José Manuel Rodríguez Ferrer

Changes in brain amino acids and nitric oxide after melatonin administration in rats with pentylenetetrazole‐induced seizures

Abstract: We examined the effect of melatonin on brain levels of amino acids and nitric oxide (NO) after pentylenetetrazole (PTZ)‐induced seizures in rats. Animals were treated with melatonin (10–160 mg/kg, i.p.) 30 min before PTZ administration (100 mg/kg, s.c.), and were killed 3 hr later. At the dose of 80 mg/kg, melatonin significantly increased the latency (5.7–12.7 min) and decreased the duration (31.2–18.4 s) of the first seizure, reducing PTZ induced mortality from 87.5 to 25%. After kill, brains were removed and neurotransmitters and nitrite levels measured in prefrontal cortex (PF), parieto‐temporal cortex (PF), striatum (ST), hippocampus (HP) and brain stem (BS) by high performance liquid chromatography. PTZ treatment increased glutamine levels in all brain areas studied, without changes in glutamate, gamma‐amino butyric acid (GABA) and glycine. Aspartate and taurine increased in PF and PT and in HS and PT, respectively. Melatonin administration displayed a dose‐dependent effect. At doses of 10–40 mg/kg, melatonin counteracted the PTZ‐induced glutamine increase and reduced both glutamate and asparatate levels in the studied areas, with minor changes in GABA and glycine content. At doses of 80 and 160 mg/kg, the levels of glutamine, and glutamate, and to a lesser extent aspartate increased, whereas serine levels did not change. These two doses of melatonin also increased taurine, GABA and glycine in most brain areas studied. Treatment with melatonin (40–160 mg/kg) significantly decreased nitrite content in PT cortex, ST and BS areas of epileptic rats, without changes in the other brain regions. The results suggest that the anticonvulsant property of melatonin involves a modulation of both brain amino acids and NO production.

Effects of agonists and antagonists of D1 and D2 dopamine receptors on self-stimulation of the medial prefrontal cortex in the rat.

The possible participation of D1 versus D2 dopamine receptors in mediating dopaminergic neurotransmission of self-stimulation (SS) in the medial prefrontal cortex (MPC) of the rat was studied neuropharmacologically. Intracerebral as well as intraperitoneal injections of agonists and antagonists of dopamine receptors were used in this study. In all experiments performed with systemic injections, spontaneous motor activity (SM) was measured parallel to self-stimulation behavior as control for non specific effects of the drugs. Intracranial injections were done unilaterally serving SS of the contralateral side (not injected or injected with 0.9% NaCl) as control in the same animals. Spiroperidol and pimozide were used as D1-D2 dopamine antagonists, while sulpiride was used as a specific D2 antagonist. Apomorphine was used as D1-D2 agonist, while bromocriptine and lergotrile were used at doses in which these ergot drugs are considered predominantly D2 agonists. Sulpiride, intraperitoneally or intracerebrally injected at the same locus at which the stimulating electrode was located produced no effect on SS. On the contrary, the D1-D2 antagonists, spiroperidol and pimozide intraperitoneally or intracerebrally injected produced a dose-dependent decrease on SS. On the basis of these data it is suggested, that the dopamine neurotransmission involved in SS of the MPC is mediated via D1 dopamine receptors. This suggestion is further emphasized by the results obtained with the agonists, apomorphine, bromocriptine and lergotrile. Apomorphine produced a dose-related decrease on SS and a decrease at lower doses and an increase at higher doses on SM. Bromocriptine and lergotrile had, on the contrary, no effect on SS and a dose-related decrease on SM.

Peptides and self-stimulation of the medial prefrontal cortex in the rat: effects of intracerebral microinjections of substance P and cholecystokinin.

The effects of intracerebral microinjections of substance P and cholecystokinin on self-stimulation of the medial prefrontal cortex of the rat were studied. Intracerebroventricular administration of substance P at doses of 2.5, 5, 10 and 20 micrograms produced a dose-related decrease in self-stimulation of the medial prefrontal cortex; spontaneous motor activity, measured as a control, was not affected. Unilateral microinjections into the medial prefrontal cortex of substance P at doses of 10 and 20 micrograms produced a decrease of self-stimulation of the ipsilateral side, but self-stimulation of the contralateral cortex, used as a control, was not affected. On the contrary, cholecystokinin in both intracerebroventricular administration at doses of 100, 200 and 400 ng, or intracortical microinjections into the medial prefrontal cortex at doses of 200, 400 and 800 ng, had no effect on self-stimulation of this cortical area. These results suggest that substance P, but not cholecystokinin, could be part of the neurochemical substrate underlying self-stimulation of the medial prefrontal cortex in the rat.

Electrophysiological and neurochemical study of the rat geniculo-cortical pathway. Evidence for glutamatergic neurotransmission

The projection from the dorsal lateral geniculate nucleus to the primary visual cortex of the rat was studied electrophysiologically. Electrical stimulation of the dorsal lateral geniculate nucleus and the optic tract produced three types of responses on neurons of area 17: excitation followed by inhibition, excitation and inhibition. These results extend and confirm, in adult rats, previous studies done in rat geniculate‐visual cortex cocultures preparations in vitro. The role of glutamate in the neurotransmission of the rat geniculo‐cortical pathway was also investigated. In a first set of experiments, the effects of kynurenate, an antagonist of glutamate receptors, on visual cortex neurons with a monosynaptic excitatory response to dorsal lateral geniculate nucleus stimulation were studied. Microiontophoresis of kynurenate in area 17 neurons selectively suppressed the excitatory response to dorsal lateral geniculate nucleus and optic tract stimulation. In a second set of experiments, the effects of electrical stimulation of the dorsal lateral geniculate nucleus and the optic tract on the release of amino acids in the rat visual cortex in vivo were studied. Using the push–pull method, we perfused a discrete region of the visual cortex with artificial cerebrospinal fluid (CSF), and the amino acid content of the perfusates was analysed by high performance liquid chromatography (HPLC). Stimulation of either the dorsal lateral geniculate nucleus or the optic tract significantly increased glutamate release in area 17. The rest of the amino acids studied did not show significant changes. The results provide evidence for the participation of glutamate in the neurotransmission of the geniculo‐cortical pathway in the rat.

Melatonin enhances neural stem cell differentiation and engraftment by increasing mitochondrial function

Neural stem cells (NSCs) are regarded as a promising therapeutic approach to protecting and restoring damaged neurons in neurodegenerative diseases (NDs) such as Parkinsons disease and Alzheimers disease (PD and AD, respectively). However, new research suggests that NSC differentiation is required to make this strategy effective. Several studies have demonstrated that melatonin increases mature neuronal markers, which reflects NSC differentiation into neurons. Nevertheless, the possible involvement of mitochondria in the effects of melatonin during NSC differentiation has not yet been fully established. We therefore tested the impact of melatonin on NSC proliferation and differentiation in an attempt to determine whether these actions depend on modulating mitochondrial activity. We measured proliferation and differentiation markers, mitochondrial structural and functional parameters as well as oxidative stress indicators and also evaluated cell transplant engraftment. This enabled us to show that melatonin (25 μM) induces NSC differentiation into oligodendrocytes and neurons. These effects depend on increased mitochondrial mass/DNA/complexes, mitochondrial respiration, and membrane potential as well as ATP synthesis in NSCs. It is also interesting to note that melatonin prevented oxidative stress caused by high levels of mitochondrial activity. Finally, we found that melatonin enriches NSC engraftment in the ND mouse model following transplantation. We concluded that a combined therapy involving transplantation of NSCs pretreated with pharmacological doses of melatonin could efficiently restore neuronal cell populations in PD and AD mouse models depending on mitochondrial activity promotion.

The basolateral limbic circuit and self-stimulation of the medial prefrontal cortex in the rat

The effects of lesions of the basolateral nucleus of the amygdala (ABL) and the mediodorsal nucleus of the thalamus (MD) on self-stimulation (SS) of the medial prefrontal cortex (MPC) were investigated. Spontaneous motor activity (SMA) was measured as a control for possible non-specific effects of the lesions. Bilateral electrolytic lesions of ABL or MD produced a parallel transient decrease of SS and SMA. However, combined lesion of ABL and MD produced clearly different effects on both parameters. SMA decreased during the 1st day post-lesion and recovered to control levels by the 3rd day post-lesion. SS, on the contrary, was significantly decreased during the first five days post-lesion and after that time SS rate recovered to control levels. These results suggest the involvement of the basolateral limbic circuit in the neural substate underlying SS behavior of the MPC.

The incidence of bifurcation among corticocortical connections from area 17 in the developing visual cortex of the cat

In newborn kittens, cells in the striate cortex (visual area 17) that project to area 18 (part of extrastriate cortex) are distributed with uniform density in the superficial and in the deep layers. During postnatal weeks 2 – 3, some of these corticocortical connections are removed to generate an adult‐like projection in which association cells are clustered mainly in the superficial layers of area 17. Axonal elimination, without cell death, is the major factor sculpting patches of corticocortical cells in superficial layers. In adult cats, few cells in area 17 (∼5%) have axons that bifurcate to multiple extrastriate areas. We have studied the possibility that the early exuberant innervation of area 18 by neurons in area 17 is largely from the transient collaterals of axons that also project to other visual areas. Kittens aged 2 – 21 days were each injected with a pair of retrogradely transported tracers, either diamidino yellow and fast blue, or diamidino yellow and a carbocyanine dye, at retinotopically corresponding points in area 18 and either area 19 or the posteromedial lateral suprasylvian cortex (PMLS). As for injections in area 18, those in area 19 and PMLS in kittens aged ≥5 days labelled cells in continuous bands in area 17; in older kittens neurons projecting from area 17 to extrastriate regions were in patches, mainly in superficial layers. In each animal, the labelling from the two injections overlapped by 51–92%. However, at all ages, never more than 4% of cells projecting to area 18 branched to PMLS; ≤6% of area 17‐to‐18 cells bifurcated to area 19 in kittens aged ≥15 days, although slightly more (10 – 12%) did so at 3 – 5 days. Thus, as in adults, we found no evidence of frequent collateralization among the axons of cells projecting from area 17 to other extrastriate areas in kittens.

Neurotensin participates in self-stimulation of the medial prefrontal cortex in the rat

The effects of intracerebral microinjections of neurotensin and xenopsin on self-stimulation of the medial prefrontal cortex of the rat were studied. Unilateral microinjections into the medial prefrontal cortex of neurotensin at doses of 0.625, 1.25, 2.5, 5 and 10 nmol produced a dose-related decrease of self-stimulation in the ipsilateral medial prefrontal cortex. Self-stimulation of the contralateral medial prefrontal contex, used as control, was not affected by the microinjections. Similar results were found with the neurotension-like octapeptide, xenopsin. Unilateral microinjections of xenoposin into the medial prefrontal cortex, at doses of 1.8, 3.6, 7.2 and 14.4 nmol produced a dose-related decrease of self-stimulation of the ipsilateral medial prefrontal cortex. Self-stimulation of the contralateral medial prefrontal cortex was not affected. These results suggest that neurotensin is part of the neurochemical substrate of self-stimulation in this cortical area.

Plasticity of an aberrant geniculocortical pathway in neonatally lesioned cats

Geniculocortical afferents to the lateral suprasylvian (LS) visual area come only from the C layers in normal adult cats. After neonatal lesions of areas 17 and 18, inputs to the LS area also arise from the A layers. We studied the plasticity of this aberrant pathway. We made lesions in areas 17 and 18 of newborn kittens, some of which were then monocularly deprived. In lesioned undeprived cats, retrograde tracing confirmed projections to the LS area from both the A and A1 as well as the C geniculate layers. In lesioned deprived cats, geniculate afferents from the A layers to the LS area came mainly from the layer receiving a normal visual input. We conclude that the development of the abnormal pathway from the A layers to the LS area is influenced by patterned visual activity.

Peptidase inhibitors potentiate the effects of neurotensin and neuromedin N on self-stimulation of the medial prefrontal cortex

The purpose of this study was to examine the possible role of endogenous peptidases in the inhibition of intracranial self-stimulation (ICSS) produced by injections of neurotensin (NT) and neuromedin N (NN) into the medial prefrontal cortex (MFC) of the rat. We studied the effects on ICSS of the MFC of the administration of thiorphan and bestatin, two specific inhibitors of the peptidases that inactivate NT and NN respectively. Microinjections into MFC of thiorphan (10 μg) and bestatin (25 μg) potentiated in inhibition of ICSS produced by the intracortical administration of NT (10 nmol) and NN (20 nmol) respectively. This potentiation affected both the amplitude and the duration of the inhibition of ICSS produced by the neuropeptides. Our data indicate that endogenous peptidases are involved in the inactivation of NT and NN in the prefrontal cortex.