Javier Yajeya

A cholinergic synaptically triggered event participates in the generation of persistent activity necessary for eye fixation.

An exciting topic regarding integrative properties of the nervous system is how transient motor commands or brief sensory stimuli are able to evoke persistent neuronal changes, mainly as a sustained, tonic action potential firing. A persisting firing seems to be necessary for postural maintenance after a previous movement. We have studied in vitro and in vivo the generation of the persistent neuronal activity responsible for eye fixation after spontaneous eye movements. Rat sagittal brainstem slices were used for the intracellular recording of prepositus hypoglossi (PH) neurons and their synaptic activation from nearby paramedian pontine reticular formation (PPRF) neurons. Single electrical pulses applied to the PPRF showed a monosynaptic glutamatergic projection on PH neurons, acting on AMPA-kainate receptors. Train stimulation of the PPRF area evoked a sustained depolarization of PH neurons exceeding (by hundreds of milliseconds) stimulus duration. Both duration and amplitude of this sustained depolarization were linearly related to train frequency. The train-evoked sustained depolarization was the result of interaction between glutamatergic excitatory burst neurons and cholinergic mesopontine reticular fibers projecting onto PH neurons, because it was prevented by slice superfusion with cholinergic antagonists and mimicked by cholinergic agonists. As expected, microinjections of cholinergic antagonists in the PH nucleus of alert behaving cats evoked a gaze-holding deficit consisting of a re-centering drift of the eye after each saccade. These findings suggest that a slow, cholinergic, synaptically triggered event participates in the generation of persistent activity characteristic of PH neurons carrying eye position signals.

GABAergic neurotransmission and new strategies of neuromodulation to compensate synaptic dysfunction in early stages of Alzheimer's disease

Alzheimers disease (AD) is a progressive neurodegenerative disease characterized by cognitive decline, brain atrophy due to neuronal and synapse loss, and formation of two pathological lesions: extracellular amyloid plaques, composed largely of amyloid-beta peptide (Aβ), and neurofibrillary tangles formed by intracellular aggregates of hyperphosphorylated tau protein. Lesions mainly accumulate in brain regions that modulate cognitive functions such as the hippocampus, septum or amygdala. These brain structures have dense reciprocal glutamatergic, cholinergic, and GABAergic connections and their relationships directly affect learning and memory processes, so they have been proposed as highly susceptible regions to suffer damage by Aβ during AD course. Last findings support the emerging concept that soluble Aβ peptides, inducing an initial stage of synaptic dysfunction which probably starts 20-30 years before the clinical onset of AD, can perturb the excitatory-inhibitory balance of neural circuitries. In turn, neurotransmission imbalance will result in altered network activity that might be responsible of cognitive deficits in AD. Therefore, Aβ interactions on neurotransmission systems in memory-related brain regions such as amygdaloid complex, medial septum or hippocampus are critical in cognitive functions and appear as a pivotal target for drug design to improve learning and dysfunctions that manifest with age. Since treatments based on glutamatergic and cholinergic pharmacology in AD have shown limited success, therapies combining modulators of different neurotransmission systems including recent findings regarding the GABAergic system, emerge as a more useful tool for the treatment, and overall prevention, of this dementia. In this review, focused on inhibitory systems, we will analyze pharmacological strategies to compensate neurotransmission imbalance that might be considered as potential therapeutic interventions in AD.

Muscarinic agonist carbachol depresses excitatory synaptic transmission in the rat basolateral amygdala in vitro.

Intracellular recordings in slice preparations of the basolateral amygdala were used to test which excitatory amino acid receptors mediate the excitatory postsynaptic potentials due to stimulation of the external capsule. These recordings were also used to examine the action of muscarinic agonists on the evoked excitatory potentials. Intracellular recordings from amygdaloid pyramidal neurons revealed that carbachol (2–20 μM) suppressed, in a dose‐dependent manner, excitatory postsynaptic responses evoked by stimulation of the external capsule (EC). This effect was blocked by atropine. The estimated effective concentration to produce half‐maximal response (EC50) was 6.2 μM. Synaptic suppression was observed with no changes in the input resistance of the recorded cells, suggesting a presynaptic mechanism. In addition, the results obtained using the paired‐pulse protocol provided additional support for a presynaptic action of carbachol. To identify which subtype of cholinergic receptors were involved in the suppression of the EPSP, four partially selective muscarinic receptor antagonists were used at different concentrations: pirenzepine, a compound with a similar high affinity for muscarinic M1 and M4 receptors; gallamine, a noncompetitive antagonist for M2; methoctramine, an antagonist for M2 and M4; and 4‐diphenylacetoxy‐N‐methylpiperidine, a compound with similar high affinity for muscarinic receptors M1 and M3. None of them independently antagonized the suppressive effect of carbachol on the evoked EPSP completely, suggesting that more than one muscarinic receptor subtype is involved in the effect. These experiments provide evidence that in the amygdala muscarinic agonists block the excitatory synaptic response, mediated by glutamic acid, by acting on several types of presynaptic receptors. Synapse 38:151–160, 2000.

Simultaneous depletion of neurokinin A, substance P and calcitonin gene- related peptide from the caudal trigeminal nucleus of the rat during electrical stimulation of the trigeminal ganglion

Abstract The central terminals of the primary sensory trigeminal ganglion (TG) neurons projecting into the caudal trigeminal nucleus (CTN) of the rat exhibit neurokinin A (NKA)‐, substance P (SP)‐, and calcitonin gene‐related peptide (CGRP)‐immunoreactivities (IRs). We stimulated the TG in the rat to induce some of the alterations which might occur during migraine (neurogenic inflammation). Under a stereotaxic apparatus and by means of a bipolar electrode, one‐side TG of the animals were electrically stimulated (7.5 Hz, 5 ms, 0.8–1.4 mA) with square pulses for 5 min. Then, using immunohistochemical methods, the lower medulla of each rat was studied for NKA‐, SP‐ and CGRP‐IRs. Light microscopic examination of brain‐stem sequencial sections revealed a simultaneous decrease in the immunoreactivities of all neuropeptides (NKA, SP and CGRP) in the CTN ipsilateral to TG stimulation in comparison with the other (not stimulated) side CTN. It is suggested that this decrease in immunoreactivity would be due to the co‐release of neuropeptides following noxious stimuli and that NKA, SP and CGRP might therefore act as co‐transmitters or co‐modulators at the first central synapses of the trigeminal sensory pathway.

Alterations in neurokinin A-, substance P- and calcitonin gene- related peptide immunoreactivities in the caudal trigeminal nucleus of the rat following electrical stimulation of the trigeminal ganglion

We have carried out an immunohistochemical study on the presence of neurokinin A (NKA) and substance P (SP) in the rat caudal trigeminal nucleus (CTN) after electrical stimulation of the trigeminal ganglion (TG), used as an experimental model to induce alterations, some of which may occur during migraine attacks (release of vasoactive peptides from perivascular trigeminal axons and neurogenic inflammation). Both unilateral, 30 min electrical stimulation (5 Hz, 5 ms, 0.1-1 mA) of the TG and 5 min stimulation with a slight increase in the stimulating parameters (7.5 Hz, 5 ms, 1.4 mA) caused a significant depletion of the NKA and SP immunoreactivities (-IR) of the TG nerve central terminals in the ipsilateral CTN. Calcitonin gene-related peptide (CGRP)-IR of the ipsilateral CTN was also studied in the CTN using the increased stimulating parameters and a marked depletion of CGRP-IR was also observed following TG stimulation. Such depletion may be due to the release of neuropeptides from the trigeminal central terminals. These findings suggest that NKA, SP and CGRP could act as neurotransmitters at the first central synapses of the trigeminal nociceptive pathway to transmit the sensory stimuli to the higher brain centers.

Immunocytochemical visualization of D-glutamate in the rat brain

Using highly specific antisera directed against conjugated d-amino acids, the distribution of d-glutamate-, d-tryptophan-, d-cysteine-, d-tyrosine- and d-methionine-immunoreactive structures in the rat brain was studied. Cell bodies containing d-glutamate, but not d-glutamate-immunoreactive fibers, were found. Perikarya containing this d-amino acid were only found in the mesencephalon and thalamus of the rat CNS. Thus, the highest density of cell bodies containing d-glutamate was observed in the dorsal raphe nucleus, the ventral part of the mesencephalic central gray, the superior colliculus, above the posterior commissure, and in the subparafascicular thalamic nucleus. A moderate density of immunoreactive cell bodies was observed in the dorsal part of the mesencephalic central gray, above the rostral linear nucleus of the raphe, the nucleus of Darkschewitsch, and in the medial habenular nucleus, whereas a low density was found below the medial forebrain bundle and in the posterior thalamic nuclear group. Moreover, no immunoreactive fibers or cell bodies were visualized containing d-tryptophan, d-cysteine, d-tyrosine or d-methionine in the rat brain. The distribution of d-glutamate-immunoreactive cell bodies in the rat brain suggests that this d-amino acid could be involved in several physiological mechanisms. This work reports the first visualization and the morphological characteristics of conjugated d-glutamate-immunoreactive cell bodies in the rat CNS using an indirect immunoperoxidase technique. Our results suggest that the immunoreactive neurons observed have an uptake mechanism for d-glutamate.

Amyloid-β induces synaptic dysfunction through G protein-gated inwardly rectifying potassium channels in the fimbria-CA3 hippocampal synapse

Last evidences suggest that, in Alzheimers disease (AD) early stage, Amyloid-β (Aβ) peptide induces an imbalance between excitatory and inhibitory neurotransmission systems resulting in the functional impairment of neural networks. Such alterations are particularly important in the septohippocampal system where learning and memory processes take place depending on accurate oscillatory activity tuned at fimbria-CA3 synapse. Here, the acute effects of Aβ on CA3 pyramidal neurons and their synaptic activation from septal part of the fimbria were studied in rats. A triphasic postsynaptic response defined by an excitatory potential (EPSP) followed by both early and late inhibitory potentials (IPSP) was evoked. The EPSP was glutamatergic acting on ionotropic receptors. The early IPSP was blocked by GABAA antagonists whereas the late IPSP was removed by GABAB antagonists. Aβ perfusion induced recorded cells to depolarize, increase their input resistance and decrease the late IPSP. Aβ action mechanism was localized at postsynaptic level and most likely linked to GABAB-related ion channels conductance decrease. In addition, it was found that the specific pharmacological modulation of the GABAB receptor effector, G-protein-coupled inward rectifier potassium (GirK) channels, mimicked all Aβ effects previously described. Thus, our findings suggest that Aβ altering GirK channels conductance in CA3 pyramidal neurons might have a key role in the septohippocampal activity dysfunction observed in AD.

Early growth hormone (GH) treatment promotes relevant motor functional improvement after severe frontal cortex lesion in adult rats.

A number of studies, in animals and humans, describe the positive effects of the growth hormone (GH) treatment combined with rehabilitation on brain reparation after brain injury. We examined the effect of GH treatment and rehabilitation in adult rats with severe frontal motor cortex ablation. Thirty-five male rats were trained in the paw-reaching-for-food task and the preferred forelimb was recorded. Under anesthesia, the motor cortex contralateral to the preferred forelimb was aspirated or sham-operated. Animals were then treated with GH (0.15 mg/kg/day, s.c) or vehicle during 5 days, commencing immediately or 6 days post-lesion. Rehabilitation was applied at short- and long-term after GH treatment. Behavioral data were analized by ANOVA following Bonferroni post hoc test. After sacrifice, immunohistochemical detection of glial fibrillary acid protein (GFAP) and nestin were undertaken in the brain of all groups. Animal group treated with GH immediately after the lesion, but not any other group, showed a significant improvement of the motor impairment induced by the motor lesion, and their performances in the motor test were no different from sham-operated controls. GFAP immunolabeling and nestin immunoreactivity were observed in the perilesional area in all injured animals; nestin immunoreactivity was higher in GH-treated injured rats (mainly in animals GH-treated 6 days post-lesion). GFAP immunoreactivity was similar among injured rats. Interestingly, nestin re-expression was detected in the contralateral undamaged motor cortex only in GH-treated injured rats, being higher in animals GH-treated immediately after the lesion than in animals GH-treated 6 days post-lesion. Early GH treatment induces significant recovery of the motor impairment produced by frontal cortical ablation. GH effects include increased neurogenesis for reparation (perilesional area) and for increased brain plasticity (contralateral motor area).

Electrophysiological study of prefrontal neurones of cats during a motor task

Abstract The prefrontal cortex is involved in many processes, some of which are related to motor activity such eye movements and speech. Experimental data exist that suggest that prefrontal cortical activity occurs in relation to attention, short-term memory, affective discrimination, and complex forms of motor behaviour, i.e. anticipatory preparation, motor sequences, programming of speech, etc. We were interested in studying participation of this cortical region in locomotion. For this purpose, recordings were made of unitary activity in the prefrontal cortex of chronically prepared cats walking on an exercise belt that was moving at a speed of 0.1 m/s. From a total of 63 neurones in the prefrontal area from which recordings were made, 37 (59%) changed their activity during locomotion, 28 of which (76%) increased and 9 of which (24%) decreased their frequency of discharge; the remaining 26 units (41%) showed no locomotor-related change in activity. The results obtained show that 59% of prefrontal units are involved in the locomotor process and it is reasonable to assume that their activity contributes to the control of the movements.

Electrophysiological and Synaptic Characterization of Transplanted Neurons in Adult Rat Motor Cortex

Lesions in specific areas of the rat motor cortex generate deficits related to fine movement performance affecting the forelimb. We have previously shown that transplants of embryonic frontal cortex ameliorate these motor deficits. Amelioration has been associated with a functional integration of the transplant due to the connections established between the host brain and the graft. In the current investigation, the electrophysiological properties of the transplanted cells and the connections both intra-transplant and with the adjacent host cortex are analyzed. For this purpose, adult rats with a motor cortical lesion plus a fetal cortical graft were used. Neurons in the transplant were recorded using sharp electrodes or whole-cell recordings in brain slices. Application of intracellular depolarizing pulses showed two patterns of cell firing: regular and burst spiking. Postsynaptic responses evoked by both, intra-transplant and adjacent host cortex stimulation were mediated by glutamic acid acting on non-NMDA and NMDA receptors, and were modulated by both cholinergic and GABAergic drugs. In some cells, supra-threshold intra-transplant stimulation generated an epileptiform-like discharge, suggesting an imbalance between excitatory and inhibitory synapses. As expected, immunohistochemistry for cholinergic and GABAergic markers confirmed the electrophysiological results. Thus we show electrophysiological and immunohistochemical evidence supporting the functional development and integration of grafted cells into the host neocortex of adult animals.