Gisela P. Pajolla

Comparative neuroanatomical and temporal characterization of FluoroJade-positive neurodegeneration after status epilepticus induced by systemic and intrahippocampal pilocarpine in Wistar rats.

The aims of this study were to characterize the spatial distribution of neurodegeneration after status epilepticus (SE) induced by either systemic (S) or intrahippocampal (H) injection of pilocarpine (PILO), two models of temporal lobe epilepsy (TLE), using FluoroJade (FJ) histochemistry, and to evaluate the kinetics of FJ staining in the H-PILO model. Therefore, we measured the severity of behavioral seizures during both types of SE and also evaluated the FJ staining pattern at 12, 24, and 168 h (7days) after the H-PILO insult. We found that the amount of FJ-positive (FJ+) area was greater in SE induced by S-PILO as compared to SE induced by H-PILO. After SE induced by H-PILO, we found more FJ+ cells in the hilus of the dentate gyrus (DG) at 12 h, in CA3 at 24 h, and in CA1 at 168 h. We found also no correlation between seizure severity and the number of FJ+ cells in the hippocampus. Co-localization studies of FJ+ cells with either neuronal-specific nuclear protein (NeuN) or glial fibrillary acidic protein (GFAP) labeling 24 h after H-PILO demonstrated spatially selective neurodegeneration. Double labeling with FJ and parvalbumin (PV) showed both FJ+/PV+ and FJ+/PV- cells in hippocampus and entorhinal cortex, among other areas. The current data indicate that FJ+ areas are differentially distributed in the two TLE models and that these areas are greater in the S-PILO than in the H-PILO model. There is also a selective kinetics of FJ+ cells in the hippocampus after SE induced by H-PILO, with no association with the severity of seizures, probably as a consequence of the extra-hippocampal damage. These data point to SE induced by H-PILO as a low-mortality model of TLE, with regional spatial and temporal patterns of FJ staining.

Cardiovascular responses to the injection of l-glutamate in the lateral hypothalamus of unanesthetized or anesthetized rats

The present experiment was designed to compare the cardiovascular effects of injections of 0.1 M L-glutamate (50, 100 or 500 nL) into the anterior (LHa), tuberal (LHt) or posterior (LHp) regions of the lateral hypothalamus (LH) of either unanesthetized or anesthetized male Wistar rats. In unanesthetized rats, L-glutamate caused significant depressor responses without significant heart rate (HR) effects. L-Glutamate caused similar depressor responses when injected into the different LH subregions. A positive trend was observed between depressor response intensity and injected volume. In urethane-anesthetized rats, L-glutamate caused either depressor responses or biphasic responses, characterized by a significant initial depressor component followed by a secondary pressor response which was significant only after the injection of L-glutamate in 500 nL. The depressor component was accompanied by significant bradycardia only when the LHa or LHt were stimulated. Similar depressor responses were observed after L-glutamate microinjection into the different LH subregions. A positive trend was observed between depressor response intensity and injected volume. The present results suggest that: 1) lateral hypothalamic L-glutamate-sensitive neurons are involved in cardiovascular control and may have a wide and homogeneous distribution throughout the LH; 2) these neurons are mainly associated to the expression of hypotensive responses in unanesthetized rats; and 3) bradycardiac responses are evidenced when L-glutamate is microinjected into the LHa and the LHt in urethane-anesthetized rats.

The Lateral Hypothalamus Is Involved in the Pathway Mediating the Hypotensive Response to Cingulate Cortex-Cholinergic Stimulation

Abstract1. The injection of acetylcholine (ACh) into the medial prefrontal cortex (MPFC) caused marked hypotensive response in either unanesthetized or anesthetized rats.2. The present experiment was designed to investigate anatomical connections of the ACh injection site in the MPFC with putative autonomic-related brain nuclei, as well as their possible involvement in the mediation of the hypotensive response to ACh.3. For the above purpose, the bidirectional neuronal tracer biotinylated dextran amine (BDA) was injected into Cg1 and Cg3 areas, within the MPFC of male Wistar rats. Five days later the animals were sacrificed and brain slices were processed and analyzed to determine neuronal projections efferent from as well afferent to the MPFC.4. Neuronal staining was more prominent in regions ipsilateral to the BDA injection site. Prominent efferent projections of the MPFC were observed in the contralateral MPFC; ipsi- and contralateral amygdala and hypothalamus; ipsilateral septal area, diagonal band, and zona incerta.5. Similar but not equal patterns of neuronal labeling were observed when BDA injections were performed within the two adjacent MPFC areas. BDA injections centered in the ACh injection site in the Cg3 area caused strong labeling in the septal area and diagonal band as well as an overall hypothalamic labeling. Within the hypothalamus an intense cortical projection was observed in the lateral hypothalamus (LH). BDA injections into the Cg1 area caused a more evident labeling of the amygdaloid complex.6. Neuronal cell bodies were evident throughout the MPFC as well as in the sensory-motor cortex when BDA was injected into the LH, thus indicating a massive ipsilateral cortical projection from the Cg3 to the LH.7. Bilateral NMDA-induced lesions within the LH caused a significant attenuation of the depressor responses to ACh injection in the MPFC, whereas unilateral lesions were marginally effective. These results indicate the involvement of the LH in the mediation of the hypotensive response to ACh injection into the MPFC as well as the bilateral distribution of the hypotensive pathway.

Neural connections between prosencephalic structures involved in vasopressin release.

Abstract1. The diagonal band (DB) and the lateral septal area (LSA) are two prosencephalic structures, which were implicated in vasopressin release.2. The present experiment was designed to investigate neural connections between the DB and the LSA and from these nuclei to the paraventricular (PVN) and supraoptic (SON) nuclei, which could be related to vasopressin release.3. For the above purpose the bidirectional neuronal tracer biotinylated dextran amine (BDA) was injected into the DB or the LSA of male Wistar rats. Five days later the animals were sacrificed and brain slices were processed and analyzed to determine neuronal projections efferent from as well as afferent to these structures.4. Neuronal staining was more prominent in regions ipsilateral to the BDA injection site.5. After BDA injections into the DB, efferent projections from the DB were observed at the LSA, the PVN, the prefrontal cortex, the mediodorsal thalamic nucleus, and throughout the anterior hypothalamus, but not at the SON. At the PVN, labeled varicose fibers were observed at the magnocellular portion. The DB was found to receive a massive input from the LSA. More discrete projections to the DB were originated at the prefrontal cortex and from hypothalamic neurons outside the PVN and the SON.6. After BDA injections into the ventral portion of the LSA, efferent projections from the LSA were intense at the DB and throughout the hypothalamus. Labeled fibers were observed at the structures surrounding the SON or the PVN but not within those nuclei.7. The results indicate a massive neural output from the LSA to the DB and the existence of a direct neural connection from the DB to the PVN. No direct connections were observed between the LSA and the magnocellular nuclei (PVN and SON) or between the DB and the SON.

Involvement of the periaqueductal gray in the hypotensive response evoked by l-glutamate microinjection in the lateral hypothalamus of unanesthetized rats

The lateral hypothalamus (LH) is involved in cardiovascular control. L-glutamate (L-glu) stimulation of the LH of unanesthetized rats evoked hypotensive responses without significant heart rate changes. The neuronal pathway that mediates this response is unknown. There is evidence that the periaqueductal gray (PAG) is involved in the mediation of hypotensive responses evoked by electrical stimulation of the LH. In the present study, we attempted to verify the effect of an acute and reversible pharmacological ablation of the PAG with lidocaine or CoCl(2) on the hypotensive response caused by L-glu injection in the LH of unanesthetized rats. Microinjection of the local anesthetic lidocaine or the unspecific synaptic blocker CoCl(2) in the PAG significantly attenuated the hypotensive effects of L-glu stimulation of the LH, indicating the involvement of local synapses within the PAG in the hypotensive pathway activated by LH glutamatergic receptors. Microinjection of the neuronal tracer biotinylated dextran amine (BDA) in the PAG labeled neuronal cell bodies in the LH, indicating the existence of direct connections between these areas. In conclusion, the present results indicate that the hypotensive response evoked by L-glu stimulation of LH may involve a synaptic relay in the dorsal PAG.

Immunoreactivity for neuronal NOS and fluorescent indication of NO formation in the NTS of juvenile rats submitted to chronic intermittent hypoxia.

Exposure to chronic intermittent hypoxia (CIH) leads to significant autonomic and respiratory changes, similar to those observed in obstructive sleep apnea. The hypertension associated with CIH is due to sympathoexcitation triggered by long-term exposure to intermittent hypoxia. However, the mechanisms underlying these effects are unknown. Changes in central regulation of sympathetic activity may underlie CIH-induced hypertension. Since NO appears to be mainly sympathoinhibitory in the nucleus of the solitary tract (NTS), we hypothesized that CIH augments sympathetic activity, in part by reducing neuronal nitric oxide synthase (nNOS) expression and consequently nitric oxide (NO) production in this brain region. To test our hypothesis, juvenile male Wistar rats were exposed to CIH for 8 h/day for 10 days and sections of perfused brainstem were either stained to reveal nNOS-immunoreactivity or loaded with DAF 2-DA to label neurons containing NO. CIH rats showed a significant increase in mean arterial pressure and heart rate compared to controls. However, there was no significant difference in the distribution, staining intensity or numbers of nNOS-immunoreactive neurons in the NTS between experimental and control rats. We also found no significant change in NO content in the DAF 2-DA-loaded sections of NTS from CIH rats. Our data show that NO is not altered in the NTS of juvenile CIH rats, suggesting that nitrergic mechanisms, at least in the NTS, are unlikely to be involved in the sympathetic excitation that generates the hypertension observed after 10 days of CIH.

Involvement of NMDA receptors in the hypotensive response to the injection of L-glutamate into the lateral hypothalamus of unanesthetized rats.

We report that microinjections of L-glutamate (L-glu) or N-methyl-D-aspartic acid (NMDA) in the lateral hypothalamus (LH) of unanesthetized rats caused a hypotensive response. Guide cannulas were stereotaxically placed in the LH 3 days before the experiments, under tribromoethanol anesthesia. One day before the experiments, the femoral artery was cannulated for pulsatile arterial pressure (PAP), mean arterial pressure (MAP) and heart rate (HR) measurements. In the first experiment, unanesthetized rats received microinjections of 2.5, 5.0 or 10.0 nmol/100 nL of L-glu in the LH. Dose-dependent hypotensive responses were observed, without significant concomitant changes in heart rate. In a second group of experiments, 5.0 nmol of L-glu was microinjected into the LH before and 10 min after pretreatment with glutamatergic antagonists. Pretreatments with the non-selective ionotropic glutamatergic-receptor antagonist kynurenic acid or the selective NMDA receptor antagonists AP-7 and LY235959 significantly reduced the hypotensive response to microinjection of L-glu in the LH. Pretreatment with the selective AMPA-receptor antagonist NBQX or with vehicle did not affect the hypotensive response. The present results suggest that the hypotensive response to the injection of L-glu into the LH is mediated by NMDA receptors.

Interaction of purinergic and nitrergic mechanisms in the caudal nucleus tractus solitarii of rats.

The interaction of purinergic and nitrergic mechanisms was evaluated in the caudal nucleus tractus solitarii (cNTS) using awake animals and brainstem slices. In awake animals, ATP (1.25 nmol/50 nL) was microinjected into the cNTS before and after the microinjection of a selective neuronal nitric oxide synthase (nNOS) inhibitor N-propyl-l-arginine (NPLA, 3 pmoles/50 nL, n=8) or vehicle (saline, n=4), and cardiovascular and ventilatory parameters were recorded. In brainstem slices from a distinct group of rats, the effects of ATP on the NO concentration in the cNTS using the fluorescent dye DAF-2 DA were evaluated. For this purpose brainstem slices (150 microm) containing the cNTS were pre-incubated with ATP (500 microM; n=8) before and during DAF-2 DA loading. Microinjection of ATP into the cNTS increases the arterial pressure (AP), respiratory frequency (f(R)) and minute ventilation (V(E)), which were significantly reduced by pretreatment with N-PLA, a selective nNOS inhibitor (AP: 39+/-3 vs 16+/-14 mm Hg; f(R): 75+/-14 vs 4+/-3 cpm; V(E): 909+/-159 vs 77+/-39 mL kg(-1) m(-1)). The effects of ATP in the cNTS were not affected by microinjection of saline. ATP significantly increased the NO fluorescence in the cNTS (62+/-7 vs 101+/-10 AU). The data show that in the cNTS: a) the NO production is increased by ATP; b) NO formation by nNOS is involved in the cardiovascular and ventilatory responses to microinjection of ATP. Taken together, these data suggest an interaction of purinergic and nitrergic mechanisms in the cNTS.