Margarita Heredia

Formation of an olfactory glomerulus: morphological aspects of development and organization.

We have studied the development of olfactory nerves in the rat from their first contact with the telencephalic vesicle until the formation of glomerular structures in the olfactory bulb at early postnatal period. The study is based on serial semithin and ultrathin sections of material prepared for electron microscopy and antibodies to label radial glial cells, glial fibrillary acidic protein and Rat-401. Beginning on embryonic day 12, developing olfactory axons from the olfactory placode are accompanied by migratory cells, also derived from the olfactory placode, that reach the prospective olfactory bulb by embryonic day 13. The mass of migratory cells accumulate superficial to the telencephalic vesicle. The cells increase in number by mitotic divisions. The majority of these cells represent precursor elements that will later develop into the ensheathing cells of the olfactory nerves and olfactory nerve layer of the adult. Some migratory cells penetrate into the prospective olfactory bulb early during development. The first synaptic contacts of olfactory axons with dendritic processes in the olfactory bulb were observed at embryonic day 18. Glomerular formation is initiated by penetration of cells from the migratory mass into the prospective glomerular layer by embryonic day 20 to postnatal day 0. These cells form walls surrounding zones of high synaptic density forming protoglomeruli. Postnatally, the peripheral processes of radial glial cells branch profusely delimiting glomerular formations and transform into periglomerular astrocytes. Rat-401 stains radial glial cells from embryonic day 14. Immunoreactivity becomes restricted to the olfactory glomeruli during the first postnatal weeks and it virtually disappears by the end of the first postnatal month. We conclude that the early penetration of cells from the migratory mass into the prospective olfactory bulb, observed immediately after the first synaptic contacts were established, initiates the formation of olfactory glomeruli which becomes completed by the transformation of radial glial cells into periglomerular astrocytes.

Transient pattern of exuberant projections of olfactory axons during development in the rat

The purpose of our study was twofold: (1) to trace the development of the olfactory axons from early embryonic stages until the mature pattern of connectivity and (2) to determine whether a transient penetration of them exists beyond the olfactory glomeruli. Two techniques were employed: DiI applied in the olfactory epithelium after aldehyde fixation, and olfactory marker protein (OMP) immunostaining. At E13 and E14 olfactory axons were observed spreading over the telencephalic vesicle and entering deeply into the prospective olfactory bulb, extending near the ventricular zone. Growth cones were seen at the end of these axons. At E15, the bundles of olfactory axons form a network, in which axons, growth cones and cells were seen. Some of these axons entered the olfactory bulb. Using OMP immunostaining olfactory axons were observed along the external plexiform layer, the mitral cell layer and in the granular layer from E19 to P6. At P9 some OMP immunoreactive axons were observed in the external plexiform layer. No OMP immunostained axons could be observed outside the glomeruli at P10. Our conclusions are that a transient immature pattern of early invasion over the telencephalic vesicle and of the olfactory bulb by olfactory axons occurs in the olfactory system. By the second postnatal week the glomerular layer reaches its mature configuration, and no olfactory fibers are seen outside the glomerular layer.

Characterization of neuronal cell varieties migrating from the olfactory epithelium during prenatal development in the rat. Immunocytochemical study using antibodies against olfactory marker protein (OMP) and luteinizing hormone-releasing hormone (LH-RH)

The development of neurons located outside the olfactory epithelium was studied by using antisera against olfactory marker protein (OMP) and luteinizing hormone-releasing hormone (LH-RH) in the rat. The study was restricted to the localization of these cells in the nasal cavity and in the region of the olfactory bulb during development. We describe groups of cells that stain positively for OMP located principally on the ventro-lateral aspect of the olfactory bulbs. A comparison is made with the LH-RH-immunoreactive system of cells which predominate on the medial aspect following the known trajectory of the nervus terminalis. OMP-immunoreactive cells appeared along the course of the olfactory fibers when they were first detected at embryonic day 16. These cells became restricted to a small group above the cribriform plate, ventral to the olfactory bulbs that seemed to disappear shortly after birth. It is concluded that these cells, which like the LH-RH cells have most probably migrated from the olfactory placode, represent a group of intervening neurons between the olfactory receptor cells and the olfactory bulb, serving as hints for olfactory axons to reach their targets during prenatal development.

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.

β-Amyloid peptide25–35 depresses excitatory synaptic transmission in the rat basolateral amygdala “in vitro”

The effects of beta-amyloid peptide25-35 on resting membrane potential, spontaneous and evoked action potential and synaptic activity have been studied in basolateral amygdaloid complex on slices obtained from adult rats. Intracellular recordings reveal that perfusion with beta-amyloid peptide25-35 at concentrations of 400 nM and less did not generate any effect on resting membrane potential. However, concentrations in the range of 800-1200 nM produced an unpredictable effect, depolarization and/or hyperpolarization, which were blocked by tetrodotoxin or 6-cyano-7-nitroquinoxaline-2,3-dione+D-(-)-2-amino-5-phosphonopentanoic acid together with bicuculline. Excitatory and inhibitory evoked responses mediated by glutamic acid or gamma-aminobutyric acid decreased in amplitude after beta-amyloid peptide25-35 perfusion. Additionally, results obtained using the paired-pulse protocol offer support for a presynaptic mode of action. To determine which type of receptors and/or channels are involved in the presynaptic mechanism of action, a specific blocker of alpha-7 nicotinic receptors (methyllycaconitine citrate) or L-type calcium channel blockers (calcicludine or nifedipine) were used. beta-amyloid petide25-35 decreased excitatory postsynaptic potentials amplitude in control conditions and also in slices permanently perfused with methyllycaconitine citrate. However, this effect was blocked in slices perfused with calcicludine or nifedipine suggesting the involvement of the L-type calcium channels. On the whole, these experiments provide evidence that beta-amyloid peptide25-35 affects neurotransmission in basolateral amygdala and its action is mediated through L-type calcium channels.

Development of the main efferent cells of the olfactory bulb and of the bulbar component of the anterior commissure

The development of the efferent cells of the main olfactory bulb and the development of the bulbar part of the anterior commissure were studied in the rat from E16 to P7. DiI was used in fixed tissues as a neuronal tracer. From E16 onwards cells located in the olfactory bulb anlage were stained in a Golgi-like appearance. The morphological changes of these cells were: from E16 to P4, re-orientation from a tangential position to a radial position, elongation of the principal dendrite and spreading out of the secondary dendrites. From P4 onwards, there was a lack of migrating mitral cells in the inner part of the bulb. At E16 some fibers of the anterior commissure reached the midline, the number of fibers increased slowly until P0/P1. At P2 there was an explosive increase in the number of fibers crossing the midline and reaching the contralateral bulb. The development in two stages is hypothesized.

Glutamatergic synaptic depression by synthetic amyloid β‐peptide in the medial septum

The medial septum/diagonal band region, which participates in learning and memory processes via its cholinergic and GABAergic projection to the hippocampus, is one of the structures affected by beta amyloid (βA) deposition in Alzheimers disease (AD). The acute effects of βA (25–35 and 1–40) on action potential generation and glutamatergic synaptic transmission in slices of the medial septal area of the rat brain were studied using current and patch‐clamp techniques. The βA mechanism of action through M1 muscarinic receptors and voltage‐dependent calcium channels was also addressed. Excitatory evoked responses decreased (30–60%) in amplitude after βA (2 μM) perfusion in 70% of recorded cells. However, the firing properties were unaltered at the same concentration. This depression was irreversible in most cases, and was not prevented or reversed by nicotine (5 μM). In addition, the results obtained using a paired‐pulse protocol support pre‐ and postsynaptic actions of the peptide. The βA effect was blocked by calcicludine (50 nM), a selective antagonist of L‐type calcium channels, and also by blocking muscarinic receptors with atropine (5 μM) or pirenzepine (1 μM), a more specific M1‐receptor blocker. We show that in the medial septal area this oligomeric peptide acts through calcium channels and muscarinic receptors. As blocking any of these pathways blocks the βA effects, we propose a joint action through both mechanisms. These results may contribute to a better understanding of the pathophysiology at the onset of AD. This understanding will be required for the development of new therapeutic agents.

PRESENCE OF LHRH (LUTEINIZING HORMONE-RELEASING HORMONE) FIBERS IN THE OPTIC NERVE, OPTIC CHIASM AND OPTIC TRACT OF THE ADULT RAT

In mammals LHRH (luteinizing hormone-releasing hormone) is synthesized and released by a set of neurons that have their embryonic origin in the olfactory placode. We have observed that, besides their classical location, LHRH fibers can also be seen in the optic nerve and optic chiasm. Some LHRH fibers could also be traced in the optic tract. The possible course of these projections, and their functional significance are discussed.

A method using DiI to study the connectivity of cortical transplants

Carbocyanine DiI is described as a suitable fluorescent tracer to investigate the connectivity of cortical transplants. DiI was applied in 2 ways: in tissue previously fixed with aldehydes, and in vivo for labelling the donor tissue prior to transplantation. When applied in fixed tissue DiI travelled anterogradely, allowing the study of efferent connections from the transplant to the host. When DiI was applied in vivo, it travelled retrogradely showing the afferents to the transplant from the host.

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.