation in the dentate gyrus when compared with animals with zero or one to three seizures. BrdU-positive cells labeled on day 2 and on day 32 were both GFAP negative. In the later group, rounded and well-defined BrdU-positive/GFAP-negative nuclei were seen in different portions of the granule cell layer. We did not observe any Fluoro-Jade or differential Timm staining in kindled animals at both killing times. However, EEG recordings showed intense epileptic activity in the hippocampus and amygdala of all animals with limbic seizures.Therefore, our data indicate that AK-induced limbic epileptogenicity is able to increase the hippocampal mitotic rate, even though it does not seem to promote neuronal death or mossy fiber sprouting in the supragranular layer of the dentate gyrus.

Sleep-Dependent Gene Expression in the Hippocampus and Prefrontal Cortex Following Long-Term Potentiation

The activity-dependent transcription factor zif268 is re-activated in sleep following hippocampal long-term potentiation (LTP). However, the activation of secondary genes, possibly involved in modifying local synaptic strengths and ultimately stabilizing memory traces during sleep, has not yet been studied. Here, we investigated changes in hippocampal and cortical gene expression at a time point subsequent to the previously reported initial zif268 re-activation during sleep. Rats underwent unilateral hippocampal LTP and were assigned to SLEEP or AWAKE groups. Eighty minutes after a long rapid-eye-movement sleep (REMS) episode (or an equivalent amount of time for awake group) animals had their hippocampi dissected and processed for gene microarray hybridization. Prefrontal and parietal cortices were also collected for qRT-PCR analysis. The microarray analysis identified 28 up-regulated genes in the hippocampus: 11 genes were enhanced in the LTPed hemisphere of sleep animals; 13 genes were enhanced after sleep, regardless of hemisphere; and 4 genes were enhanced in LTPed hemisphere, regardless of behavioral state. qRT-PCR analysis confirmed the up-regulation of aif-1 and sc-65 during sleep. Moreover, we observed a down-regulation of the purinergic receptor, P2Y4R in the LTP hemisphere of awake animals and a trend for the protein kinase, CaMKI to be up-regulated in the LTP hemisphere of sleep animals. In the prefrontal cortex, we showed a significant LTP-dependent down-regulation of gluR1 and spinophilin specifically during sleep. Zif268 was down-regulated in sleep regardless of the hemisphere. No changes in gene expression were observed in the parietal cortex. Our findings indicate that a set of synaptic plasticity-related genes have their expression modulated during sleep following LTP, which can reflect biochemical events associated with reshaping of synaptic connections in sleep following learning.

Muscarinic and Nicotinic Modulation of Thalamo-Prefrontal Cortex Synaptic Pasticity In Vivo

The mediodorsal nucleus of the thalamus (MD) is a rich source of afferents to the medial prefrontal cortex (mPFC). Dysfunctions in the thalamo-prefrontal connections can impair networks implicated in working memory, some of which are affected in Alzheimer disease and schizophrenia. Considering the importance of the cholinergic system to cortical functioning, our study aimed to investigate the effects of global cholinergic activation of the brain on MD-mPFC synaptic plasticity by measuring the dynamics of long-term potentiation (LTP) and depression (LTD) in vivo. Therefore, rats received intraventricular injections either of the muscarinic agonist pilocarpine (PILO; 40 nmol/µL), the nicotinic agonist nicotine (NIC; 320 nmol/µL), or vehicle. The injections were administered prior to either thalamic high-frequency (HFS) or low-frequency stimulation (LFS). Test pulses were applied to MD for 30 min during baseline and 240 min after HFS or LFS, while field postsynaptic potentials were recorded in the mPFC. The transient oscillatory effects of PILO and NIC were monitored through recording of thalamic and cortical local field potentials. Our results show that HFS did not affect mPFC responses in vehicle-injected rats, but induced a delayed-onset LTP with distinct effects when applied following PILO or NIC. Conversely, LFS induced a stable LTD in control subjects, but was unable to induce LTD when applied after PILO or NIC. Taken together, our findings show distinct modulatory effects of each cholinergic brain activation on MD-mPFC plasticity following HFS and LFS. The LTP-inducing action and long-lasting suppression of cortical LTD induced by PILO and NIC might implicate differential modulation of thalamo-prefrontal functions under low and high input drive.

A semi-automated algorithm for studying neuronal oscillatory patterns: a wavelet-based time frequency and coherence analysis.

In many experimental designs, animal observation is associated with local field potential (LFP) recordings in order to find correlations between behavior dynamics and neuronal activity. In such cases, relevant behaviors can occur at different times during free-running recordings and should be put together by the time of analysis. Here, we developed a MATLAB semi-automated toolbox to quantitatively analyze the temporal progression of brain oscillatory activity in multiple free-running LFP recordings obtained during spontaneous behaviors. The algorithm works by selecting LFP epochs at user-defined onset times (locked to behavior, drug injection time, etc.), calculates their time-frequency spectra, detects long-lasting oscillatory events and calculates linear coherence between pair of electrodes. As output, it generates several table-like text and tiff image files, besides group descriptive statistics. To test the algorithm, we recorded hippocampus and amygdala LFPs from rats in different behavioral states: awake (AW), sleep (SWS, slow-wave sleep and REMS, rapid-eye movement sleep) and tonic-clonic seizures. The results show that the software reliably detects all oscillatory events present in up to seven user-defined frequency bands including onset/offset time and duration. It also calculates the global spectral composition per epoch from each subject and the linear coherence (with confidence intervals) as a measure of spectral synchronization between brain regions. The output files provide an easy way to do within-subject as well as across-subject analysis. The routines will be freely available for downloading from our website http://www.neuroimago.usp.br/BPT/.

NMDA receptor blockade impairs the muscarinic conversion of sub-threshold transient depression into long-lasting LTD in the hippocampus–prefrontal cortex pathway in vivo: Correlation with gamma oscillations

Cholinergic fibers from the brainstem and basal forebrain innervate the medial prefrontal cortex (mPFC) modulating neuronal activity and synaptic plasticity responses to hippocampal inputs. Here, we investigated the muscarinic and glutamatergic modulation of long-term depression (LTD) in the intact projections from CA1 to mPFC in vivo. Cortical-evoked responses were recorded in urethane-anesthetized rats for 30 min during baseline and 4 h following LTD. In order to test the potentiating effects of pilocarpine (PILO), independent groups of rats received either a microinjection of PILO (40 nmol; i.c.v.) or vehicle, immediately before or 20 min after a sub-threshold LTD protocol (600 pulses, 1 Hz; LFS600). Other groups received either an infusion of the selective NMDA receptor antagonist (AP7; 10 nmol; intra-mPFC) or vehicle, 10 min prior to PILO preceding LFS600, or prior to a supra-threshold LTD protocol (900 pulses, 1 Hz; LFS900). Our results show that PILO converts a transient cortical depression induced by LFS600 into a robust LTD, stable for at least 4 h. When applied after LFS600, PILO does not change either mPFC basal neurotransmission or late LTD. Our data also indicate that NMDA receptor pre-activation is essential to the muscarinic enhancement of mPFC synaptic depression, since AP7 microinjection into the mPFC blocked the conversion of transient depression into long-lasting LTD produced by PILO. In addition, AP7 effectively blocked the long-lasting LTD induced by LFS900. Therefore, our findings suggest that the glutamatergic co-activation of prefrontal neurons is important for the effects of PILO on mPFC synaptic depression, which could play an important role in the control of executive and emotional functions.

Interplay of environmental signals and progenitor diversity on fate specification of cortical GABAergic neurons

Cortical GABAergic interneurons constitute an extremely diverse population of cells organized in a well-defined topology of precisely interconnected cells. They play a crucial role regulating inhibitory-excitatory balance in brain circuits, gating sensory perception, and regulating spike timing to brain oscillations during distinct behaviors. Dysfunctions in the establishment of proper inhibitory circuits have been associated to several brain disorders such as autism, epilepsy, and schizophrenia. In the rodent adult cortex, inhibitory neurons are generated during the second gestational week from distinct progenitor lineages located in restricted domains of the ventral telencephalon. However, only recently, studies have revealed some of the mechanisms generating the heterogeneity of neuronal subtypes and their modes of integration in brain networks. Here we will discuss some the events involved in the production of cortical GABAergic neuron diversity with focus on the interaction between intrinsically driven genetic programs and environmental signals during development.

Synaptic plasticity along the sleep-wake cycle: implications for epilepsy.

Activity-dependent changes in synaptic efficacy (i.e., synaptic plasticity) can alter the way neurons communicate and process information as a result of experience. Synaptic plasticity mechanisms involve both molecular and structural modifications that affect synaptic functioning, either enhancing or depressing neuronal transmission. They include redistribution of postsynaptic receptors, activation of intracellular signaling cascades, and formation/retraction of dendritic spines, among others. During the sleep-wake cycle, as the result of particular neurochemical and neuronal firing modes, distinct oscillatory patterns organize the activity of neuronal populations, modulating synaptic plasticity. Such modulation, for example, has been shown in the visual cortex following sleep deprivation and in the ability to induce hippocampal long-term potentiation during sleep. In epilepsy, synchronized behavioral states tend to contribute to the initiation of paroxystic discharges and are considered more epileptogenic than desynchronized states. Here, we review some of the current understandings of synaptic plasticity changes in wake and sleep states and how sleep may affect epileptic seizures.

Pre-ictal increase in theta synchrony between the hippocampus and prefrontal cortex in a rat model of temporal lobe epilepsy

The pathologically synchronized neuronal activity in temporal lobe epilepsy (TLE) can be triggered by network events that were once normal. Under normal conditions, hippocampus and medial prefrontal cortex (mPFC) work in synchrony during a variety of cognitive states. Abnormal changes in this circuit may aid to seizure onset and also help to explain the high association of TLE with mood disorders. We used a TLE rat model generated by perforant path (PP) stimulation to understand whether synchrony between dorsal hippocampal and mPFC networks is altered shortly before a seizure episode. We recorded hippocampal and mPFC local field potentials (LFPs) of animals with spontaneous recurrent seizures (SRSs) to verify the connectivity between these regions. We showed that SRSs decrease hippocampal theta oscillations whereas coherence in theta increases over time prior to seizure onset. This increase in synchrony is accompanied by a stronger coupling between hippocampal theta and mPFC gamma oscillation. Finally, using Granger causality we showed that hippocampus/mPFC synchrony increases in the pre-ictal phase and this increase is likely to be caused by hippocampal networks. The dorsal hippocampus is not directly connected to the mPFC; however, the functional coupling in theta between these two structures rises pre-ictally. Our data indicates that the increase in synchrony between dorsal hippocampus and mPFC may be predictive of seizures and may help to elucidate the network mechanisms that lead to seizure generation.

Genes e epilepsia I: epilepsia e alterações genéticas

INTRODUCTION Epilepsy is a neurological disorder characterized by spontaneous and recurrent seizures with an estimated prevalence of 2-3 % in the world population. Epileptic seizures are the result of paroxystic and hypersynchronous electrical activity, preferentially in cortical areas, caused by panoply of structural and neurochemical dysfunctions. Recent advances in the field have focused on the molecular mechanisms involved in the epileptogenic process. OBJECTIVES In the present review, we describe the main genetic alterations associated to the process of epileptogenesis and discuss the new findings that are shedding light on the molecular substrates of monogenic idiopathic epilepsies (MIE) and on genetically complex epilepsies (GCE). RESULTS AND CONCLUSION Linkage and association studies have shown that mutations in ion channel genes are the main causes of MIE and of predisposition for GCE. Moreover, mutations in genes involved in neuronal migration, glycogen metabolism and respiratory chain are associated to other syndromes involving seizures. Therefore, different gene classes contribute to the epileptic trait. The identification of epilepsy-related gene families can help us understand the molecular mechanisms of neuronal hyperexcitability and recognize markers of early diagnosis as well as new treatments for these epilepsies.