Expressão de zif268 no cérebro do lagarto Tropidurus Hispidus após exploração de um ambiente enriquecido.

In the present work, we investigated behavioral changes associated with the increase in Zif268 protein expression within telencephalic areas of the tropical lizard Tropidurus hispidus that correspond to the mammalian hippocampus (HC). We used 13 male individuals of this species, collected at the Federal Agrotechnical School of Rio Grande do Norte, under SISBIO license number 19561-1. Four animals had their brains removed and were submitted to a Western blot with antibodies for the Zif268 protein. The remaining animals were separated in two different groups: a control group (n=4) and an exploration group (n=5). Animals from the exploration group were exposed to an enriched environment with many sensory cues novel to them. Control group animals stayed in the environment they were already habituated to. After 90 min from the onset of exposure to the new environment, animals from both groups were submitted to intracardiac perfusion with fixative, and the brains were removed, cryoprotected and frozen. After that, brains were sectioned at 20 μm and the sections were subjected to immunohistochemistry for the Zif268 protein. We verified that the Zif268 protein is likely conserved in the brain of T. hispidus, which showed antigenicity for the antibody anti-Zif268 made in mammals. In animals from the exploration group, we detected an increase of the Zif268 protein in the Septum, Striatum, Dorsoventricular Area and in cortical areas corresponding to the HC. This increase was proportional to the amount of environmental exploration, with maximum positive correlation in the hippocampal subareas Medial Cortex (R = 0.94 and p = 0.004) and Dorsomedial Cortex (R = 0.92 and p = 0.006). The data corroborate the notion that the reptilian hippocampus, as well as the mammalian HC, plays an important role in spatial exploration.

Estimulação optogenética do septo medial no rato anestesiado e em livre comportamento

Theta rhythm consists of an electrophysiological hippocampal oscillation present in mammalian species (4-12 Hz with variations across species). This oscillation is present during active waking and is also prevalent in local field potentials (LFP) during rapid eye movement sleep (REM sleep). Several studies have shown that theta rhythm is important in cognitive tasks and that the medial septum is a key region for its occurrence. The septum sends cholinergic, GABAergic and glutamatergic projections to the hippocampus, which in turn projects axons to the septum. Besides the septum, other regions are involved in regulating theta rhythm, forming a complex network of interactions among brain areas that result in theta rhythm. Optogenetics is a recently developed method that has been widely used in various research areas. It allows us to manipulate the electrical activity of neurons through light stimulation. One of the existing techniques consists in using a viral vector to induce the neuronal expression of ion channels associated with the light-sensitive molecule rhodopsin (e.g. ChR2). Once infected, the neurons become sensitive to light of a particular wavelength. The present M. Sc. research aimed to perform luminous stimulation of the brain in anesthetized and freely behaving animals using chronically implanted electrodes and optical fibers in animals infected with a viral vector for ChR2 expression. Surgical viral injections were performed in the medial septum; histological results confirmed the expression of ChR2 by way of the presence of the eYFP reporter protein in the septum and also in hippocampal processes. Moreover, we performed acute experiments with luminous stimulation of the medial septum and LFP recordings of the septum and hippocampus of anesthetized animals. Action potentials were recorded in the septum. In these experiments we observed a significant increase in the firing rates of septal neurons during luminous stimulation (n = 300 trials). Furthermore, we found an early light-evoked response in the hippocampal LFP. Chronic experiments with luminous stimulation of the medial septum and hippocampus in freely behaving animals were also performed in combination with LFP recordings. We found that the luminous stimulation of the septum is able to induce theta rhythm in the hippocampus. Together, the results demonstrate that the luminous stimulation of the medial septum in optogenetically-modified animals causes relevant electrophysiological changes in the septum and the hippocampus.

Efeitos da nicotina no complexo do septo medial na via septo hipocampal em camundongos anestesiados por Ketamina

Nicotine administration in humans and rodents enhances memory and attention, and also has a positive effect in Alzheimer's Disease. The Medial Septum / Diagonal Band of Broca complex (MS/DBB) – a main cholinergic system – massively projects to the hippocampus through the fimbria-fornix, and this pathway is called the septohippocampal pathway. It has been demonstrated that the MS/DBB acts directly on the local field potential (LFP) rhythmic organization of the hippocampus, especially in the rhythmogenesis of Theta (4-8Hz) – an oscillation intrinsically linked to hippocampus mnemonic function. In vitro experiments gave evidence that nicotine applied to the MS/DBB generates a local network Theta rhythm within the MS/DBB. Thus, the present study proposes to elucidate the function of nicotine in the MS/DBB on the septo-hippocampal pathway. In vivo experiments compared the effect of MS/DBB microinfusion of saline (n=5) and nicotine (n=8) on Ketamine/Xylazine anaesthetized mice. We observed power spectrum density in the Gamma range (35 to 55 Hz) increasing in both structures (Wilcoxon Rank-Sum test, p=0.038) but with no change in coherence between these structures in the same range (Wilcoxon Rank-Sum test, p=0.60). There was also a decrease in power of the ketamineinduced Delta oscillation (1 to 3 Hz). We also performed in vitro experiments on the effect of nicotine on membrane voltage and action potential. We patch-clamped 22 neurons in current-clamp mode; 12 neurons were responsive to nicotine, half of them increased firing rate and other 6 decreased, and they significantly differed in action potential threshold (-47.3±0.9 mV vs. -41±1.9 mV, respectively, p=0.007) and halfwidth time (1.6±0.08 ms vs. 2±0.12 ms, respectively, p=0.01). Furthermore, we performed another set of in vitro experiments concerning the connectivity of the three major neuronal populations of MS/DBB that use acetylcholine, GABA or glutamate as neurotransmitter. Paired patch-clamp recordings found that glutamatergic and GABAergic neurons realize intra-septal connections that produce sizable currents in MS/DBB postsynaptic neurons. The probability of connectivity between different neuronal populations gave rise to a MS/DBB topology that was implemented in a realistic model, which corroborates that the network is highly sensitive to the generation of Gamma rhythm. Together, the data available in the full set of experiments suggests that nicotine may act as a cognitive enhancer, by inducing gamma oscillation in the local circuitry of the MS/DBB.