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Immunocytochemical studies performed by Storm-Mathisen et al (1983) and Ottersen and Storm-Mathisen (1984) reported that Glu is localized in pyramidal as well as nonpyramidal neurons of all cortical layers. Thereafter, a number of investigators reported Glu present only in pyramidal neurons of various cortical laminae (Conti et al 1989; Madl et al 1986). In a recent study in the visual cortex by Dori and Parnavelas (1989) Glu was found exclussively in pyramidal neurons. This disagreement was thought to be caused by differences in the specificity of the antibodies used in the various studies. The authors of the latter study, as well as Conti and colleagues (Conti et al 1989) claim that the antibodies used in their studies differ in specifity from those originally used by Storm- Mathisen (1983) and that they stain the transmitter pool rather than the metabolic pool of individual neurons (see Conti et al 1987 for extensive discussion). The pyramidal neurons labelled for Glu in the study by Dori and Parnavelas (1989) were found in layers II-VI of the visual cortex.

Glu-IR neurons are first seen at P3 (Dori PhD thesis and personal communication) but do not obtain their ulatrastructural characteristics before P20 nor demonstrate the adult pattern until the fouth postnatl

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week.

Studies using a double-labeling technique which combines the immunocytochemical localization of Glu with the presence of retrogradely transported WGA-HRP were performed by Dinopoulos et al (1989) and Dori et al (1993 ) • These studies examined the Glu containing neurons in the visual cortex that project either to the corresponding cortex in the opposite hemisphere or to a number of subcortical structures (pons, superior colliculus and dLGN) and have shown that approximately half of the neurons involved in these projections exhibit Glu immunostaining.

1.4.3b Aspartate

A number of immunocytochemical studies have demonstrated the presence of aspartate (Asp) in a population of cortical neurons (Aoki et al 1987; Conti et al 1987a). A later study in the visual cortex of the rat (Dori et al 1989) has shown Asp-immunoreactive neurons in layers II-VI with a concentration in layers II/III. Stained cells in the supragranular layers appear generally smaller in size when compared to their counterparts in infragranular layers. Layer V, in particular, is characterized by a row of especially large cells (Dori et al 1989). The same authors using both light and electron microscopic preparations demonstrated that the great majority of labeled cells are pyramidal in morphology and provided clear evidence that a small

portion of Asp-immunoreactive neurons (approximately 10%) are nonpyramidal in morphology since they receive both type I and type II axosomatic synapses.

Studies combining retrogade transport of WGA-HRP and immunocytochemistry for Asp suggest that a large number of neurons projecting to the pons, superior colliculus, dLGN as well as the contralateral visual cortex are expressing Asp (Dori et al 1992). Thus, it is shown that both Glu and Asp are used as excitatory transmitters in subcortical as well as callosal projections and that cortical projection cells, at least in the visual cortical areas, exhibit a neurochemical heterogeneity.

1.4.4 GABA

The amino acid GABA is the major inhibitory transmitter in the cerebral cortex (see review by Sillito 1984). It was originally believed that the whole amount of GABA found in the cortex originated from intrinsic interneurons, since undercutting the cortex did not seem to produce any obvious decline in GABA levels (Emson and Lindvall 1979). Some years later though, it was shown that a number of hypothalamic GABA-ergic cells project to the cortex and thus, make a small contribution to the cortical GABA concentration (Vincent et al 1983). Additionally, in a recent study by Freund and Meskenaite

(1992) it was revealed that some basal forebrain afferents use GABA as transmitter and innervate intrinsic GABA-ergic neurons that contain either Som or

parvalbumin. This could be a mechanism by which a small ascending pathway can exert a powerfull and widespread effect in the neocortex by selectively innervating GABA-ergic interneurons which, in turn, control the activity of large populations of pyramidal cells through their extensive axonal arborizations.

Early studies using immunocytochemical localization of GAD, the GABA synthesizing enzyme, in the visual cortex of the rat (Ribak 1978) showed labelled cells distributed fairly evenly throughout all cortical layers. Most neurons exhibited multipolar or bitufted morphologies except for cells in layer I and VI that sometimes showed horizontal orientation. The same author reports that immunoreactive terminals were engaged in type II synapses with the cell bodies and dendrites of pyramidal and nonpyramidal neurons, as well as with axon initial segments. In a later immunocytochemical study Lin et al (1986) argued that GAD-possitive cells are not uniformely distributed across the layers of the rat visual cortex but instead, they form a prominent band in layer IV. Additionally, GAD- immunoreactive puncta representing immunoreactive axon terminals, showed an increased concentration in layer IV as well as in layers I and V I .

The GABA-ergic population of the rat visual cortex is found to comprise roughly 15% of the total neuronal population (Ribak 1978, Meinecke and Peters 1987). It was also shown that, with the exception of the one fifth of the bipolar cell population, all nonpyramidal neurons are GABA-ergic (Meinecke and Peters 1987) and that, at least

for the rat cortex, the largest GABA- containing neurons reside superficially, in layers II/III (Lin et al 1986).

Extensive colocalization of GABA and various neuropeptides such as; cholecystokinin (CCK), somatostatin (SOM), neuropeptide Y (NPY) and substance P

(SP) has been documented (Hendry et al 1984). The GABA-ergic neurons that exhibit immunoreactivity for peptides are usually the smaller diameter GABA-ergic neurons (Naegele and Barnstable 1989 for review): a subset of the largest GABA-ergic neurons, such as large basket and chandelier cells, is consistently immunonegative for peptides. It is also interesting that neurons which colocalize GABA with peptides are usually absent from layers I and IV (Freund et al 1986).

Many GABA-ergic neurons are also immunoreactive for various calcium- binding proteins, such as parvalbumin, calbindin or calretinin (Rogers 1992 for review) all of which are suggested to play a role in buffering intracellular calcium levels. These three proteins occur in distinct cell populations with virtually no overlap.

Studies combining immunocytochemistry for GABA and [3H] thymidine labeling have shown that GABAergic neurons are generated from E14 to E20 and are, thus, cogenerated with the pyramidal neurons (Miller 1986).

In the prenatal rat and during the last week of gestation, GABA immunoreactive neurons are progressively present in the marginal, intermediate, ventricular and subventricular zones as well as in the cortical plate. Furthermore, while the number of GABAergic cells in the cortical plate increases, GABAergic neurons in these

other zones decrease in number after E19 (VanEden et al 1989). A small portion of these early differentiated neurons, however, persist through development and can be seen as interstitial cells of the adult white matter

(Luskin and Shatz 1985).

The activity of glutamic acid decarboxylase (GAD),the enzyme synthesizing GABA, measured neurochemically during the postnatal development of the rat visual cortex, showed a gradual increase during the first week, followed by a more rapid increase during the subsequent 10 days and finally a slower rate until day 24 (McDonald et al 1981). After that the number of GAD-positive neurons decreases slightly to reach adult.