Involvement of AMPA/kainate-excitotoxicity in MK801-induced neuronal death in the retrosplenial cortex.

MK801 is a prototypical non-competitive NMDA receptor-antagonist that induces behavioural changes and reversible toxicity at low doses, while at higher doses triggers neuronal death that mainly affects the retrosplenial cortex (RSC) and to a lesser extent other structures such as the posterolateral cortical amygdaloid nucleus (PLCo). The mechanism of MK801-induced neurodegeneration remains poorly understood. In this study we analysed the participation of GABA-ergic and glutamatergic neurotransmission in MK801-induced neuronal death. We used a single i.p. injection of MK801 (2.5 mg/kg) that induced moderate neuronal death in the RSC and PLCo of female rats, and combined this treatment with the i.p., i.c.v., or intra-RSC infusion of drugs that are selective agonists or antagonists of the GABA-ergic or glutamatergic neurotransmission. We found that neuronal death in the RSC, but not the PLCo, was significantly reduced by the i.p. injection of thiopental, and the i.c.v. application of muscimol, both GABA-A agonists. MK801-toxicity in RSC was abrogated by intra-RSC infusion of muscimol, but the GABA antagonist picrotoxin had no effect. HPLC-analysis showed that levels of glutamate, but not GABA, in the RSC decreased after i.p. treatment with MK801. Intra-RSC infusion of MK801 did not enhance toxicity triggered by the i.p. injection of MK801, indicating that toxicity is not due to direct blockade of NMDA receptors in RSC neurons. MK801-toxicity in the RSC was abrogated by i.c.v. and intra-RSC infusions of the AMPA/kainate antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX). Interestingly, i.c.v. application of neither muscimol or DNQX inhibited MK801-toxicity in the PLCo, suggesting that the mechanism of neuronal death in the RSC and the PLCo might be different. 1-naphthylacetyl spermine trihydrochloride (NASPM), which blocks Ca2+ permeable AMPA/kainate receptors, also reduced MK801-induced toxicity in the RSC. Intra-RSC infusion of AMPA or kainic acid alone promoted death of RSC neurons and was reminiscent of the degeneration induced by the i.p. treatment with MK801. Collectively, these experiments provide evidence for an AMPA/kainate-dependent mechanism of excitotoxicity in the death of RSC neurons after i.p. treatment with MK801.

Neurodegeneration and prolonged immediate early gene expression throughout cortical areas of the rat brain following acute administration of dizocilpine.

N-methyl-d-aspartate receptor antagonist drugs (NMDA-A), such as dizocilpine (MK801), induce long-lasting behavioral disturbances reminiscent to psychotic disorders in humans. To identify cortical structures affected by NMDA-A, we used a single dose of MK801 (10 mg/kg) that caused low and high neurodegeneration in intact and orchiectomized male rats, respectively. Degenerating somas (neuronal death) and axonal/synaptic endings (terminal degeneration) were depicted by a silver technique, and functionally affected cortical neuronal subpopulations by Egr-1, c-Fos, and FosB/DeltaFosB-immunolabeling. In intact males, MK801 triggered a c-Fos induction that remained high for more than 24 h in selected layers of the retrosplenial, somatosensory and entorhinal cortices. MK801-induced neurodegeneration reached its peak at 72 h. Degenerating somas were restricted to layer IV of the granular subdivision of the retrosplenial cortex, and were accompanied by suppression of Egr-1 immunolabeling. Terminal degeneration extended to selected layers of the retrosplenial, somatosensory and parahippocampal cortices, which are target areas of retrosplenial cortex. Induction of FosB/DeltaFosB by MK801 also extended to the same cortical layers affected by terminal degeneration, likely reflecting the damage of synaptic connectivity. In orchiectomized males, the neurodegenerative and functional effects of MK801 were exacerbated. Degenerative somas in layer IV of the retrosplenial cortex significantly increased, with a parallel enhancement of terminal degeneration and FosB/DeltaFosB-expression in the mentioned cortical structures, but no additional areas were affected. These observations reveal that synaptic dysfunction/degeneration in the retrosplenial, somatosensory and parahippocampal cortices might underlie the long-lasting impairments induced by NMDA-A.

Increased rewarding properties of morphine in perinatally protein-malnourished rats.

In the current research, we assessed the influence of a protein malnutrition schedule from the 14th day of gestation up to 40 days of age (D-rats) on the rewarding properties of morphine in adult rats by means of the conditioned place preference paradigm. Well-nourished animals (C-rats) administered with different doses of morphine (0.75, 1.5, 3, 6, 12 or 24 mg/kg i.p.) exhibited a conditioning place preference with doses of 3 and 6 mg/kg, whereas in D-rats such a conditioning effect was observed with doses of 1.5 and 3 mg/kg. No adverse effects were observed in either C- or D-rats for the higher doses of morphine. In addition, when animals of both groups were pretreated twice a day for 3 days with increasing doses of morphine (5, 10 and 20 mg/kg s.c.), only D-rats elicited sensitization to the conditioning effect with the lowest dose of morphine (0.75 mg/kg i.p.). Furthermore, sensitized D-rats showed a selective and significant increase in FosB expression in the nucleus accumbens (core and shell), basolateral amygdala and medial prefrontal cortex, brain areas that are functionally related to the rewarding neural circuit. These results demonstrate that a deficient nutritional status during the perinatal period results in adult subjects having neural alterations, leading to an increased responsiveness to morphine and/or enhanced reinforcement effects, which correlates with an overexpression of FosB in selective brain areas related to the rewarding network.

The concepts of the ventral striatopallidal system and extended amygdala.

The concepts of the ventral striatopallidal system and extended amygdala have significantly improved our understanding of basal forebrain organization. As a result of these and other advances during the last twenty years, many of the most prominent basal forebrain structures, including the nucleus accumbens, olfactory tubercle, and amygdaloid body, have all but lost their relevance as independent functional anatomical units. In order to appreciate the distinct differences that exist between the ventral striatopallidal system and the extended amygdala, and as a way of explaining the choice of the terms ventral striatopallidal system and extended amygdala, we will review the discovery and subsequent elaboration of these two systems. On the background of these discussions, we will then proceed to dispel some recently published misgivings regarding the usefulness of the extended amygdaloid concept.

The neuronal organization of the supracapsular part of the stria terminalis in the rat: the dorsal component of the extended amygdala.

In the present normal anatomical light and electron microscopic study in the rat, histochemical (Nissl, Timm, Golgi) or immunocytochemical (microtubule-associated protein type 2, glutamate decarboxylase, glutamate receptor subunit 1, synaptophysin) stains were used to analyse neurons embedded within the stria terminalis and their associated neuropil. These cells are closely related to the bed nucleus of the stria terminalis and the centromedial amygdala, and have been termed the "supracapsular part of the bed nucleus of the stria terminalis". The largest part of this neuronal complex is located in the ventrolateral part of the stria, where it appears as a round or oval "lateral pocket" in virtually any type of light microscopic preparation because of its collection of neuronal cell bodies and dense neuropil, in addition to a lacework of unmyelinated axons. A much smaller but still distinct "medial pocket" is located in the medial corner of the stria. The large lateral subdivision of the supracapsular stria terminalis is directly continuous with the lateral bed nucleus of the stria terminalis and extends to the central amygdaloid nucleus, containing a column of neurons that is only broken up into cell clusters at the most caudal levels of the stria as it drops vertically toward the amygdala. The considerably smaller medial subdivision appears, in turn, to be directly continuous with the medial part of the bed nucleus of the stria terminalis. The medial column tapers off more rapidly than the lateral part, so that as the middle levels are approached, only small interrupted clusters of cells are seen. Solitary neurons can also be found in practically every part of the stria terminalis except among the ventrally located axons of the commissural component. Most of the neurons are small to medium in size, as viewed in transverse sections of the stria, but larger neurons are also encountered. In sections parallel to the stria, many neurons are fusiform in appearance. The dendrites are often aligned in a longitudinal fashion; many of the dendrites related to the cells in the lateral pocket are moderately to densely spined, whereas those in the medial pocket are more sparsely spined. The neuropil in both the lateral and medial pockets is characterized by boutons, bundles of unmyelinated axons, and dendrites. Based on their vesicle content, the boutons are divided into three major types: (A) round or slightly oval, agranular vesicles of uniform size; (B) pleomorphic, agranular vesicles, many of which are flattened; and (C) pleomorphic agranular vesicles, some of which are considerably larger than the ones in type B boutons. Type A boutons establish contacts with both dendritic spines and shafts, whereas types B and C usually contact dendritic shafts and sometimes somata. These synaptic components are similar to those described earlier for the central and medial amygdaloid nuclei. Overall, our results support the contention advanced in 1923 by Johnston [J. comp. Neurol. 35, 337481] that the cells accompanying the stria terminalis are interconnecting columns of a macrostructure encompassing the bed nucleus of the stria terminalis and centromedial amygdala. More recently, it has been appreciated that columns of neurons below the globus pallidus also belong to this macrostructure [Alheid G. F. et al. (1995) In The Rat Nervous System, 2nd edn, pp. 495 578, Academic, San Diego; de Olmos J. S. et al. (1985) In The Rat Nervous System, pp. 223-334, Academic, Sydney], which has been named the "extended amygdala".

The accumbens: beyond the core-shell dichotomy.

This article highlights recent discoveries related to the accumbens and closely associated structures, with special reference to their importance in neuropsychiatry. The development of "striatal patches" in the accumbens is reviewed in a series of pictures. Neuronal ensembles are discussed as potentially important functional-anatomical units. Attention is also drawn to recent discoveries related to the neuronal circuits that the primate accumbens establishes with the mesencephalic dopamine system. On the basis of histological and neurochemical differences, the accumbens has been divided into core and shell compartments. In the context of this article, the shell, which is an especially diversified part of the accumbens, is the subject of special attention because of its close relation to the extended amygdala and distinctive response to antipsychotic and psychoactive drugs.

Use of an amino-cupric-silver technique for the detection of early and semiacute neuronal degeneration caused by neurotoxicants, hypoxia, and physical trauma.

A new amino-cupric silver protocol is described for detection of neuronal degeneration. We describe its selectivity in visualizing both early and semiacute degeneration after intracerebral or systemic administration of a variety of neurotoxicants in rats, and after transient ischemic episodes in gerbils. As early as 5 min after physical trauma, or 15 min following either intrastriatal injections of glutamate analogs or exposure to ischemic episodes, neuronal silver staining was evident at primary sites of trauma (i.g. injection sites) and at hodologically related secondary sites. With intoxication by peripheral injections of trimethyltin (IP) or intracerebral injections of Doxorubicin, reproducible patterns of degeneration are demonstrable after 24 h or after 9-13 days, respectively. The amino-cupric silver method permits simultaneous detection of all neuronal compartments against a clear background. Degeneration in the neuronal cell bodies, dendrites, axons and terminals, as well as the recruitment of new structures in a progressive pathologic process, could be accurately followed. The inclusion of new reagents increased the sensitivity vis-à-vis previous versions of the cupric-silver method. The advantages and disadvantages of the current method in comparison with other means of neurotoxic assessment are discussed in detail, with special emphasis on its unique ability to discriminate irreversible degenerative phenomena and degeneration of axonal components in cases where the cell body remains apparently intact. The amino-cupric silver method is an especially useful tool for surveying neuronal damage in basic neuroscience investigations and in neuropathologic and neurotoxic assessment.

Silver staining as a tool for neurotoxic assessment.

There is no denying that the silver methods lost their dominant role as tract-tracing methods in the past 10 to 15 years. But it seems equally clear that the silver technique is headed for a dramatic revival in many fields of neuroscience, where the scope and localization of neuronal degeneration are a central issue. Together with the immunostaining of proteins formed or altered in traumatized neurons, the modern silver techniques provide neurotoxicologists and neuropathologists with unparalleled opportunities to detect and study injured and dying neurons. Characterized by great sensitivity and distinct rendition of the morphology of degenerating neurons and their processes, the reduced silver methods constitute the ideal tool for screening irreversible neuronal damage caused by neurotoxic substances including drugs of abuse. Those interested in the rapidly expanding fields of "excitotoxicity" and neurodegenerative disorders (Taylor 1991) are also likely to find increasing use for the silver methods. The pattern of degeneration in so-called "system degenerations" may be predetermined by the neuronal connections (Saper et al. 1987), and as the disease progresses from the destruction of the originally affected neuron population, closely related systems and pathways may be recruited into the pathophysiologic cascade. Any type of trauma to the CNS has the potential to produce this type of "domino effect" of degeneration, through which additional systems are progressively recruited into a degenerative chain reaction of transneuronal degeneration. In other words, even longstanding disorders may exhibit signs of more recent degeneration, and the proper use of silver methods at autopsy may give some important clues regarding the etiology of disease; it may also provide new insights about the anatomy of the human brain. Little can be said at present about the chemical basis of argyrophilia in degenerating and "reactive" neurons, but there is every reason to pay more attention to this subject. One can expect that a continuing and concerted effort will result in a rational understanding of the molecular biological and physicochemical events that fortuitously provide the basis for the selective impregnation of degenerating neuronal elements. This knowledge can be the basis for the development of even more reliable and simple, yet sensitive, silver methods suited for neurotoxic risk assessment on a large scale.

A modified cupric-silver technique for the impregnation of degenerating neurons and their processes.

A rapid version of the de Olmos-Ingram cupric-silver technique with higher sensitivity and affinity for degenerative changes is introduced for the staining of mechanically and chemically induced degeneration. Emphasis is laid on induction of degeneration with experimental approaches sparing 'fibers of passage'.

An improved HRP method for the study of central nervous connections.

The HRP tract-tracing method has been modified by the introduction of cryoprotective agents, thereby allowing the enzymatic reaction to take place at sub-zero temperatures. By using cold temperatures the enzymatic reaction can be better controlled, which in turn makes it possible to "push" the method to its maximum without obtaining excessive crystallization. The addition of antifreeze agents, however, changes the acid-base phenomena, which in turn have to be adjusted by the use of appropriate buffers. The overall result is a considerable increase in efficiency as compared with the commonly used HRP procedure.

Autoradiographic studies of the projections of the midbrain reticular formation: ascending projections of nucleus cuneiformis.

The ascending projections of the cuneiform nucleus in the cat were traced by autoradiography in the transverse and sagittal planes following stereotaxically placed injections of (3)H-leucine. The ascending fibers are almost exclusively ipsilateral and enter the diencephalon as a wide radiation. At the mesodiencephalic junction fibers enter the nucleus of the posterior commissure and pretectal nuclei, and others cross in the posterior commissure to distribute to these structures on the contralateral side. More ventrally directed fibers distribute to the fields of Forel and then spread into the posterior hypothalamus and zona incerta. At the caudal level of the ventral thalamic group, the ascending fibers diverge and follow two separate courses. One division of fibers continues forward beneath the ventral thalamic group and distributes to the zpna incerta and dorsal hypothalamic area. It rapidly diminishes in size as it attains more rostral levels where it is found in the bed nuclei of the stria terminalis and the anterior commissure. Other fibers of this division spread laterally to innervate the ventral lateral geniculate nucleus, the lateral hypothalamus, and preoptic area, and still others follow the entire confirmation of the thalamic reticular nucleus. The second division of fiber ascends through midline and intralaminar nuclei, completely encircling the mediodorsal nucleus, which is uninnervated except for a small ventral region. The distribution of this division is heaviest to the paraventricular, parafascicular, and central dorsal nuclei. Neither division is conspicuous rostral to the anterior commissure. No projections to neostriatum or specific thalamic nuclei were evident.