Convergence and Plasticity of Monoaminergic Systems in the Medial Prefrontal Cortex during the Postnatal Period: Implications for the Development of Psychopathology

Francine M. Benes, Jill Bolte Taylor and Miles C. Cunningham

Laboratory for Structural Neuroscience, McLean Hospital, Belmont, MA: Program in Neuroscience and Department of Psychiatry, Harvard Medical School, Boston, MA, USA


    Abstract
 Top
 Abstract
 Introduction
 Interactions of Dopamine and...
 Postnatal Development of...
 Conclusions
 Notes
 References
 
A variety of observations have suggested that the dopamine and serotonin systems may play a role in the pathophysiology and treatment of major mental disorders of childhood, adolescence and early adulthood. A recent triple immunofluorescence study has demonstrated a convergence of serotonin and dopamine fibers onto both pyramidal cells and GABAergic interneurons in the rat medial prefrontal cortex (mPFCx). These findings are consistent with the results of an electrophysiological study conducted in another laboratory that suggested such a relationship exists in the pyriform cortex of the rodent brain. During postnatal development, the dopamine system shows a progressive ingrowth of fibers into this region that continues until the early adult period. In contrast, GABAergic neurons appear to complete their postnatal maturation by the fourth postnatal week (the early post-weanling period). As dopamine fibers infiltrate the rat mPFCx, they progressively increase their interaction with neural elements within the neuropil and with the cell bodies of both pyramidal cells and GABAergic interneurons. This process appears to be influenced by the serotonin system, since lesioning of the nucleus raphe dorsalis during the neonatal period results in a significant increase of dopamine fibers. This finding suggests that lesions of the serotonin system induce plasticity of the cortical dopamine system; however, it is not known whether this inferred suppressive effect of serotonin fibers occurs at brainstem levels or within the mPFCx itself. Taken together, these various studies suggest that the convergence of dopamine and serotonin fiber systems on intrinsic cortical neurons shows considerable plasticity during postnatal life that could theoretically contribute to the development of ‘miswired’ circuits in individuals with neuropsychiatric disorders.


    Introduction
 Top
 Abstract
 Introduction
 Interactions of Dopamine and...
 Postnatal Development of...
 Conclusions
 Notes
 References
 
The past decade has been characterized by a significant change in how we conceptualize the etiology of mental illness during childhood, adolescence and adulthood (Benes, 1995). Among these disorders, schizophrenia has received the most attention, with recent post-mortem studies having provided compelling evidence for a defect of GABAergic neurotransmission playing a role in its pathophysiology (Bird et al., 1979Go; Hanada et al., 1987Go; Simpson et al., 1989Go; Reynolds et al., 1990Go; Benes et al., 1991Go, 1992Go, 1996aGo,bGo, 1997bGo; Akbarian et al., 1995Go; Beasley and Reynolds, 1996Go; Woo et al., 1997; Todtenkopf and Benes, 1998Go). Taken together, these various neurochemical and microscopic findings reported to date are consistent with the idea that there may be a decrease of GABAergic cells and/or activity in this disorder. Since the mechanism of action of antipsychotic medication involves blockade of both dopamine and serotonin receptors (Meltzer, 1994Go), a key question is how GABA cells interact with these monoaminergic systems in corticolimbic regions of schizophrenic brain. Thus far, studies of the dopamine (Mackay et al., 1978Go; Owen et al., 1978Go; Lee and Seeman, 1980Go; Cross et al., 1981Go; Mackay et al., 1982Go; Cross et al., 1983Go; Joyce et al., 1988Go; Kornhuber et al., 1989aGo,bGo; Seeman and Niznik, 1990Go; Ohara et al., 1993Go; Seeman et al., 1993aGo,bGo; Akil and Lewis, 1997Go; Benes et al., 1997aGo,bGo; Joyce and Meador-Woodruff, 1997Go; Meador-Woodruff et al., 1997Go) and serotonin (Bennett et al., 1979Go; Whitaker et al., 1981Go; Mita et al., 1986Go; Hashimoto et al., 1991Go; Joyce et al., 1993Go) systems have failed to demonstrate consistent abnormalities that can be convincingly distinguished from neuroleptic effects. A recent report, however, has suggested that a subtle ‘miswiring’ of the dopamine system with respect to pyramidal neurons and GABA cells may be present in the anterior cingulate cortex of subjects with schizophrenia (Benes, 1997aGo,bGo; Benes et al., 1997aGo,bGo). Such an abnormality could be present without there being any associated changes in the levels of biochemical and molecular markers for the dopamine system. If this latter hypothesis is correct, it will be important to gain some insight into how aberrant connections between monoaminergic fibers and intrinsic cortical neurons may arise and how such changes may influence corticolimbic function.

It is now broadly believed that schizophrenia is a neurodevelopmental disorder (Jakob and Beckmann, 1986Go; Weinberger, 1987Go; Benes, 1988Go), one in which normal maturational changes in the corticolimbic system during late adolescence may ‘trigger’ its onset in susceptible individuals (Benes, 1988Go, 1989Go; Benes et al., 1994Go). In order to understand further the implications of this hypothesis, not only for schizophrenia but also for other neuropsychiatric disorders that present during childhood and adolescence, the following discussion will examine the anatomic relationship of the monoaminergic systems to intrinsic cortical neurons, particularly GABAergic cells, and will consider how the development of these interactions could potentially go awry during the postnatal period.


    Interactions of Dopamine and Serotonin Fibers with Cortical Neurons
 Top
 Abstract
 Introduction
 Interactions of Dopamine and...
 Postnatal Development of...
 Conclusions
 Notes
 References
 
It is now well-established that the activity of cortical neurons is probably modulated by both the dopamine and serotonin systems. In the rat medial prefrontal cortex (mPFCx), a homologue of the anterior cingulate cortex of human brain, serotoninergic fibers are abundantly present in both superficial and deep laminae (Lidov et al., 1980Go; Reader, 1981Go), while dopamine fibers are most densely distributed in layers V and VI (Emson and Koob, 1978Go; Lindvall and Bjorklund, 1984Go). Recent studies have demonstrated that both pyramidal (Seguela et al., 1988Go; Goldman-Rakic et al., 1989Go; Verney et al., 1991Go) and nonpyramidal (Verney et al., 1991Go; Benes et al., 1993a) neurons of this region receive inputs from dopamine fibers. In some studies, both of these neuronal subtypes showed D1 and D2 receptor binding activity (Vincent et al., 1993Go, 1995aGo) and their respective messenger RNAs (Huntley et al., 1992Go) localized to their cell bodies. Rodent studies in which in situ hybridization has been used to localize mRNA for the two subtypes have demonstrated that projection cells of various laminae may express one or the other subtype (Gaspar et al., 1995Go), although the D2 receptor seemed to be principally associated with those in layer V. More recent work has demonstrated that mRNA for the D1 and D2 subtypes are also expressed by cortical interneurons subtype (LeMoine and Gaspar, 1998Go); but the cells showing mRNA for both receptors were principally those containing parvalbumin, while those showing calbindin-immunoreactivity seemed to preferentially express the D1. It is important to emphasize, however, that the ability to localize mRNA for a particular protein is limited by the degree to which this nucleic acid is being expressed at any given time by a subpopulation of cells. Thus, the absence of a particular mRNA in subpopulations of neurons in situ hybridization studies does not exclude the possibility that the receptor in question is actually being synthesized and utilized by these cells. Some immunocytochemical studies in primates have preferentially localized the D1 receptor to pyramidal neurons (Smiley et al., 1994Go; Bergson et al., 1995Go), while others have found it in interneurons (Muly et al., 1998Go). It appears likely that different antibody preparations may yield different localization patterns. Using a high resolution Scatchard analysis of the distribution of D1 receptor binding activity, this receptor was found to be expressed by both projection cells and interneurons in rodent mPFCx (Davidoff and Benes, 1998Go). This latter technique, like the ones employing fluorescently tagged ligands for the D1 and D2 receptors (Vincent et al., 1993Go, 1995aGo), have the advantage of localizing the high affinity binding activity and are much less likely to be distorted by indeterminate losses of mRNA or immunoreactivity that are inherent to in situ hybridization and immunocytochemistry, respectively. In any case, it seems likely that D1 and D2 receptors are employed by many different neuronal populations to mediate the effects of dopamine in the cortex.

In the pyriform cortex, the activity of both pyramidal and nonpyramidal neurons can also be manipulated with either agonists or antagonists of serotonin receptors (Sheldon and Aghajanian, 1990Go; Gellman and Aghajanian, 1993Go). Both pyramidal cells (Jakab and Goldman-Rakic, 1998Go; Wu et al., 1998Go) and GABA neurons (Gellman and Aghajanian, 1993Go; Morilak et al., 1993Go) in the prefrontal cortex express the serotonin (5-HT)2A, whereas pyramidal neurons in the hippocampus (Chalmers et al., 1993Go) and pyriform cortex (Sheldon and Aghajanian, 1991Go) have been associated with the 5HT1A and 5HT1C subtypes, respectively. Thus, the pattern of expression for various receptor subtypes may vary from one region to another.

The above observations suggest that both projection cells and local circuit cells may be potentially influenced by the dopaminergic and serotoninergic projections to the mPFCx (Benes, 1995aGo,bGo). It is noteworthy that a convergence of these fiber systems onto intrinsic cortical neurons could play an important role in the cortical stress response. Exposure to stress has not only been associated with changes in dopamine (Thierry et al., 1976Go; Roth et al., 1988Go) and serotonin in both the prefrontal cortex (Thierry et al., 1986Go) and hippocampus (Kalen et al., 1989Go) [for a review see Stanford (Stanford, 1993Go)] systems, but has also been implicated in the regulation of pyramidal cells (Chalmers et al., 1994Go) and GABAergic interneurons (Corda and Biggio, 1986Go; Schwartz et al., 1987Go).

To understand how the dopamine and serotonin systems may be interacting with intrinsic cortical neurons, it is important to know whether the respective fiber systems project to mutually exclusive neuronal subpopulations or whether perhaps there is a significant degree of overlap in the neurons receiving inputs from these two systems. In order to investigate this question, these two transmitter systems have been localized using a combination of single, double and triple immunocytochemical approaches (Taylor and Benes, 1996Go). Using a single immunoperoxidase technique, the proportion of 5-HT-IR varicosities that are in apposition with neuronal cell somata in layers II and VI is ~35 and 18%, respectively, while the majority (65 and 82%, respectively) are found in the neuropil of these two laminae (Table 1Go). For tyrosine hydroxylase (TH)-IR varicosities, a similar pattern is observed, although the percentages associated with cell bodies (23 and 13%, respectively) is somewhat lower for 5-HT-IR varicosities (Table 1Go). In order to assess whether these interactions might be random in nature, a ‘predicted’ number of varicosities in neuropil versus on cell bodies was computed by calculating the number of varicosities that would be found in each compartment according to their respective areal percentages. Using this so-called Poisson analysis (Benes et al., 1993Go), the ‘observed’ frequencies for 5-HT-IR varicosities in apposition with cell bodies in layers II and VI is 2–3 times higher than the ‘predicted’ ({chi}2 = 93.7, P = 0.0001 and {chi}2 = 19.5, P = 0.0001, respectively). The ‘observed’ frequency with which TH-IR varicosities form appositions with cell bodies shows a similar pattern. In the rat mPFCx, somata <100 µm2 are believed to be primarily nonpyramidal in nature, while those >100 µm2 are probably pyramidal cells (Vincent et al., 1993Go). Using these criteria, both ‘nonpyramidal’ and ‘pyramidal’ neurons in layers II and VI show an approximately equal distribution of 5-HT-IR and TH-IR in apposition with their somata. Based on these findings, the probability that both 5-HT- and TH-IR fibers would be found simultaneously on individual somata was found to be 23 and 25% for neurons in layers II and VI, respectively, that were >100 µm2 in size and 36 and 26%, respectively, for neurons that were <100 µm2 in size (Table 2Go). Accordingly, these latter data had suggested that a convergence of 5-HT and dopamine fibers might be a relatively frequent occurrence in the rat mPFCx and that such events probably occur to an equal degree for both pyramidal neurons and interneurons.


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Table 1 {chi}2 analysis of the randomness of 5HT and TH varicosity interactions with cell bodies and in neuropil
 

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Table 2 Percent of large (>=100 µm2) and small (<100 µm2) neurons with varicosities plus probability of convergence
 
When sections are processed simultaneously with antibodies against 5-HT, TH and the 67 kDa isoform of glutamate decarboxylase [GAD67], two patterns of interaction have been observed (Figure 1A–DGo). On the one hand, both 5-HT-IR and TH-IR varicosities are found in apposition with the same GAD67-IR somata (Figure 1A–CGo), with ~25% of GAD67-IR somata showing a convergence of these two fiber systems. This latter frequency agrees remarkably well with the ‘predicted’ frequency that was computed using the number of contacts observed for each fiber system in single immunocytochemical preparations (Table 2Go). The second pattern observed with the triple localization involved appositions of 5-HT-, TH- and GAD67-IR fibers with ‘ghosts’ of pyramidal neuron cell bodies visualized by the absence of cytoplasmic or nuclear fluorescence. This so-called ‘trivergence’ of 5TH-IR and TH-IR varicose fibers, together with GAD67-IR puncta forming ‘classical’ rings of axosomatic synapses (Figure 1DGo), suggests that pyramidal neurons may receive not only traditional synaptic inputs from GABAergic terminals, but also modulatory ones from the DA and 5-HT systems. Taken together, it appears that both the dopamine and serotonin systems may interact extensively with both pyramidal cells and interneurons. Although some of these interactions may be present at the level of the cell body, it is likely that the majority occur within the neuropil area where the dendritic branches of both cell types are localized (see Table 1Go).



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Figure 1.  A set of co-registered digital confocal images of 5-HT-, GAD- and TH-IR staining. (A–C) Three deconvolved images showing a convergence of monoaminergic inputs (arrows = 5-HT-IR; arrrowheads = TH-IR) to GAD-IR cell bodies in the rat mPFCx. (D) 5-HT- and TH-IR varicose fibers converge on a pyramidal neuron (P) with GAD-IR terminals forming ‘classic’ axosomatic contacts. There also appear to be appositions between GAD-IR terminals and 5-HT-IR varicosities (*). Similar interactions also seem to be occur between GAD-IR terminals and TH-IR varicosities. Bar = 10 µm. Reproduced with permission (Taylor and Benes, 1996Go).

 
It is important to point out that the double and triple localizations discussed above do not have the spatial resolution needed to determine whether the contacts observed are synaptic in nature. Studies from other laboratories, however, have suggested that both dopamine and serotonin fibers primarily form synaptic contacts with dendrites. For example, at the ultrastructural level, DA-containing varicosities have been shown to form synapses with shafts and spines of distal dendritic branches; although consistent with the findings discussed above and elsewhere (Benes et al., 1993Go), some also form contacts with neuron somata having a morphological appearance similar to that of inhibitory basket cells (Goldman-Rakic et al., 1989Go). Based on primate studies, serotoninergic fibers form appositions predominantly on interneurons and, like dopamine fibers, most do not show synaptic profiles (Smiley and Goldman-Rakic, 1993Go, 1996Go). The appositions formed by DA varicosities on neuron somata are also typically nonsynaptic in nature and do not show glial processes interposed between them (Verney et al., 1990Go). Approximately 90% of all varicosities show a small area specialized for synaptic transmission (Seguela et al., 1988Go), although it is not clear whether this is also true for varicosities in apposition with cell bodies. Similar data are lacking for serotoninergic contacts on neuronal cell bodies. The responses associated with activation of the dopamine system are typically modulatory in nature and show a much longer duration than is typically observed with ‘classic’ synaptic inputs (Reader et al., 1979Go; Bunney and Chiodo, 1984Go; Mantz et al., 1988Go; Williams and Goldman-Rakic, 1995Go; Yang and Seamans, 1996Go; Gulledge and Jaffe, 1998Go). In contrast, those responses associated with serotoninergic inputs to the pyriform cortex in the rodent brain have the properties of synaptic inputs that primarily influence GABAergic cells and, in turn, exert a secondary influence on pyramidal neurons (Sheldon and Aghajanian, 1991Go). In the medial prefrontal cortex, the action of serotonin appears to be a presynaptic one on a subpopulation of glutamatergic terminals (Marek and Aghajanian, 1998Go). Clearly, the action of the monoaminergic systems on intrinsic cortical neurons varies on a region-by-region basis. Further study is needed to identify how the somal contacts made by dopamine and serotonin fibers influence the activity of these intrinsic neurons.


    Postnatal Development of Monoaminergic Fibers and Intrinsic Cortical Neurons
 Top
 Abstract
 Introduction
 Interactions of Dopamine and...
 Postnatal Development of...
 Conclusions
 Notes
 References
 
It has long been suspected that the development of the corticolimbic regions of the human brain may continue well beyond birth (Flechsig, 1920Go; Yakovlev and Lecours, 1967Go; Benes, 1989Go; Benes et al., 1994Go). Recently, this idea has received increased attention with the growing realization that normal maturational changes probably play an important role in the appearance of various neuropsychiatric diseases at specific stages of postnatal life (Weinberger, 1987Go; Benes, 1988Go). In other words, a normal ontogenetic change at a critical stage of development could potentially act as a ‘trigger’ for the onset of a given disorder at that stage. Consistent with this concept, many studies in the rodent brain have demonstrated that there are significant changes in several key neurotransmitter systems at key stages of the postnatal period [for comprehensive reviews see Johnston and Parnavelas et al. (Johnston, 1988Go; Parnavelas et al., 1988Go)].

The Dopamine System

Dopaminergic projections to the rat mPFCx have been found to increase progressively beyond the weanling stage until to the early adult period (Verney et al., 1982Go; Kalsbeek et al., 1988Go). During the first 2 weeks of postnatal life (Figure 2Go, P11) the relative distribution of DA-IR varicose fibers in the rat mPFCx is quite low, but shows the highest density in deeper laminae (Benes et al., 1993a). During the third postnatal week, however, the density of such fibers shows discernible increases (Figure 2Go, P20), and this pattern continues until adulthood (Figure 2Go, P45, P52 and Adult). As described by others (Lindvall et al., 1978Go), the density of DA-containing fibers in the rat mPFCx is greatest in layer VI and shows a progressive decrease toward layer I. Unlike noradrenergic fibers in the mPFCx (Lindvall and Bjorklund, 1984Go), the dopamine system does not show long, vertical fibers travelling either vertically toward layer I or horizontally within this lamina. Together with the fact that the densest distribution of fibers observed is in deeper layers, it seems unlikely that noradrenergic axons have been included in these analyses, particularly since the antibody preparation employed in one of these studies (Benes et al., 1993a) is 50 times less selective for norepinephrine–glutaraldehyde–protein conjugates than for those made with dopamine (Geffard et al., 1984Go). Thus, it seems unlikely that a significant number of noradrenergic fibers were included in the count of DA-IR varicosities.



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Figure 2.  A set of low power, darkfield photomicrographs of dopamine-IR varicose fibers in the rat mPFCx at five different postnatal stages (P11, P20, P45, P52 and early adult). At P11, there is a paucity of dopamine-containing fibers in the cortical mantle, but by P20, these fibers have become much more common. At P45 and P52, dopamine-IR fibers show a typical laminar distribution, with the highest density occurring in deeper laminae V and VI and a progressive decrease in density occurring in a gradient fashion toward layer I. In adult rats, this unique laminar distribution is still present, even though there is a marked increase in the density of fibers in each of the layers. Bar = 100 µm. Reproduced with permission (Benes et al., 1996).

 
A similar pattern of fiber staining can be distinguished at all postnatal stages examined. The increase of fiber density occurs to a proportionate degree across layers VI–II and does not progress in a distinct ‘inside-out’ manner. The size of the DA-IR varicosities also increases from ~1.2 µm at P20 to ~2.4 µm by P60; however, it is not likely that this change in size accounts for the increase in the density of fibers during the postnatal period, because the dimensions of the largest varicosities are still quite small relative to the thickness of the sections (40 µm). The size of varicosities reported in this study is larger than that previously described in an ultrastructural analysis (Seguela et al., 1988Go); however, this apparent discrepancy is probably related to shrinkage incurred during the fixation, dehydration and imbedding that is typically required for electron microscopic studies. Moreover, using a correction for particle size in which the numerical density, Na, is divided by section thickness, t, plus the average diameter of the particles, D (Abercrombie, 1946Go; Weibel, 1979Go), the density of fibers in post-weanling mPFCx is ~3.5-fold higher than in that of pre-weanling rats. For sections in which single immunoperoxidase-processing is combined with cresyl violet staining, DA-containing varicose fibers can be observed throughout the neuropil, but very commonly, such fibers course toward neuronal cell bodies (Benes et al., 1993Go). Many cell bodies have one or more varicosities in close apposition, and such contacts are observed in both superficial and deep laminae at all stages of postnatal life examined, although the density of varicosities in layers II and III is characteristically quite low.

Postnatal increases in the density of dopaminergic projections to the rat mPFCx (Verney et al., 1982Go; Kalsbeek et al., 1988Go) are paralleled by an increase of D2 receptor binding activity, which begins prenatally (Bruinink et al., 1983Go) and continues until the fourth postnatal week (Deskin et al., 1981Go). Interestingly, administration of 6-OH-dopamine prevents this latter increase of D2 receptor binding (Deskin et al., 1981Go), an effect that is associated with dystrophic changes in the basal dendrites of pyramidal neurons (Kalsbeek et al., 1988Go). Lesions induced in the prefrontal cortex of adult monkeys using 6-OH-dopamine result in an impaired performance of the spatial delayed alternation task (Brozoski et al., 1979Go) and it seems likely that this functional deficit would be associated with changes in the D2 receptor on pyramidal neurons.

The GABA System

GABA has long been considered the most important inhibitory neurotransmitter in the mammalian brain and extensive neurochemical studies of its development in rodent brain suggest that its maturation continues well into the postnatal period [for a review see Johnston (Johnston, 1988Go)]. For example, GABA- accumulating cells show a progressive increase in numerical density until P11 (Chronwall and Wolff, 1980Go). In contrast, the concentration of GABA and the specific activity of glutamate decarboxylase (GAD) (Coyle and Enna, 1976Go), as well as GABA receptor binding activity (Coyle and Enna, 1976Go; Palacios et al., 1979Go) and the messenger [m]RNA for this receptor (Gambarana et al., 1990Go), all increase until the third postnatal week.

At birth, the numerical density of GAD-IR somata in the anterior cingulate cortex reaches a peak at approximately postnatal day 5 (PN5) then diminishes until PN20, when the thickness of the cortical mantle is maximal (Vincent et al., 1995bGo). During this same period, the relative amount of neuropil surrounding all cell bodies in this region is expanding as dendritic and axonal fibers are increasing. As shown in Figure 3Go, not only do GABAergic cell bodies show a gradual increase in their size but, by P20–40, primary, secondary and tertiary branches of their dendritic tree can also be visualized. The expansion of neuropil in the rat mPFCx probably involves an increase of both dendritic branches and terminals of GABAergic neurons, a process that continues in the superficial layers until P25 (Vincent et al., 1995bGo), when the efficacy of GABAergic synaptic transmission also becomes optimal (Luhmann and Prince, 1991Go). In general terms, the maturation of the GABA system continues for ~2–3 weeks postnatally. As such, its full maturation within the mPFCx is probably complete before the dopamine system attains its full postnatal profile. Presumably, then, the dendritic branches of GABAergic interneurons lay in waiting for ingrowing dopamine fibers to target them for the formation of functional interactions.



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Figure 3.  A set of Nomarsky photomicrographs showing GABA-IR cell bodies in the rat mPFCx at various postnatal ages (P1, P5, P10, P20 and P41). (A,B) At early postnatal stages, GABA-IR cells have an elongated shape and a vertical orientation with respect to the pial surface. These cells also show thick apical dendrites and thinner basal processes (not in plane of focus). (C,D) At intermediate stages, some neuron somata become round or oval in shape and their dendritic processes show a reduction in caliber. (E) At later stages of the postnatal period, most GABA-IR somata show a round or oval shape and the dendrites exhibit secondary and tertiary branching (arrowheads). Scale bar = 10 µm. Reproduced with permission (Vincent et al., 1995).

 
Postnatal Development of Dopamine–GABA Interactions

As previously reported (Benes et al., 1996), specimens of the rat mPFCx processed with a double-immunostaining technique that localizes both DA-immunofluorescent (-IF) varicosities and GABA-IF cell bodies show a progressive increase in the interaction of these two neuronal elements between the pre-weanling period (Figure 4Go, P20) and the early stages of the post-weanling period (Figure 4Go, P25). An increasing number of such varicosities form contacts with GABA-IF neurons as the post-weanling period progresses, and this becomes most apparent at the beginning of the adult period (Figure 4Go, P60). When the latter double-IF preparations are subjected to blind, semiquantitative analysis, a progressive linear increase in the percentage of GABA-IF cell bodies (Figure 5Go, upper panel) with apposed DA-IR varicosities occurs between P0 and P60, and these data best fit a first-order polynomial equation (r = 0.75, P = 0.0005). During the pre-weanling period, any given GABA-IR cell body, on average, can show approximately one apposed varicosity. During the post-weanling period, however, the number of DA-IR varicosities (Figure 5Go, lower panel) in contact with GABA-IR cell bodies shows a curvilinear rise through P60 (r = 0.81, P = 0.0005). Some neurons have no varicosities forming appositions with their somata, while other have more than one, making the average number per cell >1.0. For these latter data, a second-order polynomial equation provides the best fit. When an index of interaction similar to that described in Table 4 is computed by multiplying the percentage of GABA cell somata having apposed dopamine varicosities and the number of such varicosities in contact with any given GABA cell body, post-weanling rats have an index that is 1.5 times higher than that seen in pre-weanling animals. By adulthood, this index increases 1.8 times with respect to post-weanling rats and 2.5 times when compared with pre-weanling animals (Benes et al., 1996).



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Figure 4.  A set of digital confocal photomicrographs of a double-immunofluorescence localization of dopamine-IR fibers (yellow) and GABA-IR (green) somata in layer VI of the rat mPFCx at three different postnatal ages (P20, P25 and P66). By P20, dopamine-IR varicosities are already forming contacts with GABAergic cell bodies (arrows). Within a few days of weaning (P25), there are still no obvious changes in the extent of interaction, although some varicosities may be forming contacts with a dendritic shaft (arrowhead). By P66, however, there are several varicosities in apposition with the GABA-containing cell body. Magnification = x1238.

 


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Figure 5.  A set of bivariate plots showing the interaction of dopamine-IF varicosities with GABA-IF somata in the rat mPFCx at different postnatal ages. Upper panel: the percentage of GABA-IF cell bodies having an apposed dopamine-IF varicosity. Lower panel: the number of dopamine-IF varicosities per GABA-IF cell body. Between P5 and P60, there is a progressive increase in the percentage of GABA-IF cell bodies in apposition with dopamine-IF varicositeis (r = 0.75, P = 0.0005 using a first-order polynomial equation). During this same period, there is, on average, only one dopamine-IF varicosity in apposition with any given GABA-IF neuron; however, after weaning (P24), the number of varicosities per cell shows a curvilinear rise toward adult levels (r = 0.81, P = 0.0005 using a second-order polynomial equation). Repoduced with permission (Benes et al., 1996).

 
In primates, dopaminergic inputs to neuron somata appear to be minimal (Goldman-Rakic et al., 1989Go), while in the human cortex, TH-IR varicosities have been shown to form appositions with the cell bodies of both pyramidal and nonpyramidal neurons with a remarkable degree of consistency across many cases (Todtenkopf and Benes, 1998Go). Using a Poisson analysis (Benes et al., 1993Go) of a large number of varicosities (>10 000) counted in 15 normal human cases, neurons in layers II (P = 0.0001), III (P = 0.004), V (P = 0.0001) and VI (P = 0.0001) of the anterior cingulate cortex were found to have non-random contacts with TH-IR varicosities (unpublished observations). The data obtained in a parallel analysis of a schizophrenic cohort (n = 10) were remarkably similar. Overall, the ‘observed’ percentage of varicosities in contact with cell bodies ranged from ~3 to 7% and is much lower than that seen for DA-IR (Benes et al., 1993Go) and TH-IR (Taylor and Benes, 1996Go) in the rat mPFCx. Although the majority are clearly associated with the neuropil, the proportion on cell somata is nevertheless quite significant in a Poisson sense. It is not clear, however, why the primate and human brain show such a lower density, although even a small number of varicosities could potentially exert a significant modulatory influence at the level of the cell body.

Taken together, the somata of GABAergic neurons probably act as a site with which sprouting dopaminergic fibers may form appositions. In this process, GABA cells may be a ‘passive’ target for the formation of interactions, or they may exert an ‘active’ neurotrophic influence on fiber sprouting and/or contact formation (Spoerri, 1988Go). Either way, it seems likely that dopaminergic fibers are capable of exerting an increasing modulatory influence on the activity of inhibitory interneurons during the postnatal period, particularly since DA receptors are localized on nonpyramidal cell bodies in the rat mPFCx (Vincent et al., 1993Go; Vincent and Benes, 1995Go). Moreover, both agonists and antagonists of DA can alter the postsynaptic potentials recorded in GABAergic interneurons in the pyriform (Gellman and Aghajanian, 1993Go) and frontal (Zhou and Hablitz, 1999Go) cortices. Organically synthesized agonists of the D2 receptor (i.e. RU24926 and LY171555) have been found to inhibit the release of [3H]GABA (Tam and Roth, 1990Go; Retaux et al., 1991aGo,bGo) and dopamine itself can influence the firing of GABAergic neurons (Penit-Soria et al., 1987Go).

The Influence of Serotonin on Dopamine Fiber Ingrowth

Recent evidence from studies of the anterior cingulate region of post-mortem brain has suggested that the interaction of dopamine fibers with intrinsic cortical neurons may be abnormal, as the distribution of TH-IR varicosities appears to be shifted from pyramidal to nonpyramidal neurons in layer II of schizophrenics (Benes et al., 1997aGo,bGo). Although many different neurotrophic mechanisms could potentially contribute to the induction of such a change, a facilitatory effect associated with the serotonin system presents an intriguing possibility because these latter fibers have been found to promote the ingrowth of afferents originating in the thalamus during cortical development (D'Amato et al., 1987Go). This possibility seems particularly intriguing because a significant number of cortical neurons probably receive a convergence of these two monoaminergic systems (see above). To assess the nature of this relationship, a series of experiments in which the 5-HT projections from the nucleus raphe dorsalis (NRD) were lesioned during the neonatal period using the selective toxin, 5,7-dihydroxytryptamine (5,7-DHT) were recently undertaken (Taylor et al., 1998Go). As shown in Figure 6Go (left), the sham-lesioned rats in which only vehicle was injected (n = 4) showed a dense distribution of 5-HT-IR neuronal cell bodies in ventral portions of the peri-aqueductal gray (PAG) at the level of the decussation of the superior cerebellar peduncle. The NRD is particularly prominent at this brainstem level and lesioned rats show a marked reduction in the number of immunoreactive cells (Figure 1Go, right). At more rostral levels of the midbrain, both sham and lesioned rats show abundant TH-IR fibers in the substantia nigra (SN) and ventral tegmental area (VTA). It seems unlikely that 5,7-DHT adversely affects dopamine neurons, since the rats have been treated with nomifensine, which blocks its uptake into these latter cells. It is hypothetically possible that this treatment could potentially have had an adverse effect on other neuronal populations, particularly those that receive an abundant serotoninergic innervation; however, there was no obvious indication of this. Rather than being decreased, TH-IR fibers appeared to be increased in dopaminergic nuclei of 5,7-DHT-lesioned rats, suggesting that the NRD may exert a suppressive effect on the projection neurons of the SN and VTA. In the mPFCx of sham-lesioned rats, 5-HT-IR varicose fibers are distributed throughout the cortical mantle (Figure 7Go, left), while the 5,7-DHT-lesioned rats show almost a complete absence of stained fibers (Figure 7Go, right). When TH-IR is visualized in this region, a rich plexus of varicose fibers is seen throughout the cortical mantle in both the sham- and 5,7-DHT-lesioned groups (Figure 8Go, left and right, respectively), particularly in layers V and VI, where DA fibers are typically most abundant. As with the SN and VTA, visual inspection suggests that the lesioned rats may have a higher density of TH-IR fibers in layer V and possibly also layer VI. Consistent with this impression, a computer-assisted microscopic analysis (Figure 9Go) has revealed that the density of TH-IR fibers in layer II showed no difference in the sham- versus 5,7-DHT-lesioned groups, while a twofold increase on neuronal cell bodies (t = 5.35; P = 0.0007) and in the neuropil (t = 4.08; P = 0.0035) was observed in layer V (Figure 5Go); a significant increase has also been observed in the neuropil of layer VI (t = 2.63; P = 0.03). When the distribution of TH-IR fibers is compared for different neuronal subtypes in layer V, the density is increased by 100% on neuron somata >100 µm2 in size (P = 0.002) and 60% on those <100 µm2 in size (P = 0.02) in the mPFCx of the lesioned group (not shown). There were no obvious differences in the size of varicosities, suggesting that an increase in the content of TH per varicosity cannot explain the current findings. In a controlled series of experiments in the frog neuromuscular junction, the size of axon terminals was found to increase in proportion to the number of synaptic vesicles and their content of neurotransmitter-synthesizing enzyme (Benes and Barrnett, 1978Go).



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Figure 6.  Brightfield photomicrographs of 5-HT-IR neuronal cell bodies in the NRD in ventral portions of the PAG of sham- (left) and 5,7-DHT-lesioned (right) rats.

 


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Figure 7.  Darkfield photomicrographs of 5-HT-IR fibers in the mPFCx of a sham- (left) and 5,7-DHT-lesioned rats (right). There are abundant fibers present in the specimen from the sham-lesioned rat, while there is a paucity of such fibers in the 5,7-DHT-lesioned animals. Reproduced with permission (Taylor et al., 1998Go).

 


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Figure 8.  Darkfield photomicrographs of TH-IR varicose fibers in the mPFCx of sham- (left) and 5,7-DHT-lesioned (right) rats. There are abundant fibers present in the cortical mantle with both treatments; however, the mPFCx of a 5,7-DHT-lesioned rat appears to have a higher density of TH-IR fibers in layer V and possibly also layer VI. Reproduced with permission (Taylor et al., 1998Go).

 


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Figure 9.  A set of bar graphs showing the density of TH-IR varicosites in apposition with neuronal cell bodies (upper panel) and in the neuropil (lower panel) of sham and 5,7-DHT lesioned rats. The data are expressed as the average number of varicosities per µm2 ± SEM in the sham versus lesioned groups. Although there are no differences in layer II, both layers V and VI show a marked increase in the density of TH-IR varicosities, particularly in neuropil. Reproduced with permission (Taylor et al., 1998Go).

 
Consistent with the above findings, DA fibers are known to interact extensively with dendritic processes throughout the neuropil (Seguela et al., 1988Go; Goldman-Rakic et al., 1989Go; Verney et al., 1990Go; Smiley and Goldman-Rakic, 1993Go), although the somata of both pyramidal and nonpyramidal neurons probably also serve as non-random targets for these fibers in the rat mPFCx (Verney et al., 1990Go; Huntley et al., 1992Go; Benes et al., 1993Go; Vincent et al., 1993Go, 1995aGo; Taylor and Benes, 1996Go; Davidoff and Benes, 1998Go; Davidoff et al., 2000Go). In the primate PFCx, TH-IR varicosities have not been found on neuron somata (Krimer et al., 1997Go); however, in the PFCx of the human brain, TH-IR varicosities are present on the somata of both pyramidal and nonpyramidal neurons (Todtenkopf and Benes, 1998Go). As noted above, it appears that the association of dopamine fibers with neuronal cell bodies may vary in degree from one species to another. Based on rodent studies, serotoninergic fibers appear to have a distribution that is similar to that of TH-IR fibers, although the latter also include some noradrenergic elements, particularly in the superficial layers where DA fibers are quite sparse. Serotonergic fibers probably interact with both projection cells and interneurons (Sheldon and Aghajanian, 1991Go; Morilak et al., 1993Go; Smiley and Goldman-Rakic, 1996Go; Taylor and Benes, 1996Go).

It is noteworthy that thermal ablation of the VTA in neonatal rats has been associated with a 30% decrease of basal dendritic branches of pyramidal neurons (Kalsbeek et al., 1989bGo). This latter treatment resulted in a depletion not only of dopamine levels, but also those of serotonin (Kalsbeek et al., 1989aGo). It seems likely that an interruption of fibers originating in the raphe nuclei and traveling en passage through the midbrain could have contributed to this change in the cortical serotoninergic projections. If so, it is uncertain as to whether one or both monoaminergic systems contributed to the observed decrease in pyramidal cell dendrites. It is important to emphasize that in the studies described herein, nomifensine was co-administered with 5,7-DHT to prevent the uptake of this latter toxin into midbrain dopamine cells. This pharmacologic strategy has made it possible to lesion the serotoninergic projections to the mPFCx, whilst preserving the dopaminergic ones originating in the VTA.

Overall, the present findings are not consistent with the idea that 5-HT may act trophically to facilitate the ingrowth of DA fibers during the late post-weanling and early adult periods. Rather, it seems more likely that the opposite is the case, i.e. the 5-HT system seems to be exerting an inhibitory trophic effect on the normal postnatal ingrowth of TH-IR fibers. One interpretation of the findings described above is that the 5-HT and DA systems may be competing with one another for functional territory on the surface of intrinsic cortical neurons within the rat mPFCx. An interaction of this type would tend to produce a reciprocal relationship between the two systems. An alternative possibility, however, is that the 5-HT and DA systems mainly influence one another at the level of their respective brainstem nuclei. Accordingly, lesioning of the NRD may result in a stimulation or release of dopaminergic neurons within the VTA to sprout the distal portion of their fiber projections in various termination sites, such as the mPFCx. Consistent with this idea, an increase of TH-IR staining was observed in the SN, VTA and PAG of 5,7-DHT-lesioned rats. Physiological studies have yielded contradictory results regarding the manner in which the DA and 5-HT systems may be influencing one another. On the one hand, some believe that 5-HT can increase the release of DA in the nucleus accumbens (Van Bockstaele et al., 1994Go; Broderick and Phelix, 1997Go), corpus striatum (Gudelsky and Nash, 1996Go; West and Galloway, 1996Go; Broderick and Phelix, 1997Go) and prefrontal cortex (Gudelsky and Nash, 1996Go; Iyer and Bradberry, 1996Go). Contrariwise, some studies suggest that 5-HT may actually decrease the release of DA, since exposure to selective 5-HT receptor antagonists has been associated with an increase of extracellular DA concentrations (Pehek, 1996Go; Howell et al., 1997Go). The latter pattern is consistent with the idea that there may be a competitive interactive between these two monoaminergic systems. This idea is a particularly appealing one because the VTA receives a direct input from serotoninergic fibers (Van Bockstaele et al., 1994Go).


    Conclusions
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 Abstract
 Introduction
 Interactions of Dopamine and...
 Postnatal Development of...
 Conclusions
 Notes
 References
 
The studies described above provide evidence in support of the idea that the dopamine and serotonin systems show a significant degree of convergence and plasticity in the rat mPFCx, and the degree to which this occurs is probably similar for both pyramidal cells and GABAergic interneurons. Particularly noteworthy is the fact that the dopamine system may be capable of considerable plasticity, at least until the start of the early adult period. If the dopamine system in the human brain exhibits similar characteristics, the maturation of the limbic cortex during adolescence and early adulthood may potentially provide ‘a window of opportunity’ for the induction of abnormal interactions of the monoaminergic systems with one another and with their intrinsic cortical targets. Indeed, some experimental evidence suggests that exposure to adrenal steroids during the postnatal period can result in an increase of dopamine-IR varicosities on interneurons in the mPFCx of rats also exposed to these hormones prenatally (Benes, 1997). Based on the studies discussed above, an important question to ask is whether pre- and/or postnatal stress might also result in an altered distribution of serotoninergic projections to the rat mPFCx [for a review see Stanford (Stanford, 1993Go)], one that is reciprocal in nature to that observed for the dopamine system. Since a combination of pre- and postnatal stress is believed to play a central role in the pathophysiology of some neuropsychiatric disorders (Benes, 1997; Walker and DiFiorio, 1997Go), it is plausible that changes in the way these two monoaminergic systems interact with one another might ultimately influence the activity of the individual cortical neurons upon which they both converge. Future studies will be directed at identifying further what effect dopaminergic fibers in the mPFCx might have on convergent serotoninergic inputs and their shared target neurons, and how this interaction may be influenced by psychotropic drugs.


    Notes
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 Abstract
 Introduction
 Interactions of Dopamine and...
 Postnatal Development of...
 Conclusions
 Notes
 References
 
The authors would like to thank Ms Maureen Medeiros for her help in the preparation of this manuscript. The work described herein was supported by grants from the National Institutes of Mental Health (MH00423, MH42261, MH/NS31862 and MH31154) and the Stanley Foundation.

Address correspondence to: Francine M. Benes, MD, PhD, McLean Hospital, 115 Mill Street, Belmont, MA 02478, USA. Email: benesf{at}mclean.harvard.edu.


    References
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 Interactions of Dopamine and...
 Postnatal Development of...
 Conclusions
 Notes
 References
 
Abercrombie M (1946) Estimation of nuclear population from microtomic sections. Anat Rev 94:239.

Akbarian S, Huntsman MM, Kim JJ, Tafazzoli A, Potkin SG, Bunney WE, Jones EG (1995) GABAA receptor subunit gene expression in human prefrontal cortex: comparison of schizophrenics and controls. Cereb Cortex 5:550–560.[Abstract]

Akil M, Lewis DA (1997) Cytoarchitecture of the entorhinal cortex in schizophrenia. Am J Psychiat 7:1010–1012.

Beasley CL, Reynolds GP (1996) Parvalbumin-immunoreactive neurons are reduced in the prefrontal cortex of schizophrenics. Schizophr Res 24:349–355.[ISI]

Benes FM (1988) Post-mortem structural analyses of schizophrenic brain. Study designs and the interpretation of data. Psychiat Develop 6: 213–226.

Benes FM (1989) Myelination of cortical-hippocampal relays during late adolescence: anatomical correlates to the onset of schizophrenia. Schizophr Bull 15:585–594.[ISI][Medline]

Benes FM (1995a) Is there a neuroanatomic basis for schizophrenia? The Neuroscientist 1:112–120.

Benes FM (1995b) A neurodevelopmental approach to the understanding of schizophrenia and other mental disorders. In: Developmental psychopathology, Vol. 1. Theory and methods (Cicchetti D, Cohen DJ, eds), pp. 227–253. New York: John Wiley & Sons.

Benes FM (1997a) A comparison of pyramidal and nonpyramidal neuron density in anterior cingulate cortex of schizophrenic and manic depressive subjects. Int Rev Psychiat (in press).

Benes FM (1997b) The role of stress and dopamine–GABA interactions in the vulnerability for schizophrenia. J Psychiat Res 31:257–275.[ISI][Medline]

Benes FM, Barrnett RJ (1978) Biochemical and morphometric studies of the relationship of acetylcholine synthesis and vesicle numbers after stimulation of frog neuromuscular junctions: the effect of a choline-o-acetyltransferase inhibitor. Brain Res 150:277–293.[ISI][Medline]

Benes FM, McSparren J, Bird ED, Vincent SL, SanGiovanni JP (1991) Deficits in small interneurons in prefrontal and anterior cingulate cortex of schizophrenic and schizoaffective patients. Arch Gen Psychiat 48:996–1001.[Abstract]

Benes FM, Sorensen I, Vincent SL, Bird ED, Sathi M (1992) Increased density of glutamate-immunoreactive vertical processes in superficial laminae in cingulate cortex of schizophrenic brain. Cereb Cortex 2: 502–512.

Benes FM, Vincent SL, Molloy R (1993) Dopamine-immunoreactive axon varicosities form nonrandom contacts with GABA-immunoreactive neurons in rat medial prefrontal cortex. Synapse 15:285–295.[ISI][Medline]

Benes FM, Turtle M, Khan Y, Farol P (1994) Myelination of a key relay zone in the hippocampal formation occurs in human brain during childhood, adolescence and adulthood. Arch Gen Psychiat 51: 477–484.[Abstract]

Benes FM, Khan Y, Vincent SL, Wickramasinghe R (1996a) Differences in the subregional and cellular distribution of GABAA receptor binding in the hippocampal formation of schizophrenic brain. Synapse 22: 338–349.[ISI][Medline]

Benes FM, Vincent SL, Marie A, Khan Y (1996b) Upregulation of GABAA receptor binding on neurons of prefrontal cortex in schizophrenic subjects. Neuroscience 75:1021–1031.[ISI][Medline]

Benes FM, Vincent SL, Molloy R, Khan Y (1996c) Increased interaction of dopamine-immunoreactive varicosities with GABA neurons of rat medial prefrontal cortex occurs during the postweanling period. Synapse 23:237–245.[ISI][Medline]

Benes FM, Todtenkopf MS, Taylor JB (1997a) Differential distribution of tyrosine hydroxylase fibers on neuronal subtypes in layer II of anterior cingulate cortex of schizophrenic brain. Synapse 25:80–92.[ISI][Medline]

Benes FM, Wickramasinge R, Vincent SL, Khan Y, Todtenkopf MS (1997b) Uncoupling of GABAA and benzodiazepine receptor binding activity in the hippocampal formation of schizophrenic brain. Brain Res 755:121–129.[ISI][Medline]

Bennett JP, Enna SJ, Bylund DB, Gillin JC, Wyatt RJ, Snyder SH (1979) Neurotransmitter receptors in frontal cortex of schizophrenics. Arch Gen Psychiat 36:927–934.[Abstract]

Bergson C, Mrzljak L, Smiley JF, Pappy M, Levenson R, Goldman-Rakic PS (1995) Regional, cellular, and subcellular variations in the distribution of D1 and D5 dopamine receptors in primate brain. J Neurosci 15: 7821–7836.[Abstract]

Bird ED, Spokes EGS, Iversen LL (1979) Increased dopamine concentration in limbic areas of brain from patients dying with schizophrenia. Brain 102:347–360.[ISI][Medline]

Broderick PA, Phelix CF (1997) I. Serotonin (5-HT) within dopamine reward circuits signals open-field behavior. II. Basis for 5-HT–DA interaction in cocaine dysfunctional behavior. Neurosci Biobehav Rev 21:227–260.[ISI][Medline]

Brozoski T, Brown RM, Rosvold HE, Goldman PS (1979) Cognitive deficit caused by depletion of dopamine in prefrontal cortex of rhesus monkey. Science 205:929–931.[ISI][Medline]

Bruinink A, Lichtensteiner W, Schlumpf M (1983) Pre- and postnatal ontogeny and characterization of dopaminergic D2, serotonergic S2, and spirodecanone binding sites in rat forebrain. J Neurochem 40:1227–1237.[ISI][Medline]

Bunney BS, Chiodo LA (1984) Mesocortical dopamine systems: further electrophysiological and pharmacological characteristics. In: Monoamine innervation of the cerebral cortex (Descarries L, Reader TA, Jasper HH, eds), pp. 263–277. New York: Alan R. Liss.

Chalmers DT, Kwak S, Mansour A, Akil H, Watson S (1993) Corticosteroids regulate brain hippocampal 5-HT1A receptor mRNA expression. J Neurosci 13:914–923.[Abstract]

Chalmers DT, Lopez JF, Vazquez DM, Akil H, Watson SJ (1994) Regulation of hippocampal 5-HT1A receptor gene expression by dexamethasone. Neuropsychopharmacology 10:215–222.[ISI][Medline]

Chronwall B, Wolff JR (1980) Prenatal and postnatal development of GABA-accumulating cells in the occipital neocortex of rat. J Comp Neurol 190:187–208.[ISI][Medline]

Corda MG, Biggio G (1986) Stress and GABAergic transmission: biochemical and behavioural studies. In: GABAergic transmission and anxiety (Biggio G, Costa E, eds), pp. 121–135. New York: Raven Press.

Coyle JT, Enna S (1976) Neurochemical aspects of the ontogenesis of GABAnergic neurons in the rat brain. Brain Res 111:119–133.[ISI][Medline]

Cross AJ, Crow TJ, Owen F (1981) 3H-Flupenthixol binding in post-mortem brains of schizophrenics: evidence for a selective increase in dopamine D2 receptors. Psychopharmacology 74:122–124.[ISI][Medline]

Cross AJ, Crow TJ, Ferrier IN, Johnstone EC, McCreadie RM, Owen F, Owens DGC, Poulter M (1983) Dopamine receptor changes in schizophrenia in relation to the disease process and movement disorder. J Neural Transmiss (Suppl) 18:265–272.

D'Amato RJ, Blue M, Largent B, Lynch D, Leobetter D, Molliver M, Snyder S (1987) Ontogeny of the serotonergic projection of rat neocortex: transient expression of a dense innervation of primary sensory areas. Proc Natl Acad Sci USA 84:4322–4326.[Abstract]

Davidoff SA, Benes FM (1998) High resolution analysis shows D1 receptor binding on pyramidal and nonpyramidal neurons. Synapse 28:83–90.[ISI][Medline]

Davidoff SA, Chu HM, Benes FM (2000) Acute administration of SCH23390 increase D1 receptors on nonpyramidal neurons in rat mPFC. Synapse 35:173–181.[ISI][Medline]

Deskin R, Seidler FJ, Whitmore WL, Slotkin TA (1981) Development of noradrenergic and dopaminergic receptor systems depends on maturation of their presynaptic nerve terminals in the rat brain. J Neurochem 36:1683–1690.[ISI][Medline]

Emson PC, Koob GF (1978) The origin and distribution of dopamine containing afferents to rat frontal cortex. Brain Res 142:249–267.[ISI][Medline]

Flechsig P (1920) Anatomie des menschlichen Gehirns und Ruckenmarks auf myelogenetischer Gundlange. Leipzig: G. Thieme.

Gambarana C, Pittman R, Siegel RE (1990) Development expression of the GABA-A receptor g1 subunit mRNA in the rat brain. J Neurobiol 21:1169–1179.[ISI][Medline]

Gaspar PP, Bloch B, LeMoine C (1995) D1 and D2 receptor gene expression in the rat frontal cortex: cellular localization in different classes of efferent neurons. Eur J Neurosci 7:1050–1063.[ISI][Medline]

Geffard M, Buijs RM, Seguela P, Pool CW, Le Moal M (1984) First demonstration of highly specific and sensitive antibodies against dopamine. Brain Res 294:161–165.[ISI][Medline]

Gellman RL, Aghajanian GK (1993) Pyramidal cells in piriform cortex receive a convergence of inputs from monoamine activated GABA- ergic interneurons. Brain Res 600:63–73.[ISI][Medline]

Goldman-Rakic PS, Leranth C, William SM, Mons N, Geffard M (1989) Dopamine synaptic complex with pyramidal neurons in primate cerebral cortex. Proc Natl Acad Sci USA 86:9015–9019.[Abstract]

Gudelsky GA, Nash JF (1996) Carrier-mediated release of serotonin by 3,4-methylenedioxymethamphetaine: implications for serotonin– dopamine interactions. J Neurochem 66:243–249.[ISI][Medline]

Gulledge AT, Jaffe DB (1998) Dopamine decreases the excitability of layer V pyramidal cells in the rat prefrontal cortex. J Neurosci 18:9139–9151.[Abstract/Free Full Text]

Hanada S, Mita T, Nishinok N, Tankaka C (1987) 3H-Muscimol binding sites increased in autopsied brains of chronic schizophrenics. Life Sci 40:259–266.[ISI][Medline]

Hashimoto T, Nishino N, Nakai H, Tanaka C (1991) Increase in serotonin 5-HT1A receptors in prefrontal and temporal cortices of brains from patients with chronic schizophrenia. Life Sci 48:355–363.[ISI][Medline]

Howell LL, Czoty PW, Byrd LD (1997) Pharmacological interactions between serotonin and dopamine on behavior in the squirrel monkey. Psychopharmacology 131:40–48.[ISI][Medline]

Huntley GW, Morrison JH, Prikhozhan A, Sealfon SC (1992) Localization of multiple dopamine receptor subtype mRNAs in human and monkey motor cortex and striatum. Mol Brain Res 15:181–188.[ISI][Medline]

Iyer RN, Bradberry CW (1996) Serotonin-mediated increase in pre- frontal cortex dopamine release: pharmacological characterization. J Pharmacol Exp Ther 277:40–47.[Abstract]

Jakab RL, Goldman-Rakic PS (1998) 5-Hydroxytryptamine2A serotonin receptors in the primate cerebral cortex: possible site of action of hallucinogenic and antipsychotic drugs in pyramidal cell apical dendrites. Proc Natl Acad Sci USA 95:735–740.[Abstract/Free Full Text]

Jakob H, Beckmann H (1986) Prenatal developmental disturbances in the limbic allocortex in schizophrenics. J Neural Transm 65:303–326.[ISI]

Johnston MV (1988) Biochemistry of neurotransmitters in cortical development. In: Cerebral cortex, Vol. 7. Development and maturation of cerebral cortex (Peter A, Jones EG, eds), pp. 211–236. New York: Plenum Press.

Joyce JN, Meador-Woodruff JH (1997) Linking the family of D2 receptors to neuronal circuits in human brain: insights into schizophrenia. Neuropsychopharmacology 6:375–384.

Joyce JN, Lexow N, Bird E, Winokur A. (1988) Organization of dopamine D1 and D2 receptors in human striatum: receptor autoradiographic studies in Huntington's disease and schizophrenia. Synapse 2: 546–557.[ISI][Medline]

Joyce JN, Shane A, Lexow N, Winokur A, Casanova MF, Kleinman JE (1993) Serotonin uptake sites and serotonin receptors are altered in the limbic system of schizophrenics. Neuropsychopharmacology 8:315–336.[ISI][Medline]

Kalen P, Rosengren E, Lindvall O, Bjorklund A (1989) Hippocampal noradrenaline and serotonin release over 24 h as measured by the dialysis technique in freely moving rats: correlation to behavioural activity state, effect of handling and tail-pinch. Eur J Neurosci 1: 181–188.[ISI][Medline]

Kalsbeek A, Voorn P, Buijs RM, Pool CW, Uylings HB (1988) Development of the dopaminergic innervation in the prefrontal cortex of rat. J Comp Neurol 269:58–72.[ISI][Medline]

Kalsbeek A, DeBruin JP, Mathijssen MA, Uylings HB (1989a) Ontogeny of open field activity in rats after neonatal lesioning of the mesocortical dopaminergic projection. Behav Brain Res 32:115–127.[ISI][Medline]

Kalsbeek A, Matthijssen MA, Uylings HB (1989b) Morphometric analysis of prefrontal cortical development following neonatal lesioning of the dopaminergic mesocortical projection. Exp Brain Res 78:279–289.[ISI][Medline]

Kornhuber J, Mack-Burkhardt F, Riederer P, Hebenstreit GF, Reynolds GP, Andrews HB, Beckman H (1989a) [3H]MK-801 binding sites in post-mortem brain regions of schizophrenic patients. J Neural Transm 77: 231–236.[ISI]

Kornhuber J, Riederer P, Reynolds GP, Beckmann H, Jellinger K, Gabriel E (1989b) 3H-Spiperone binding sites in post-mortem brains from schizophrenic patients: relationship to neuroleptic drug treatment, abnormal movements, and positive symptoms. J Neural Transm 75:1–10.[ISI]

Krimer LS, Jakab RL, Goldman-Rakic PS (1997) Quantitative three-dimensional analysis of the catecholaminergic innervation of identified neurons in the macaque prefrontal cortex. J Neurosci 17:7450–7461.[Abstract/Free Full Text]

Lee T, Seeman P (1980) Elevation of brain neuroleptic/dopamine receptors in schizophrenia. Am J Psychiat 137:191–197.[Abstract]

LeMoine C, Gaspar P (1998) Subpopulations of cortical GABAergic interneurons differ by their expression of D1 and D2 dopamine receptor subtypes. Mol Brain Res 15:231–236.

Lidov HGW, Grzanna R, Molliver ME (1980) The serotonin innervation of the cerebral cortex in the rat — an immunocytochemical analysis. Neuroscience 5:207–227.[ISI][Medline]

Lindvall O, Bjorklund A (1984) General organization of cortical monoamine systems. In: Monoamine innervation of cerebral cortex (Descarries L, Reader TR, Jasper HH, eds), pp. 9–40. New York: Alan R. Liss.

Lindvall O, Bjorklund A, Divac I (1978) Organization of catecholamine neurons projecting to frontal cortex in the rat. Brain Res 142:1–24.[ISI][Medline]

Luhmann HJ Prince DA (1991) Postnatal maturation of the GABAergic system in rat neocortex. J Neurophysiol 65:247–263.[Abstract/Free Full Text]

Mackay AVP, Doble A, Bird ED, Spokes EG, Quick M, Iversen LL (1978) 3H-Spiperone binding in normal and schizophrenic post-mortem human brain. Life Sci 23:527–532.[Medline]

Mackay AVP, Iversen LL, Rossor M, Spokes E, Bird E, Arregui A, Creese I, Snyder SH (1982) Increased brain dopamine and dopamine receptors in schizophrenia. Arch Gen Psychiat 39:991–997.[Abstract]

Mantz J, MIlla C, Glowinski J, Thierry AM (1988) Differential effects of ascending neurons containing dopamine and noradrenaline in the control of spontaneous activity and of evoked responses in the rat prefrontal cortex. Neuroscience 27:517–526.[ISI][Medline]

Marek GJ, Aghajanian GK (1998) The electrophysiology of prefrontal serotonin systems: therapeutic implications for psychosis. Biol Psychiat 44:1118–1127.[ISI][Medline]

Meador-Woodruff JH, Haroutunian V, Powchik P, Davidson M, Davis KL, Watson SJ (1997) Dopamine receptor transcript expression in striatum and prefrontal and occipital cortex. Focal abnormalities in orbitofrontal cortex in schizophrenia. Arch Gen Psychiat 54: 1089–1095.[Abstract]

Meltzer HY (1994) An overview of the mechanism of action on clozapine. J Clin Psychiat 55(Suppl B): 47–52.[ISI][Medline]

Meltzer HY, Stahl SM (1976) The dopamine hypothesis of schizophrenia: a review. Schizophr Bull 2:19–76.[ISI][Medline]

Mita T, Hanada S, Nishino N, Kuno T, Nakai H, Yamadori T, Mizoi Y, Tanaka C (1986) Decreased serotonin S2 and increased dopamine D2 receptors in chronic schizophrenics. Biol Psychiat 21:1407–1414.[ISI][Medline]

Morilak DA, Garlow SJ, Ciaranello RD (1993) Immunocytochemical localization and description of neurons expressing 5-HT-2 receptors in the rat brain. Neuroscience 54:701–717.[ISI][Medline]

Muly EC, Szigeti K, Goldman-Rakic PS (1998) D1 receptor in interneurons of macaque prefrontal cortex: distribution and subcellular localization. J Neurosci 18:10553–10565.[Abstract/Free Full Text]

Ohara K, Ulpian C, Seeman P, Sunahara R K, Van Tol HHM, Niznik HB (1993) Schizophrenia: dopamine D1 receptor sequence is normal, but has DNA polymorphisms. Neuropsychopharmacology 8:131–135.[ISI][Medline]

Owen F, Crow TJ, Poulter M, Cross AJ, Longden A, Riley GJ (1978) Increased dopamine-receptor sensitivity in schizophrenia. Lancet ii:223–225.

Palacios JM, Niehoff DL, Kuhar MJ (1979) Ontogeny of GABA and benzodiazepine receptors: effects of Triton X-100, bromide and muscimol. Brain Res 179:390–395.[ISI][Medline]

Parnavelas JG, Papadopoulos GC, Cavanagh ME (1988) Changes in neurotransmitters during development. In: Cerebral cortex, Vol. 7. Development and maturation of cerebral cortex (Peters A, Jones EG eds), pp. 177–209. New York: Plenum Press.

Pehek EA (1996) Local infusion of the serotonin antagonist ritanserin or ICS 205,930 increases in vivo dopamine release in the rat medial prefrontal cortex. Synapse 24:12–18.[ISI][Medline]

Penit-Soria J, Audinat E, Crepel F (1987) Excitation of rat prefrontal cortical neurons by dopamine: an in vitro electrophysiological study. Brain Res 425:363–374.

Reader TA (1981) Distribution of catecholamines and serotonin in the rat cerebral cortex: absolute levels and relative proportions. J Neural Transm 50:13–27.[ISI]

Reader TA, Ferron A, Descarries L, Jasper HH (1979) Modulatory role for biogenic amines in the cerebral cortex: microiontophoretic studies. Brain Res 160:217–229.[ISI][Medline]

Retaux S, Besson MJ, Penit-Soria J (1991a) Opposing effects of dopamine D2 receptor stimulation on the spontaneous and the electrically evoked release of [3H]GABA on rat prefrontal cortex slices. Neuroscience 42:61–71.[ISI][Medline]

Retaux S, Besson MJ, Penit-Soria J (1991b) Synergism between D1 and D2 dopamine receptors in the inhibition of the evoked release of [3H]GABA in the rat prefrontal cortex. Neuroscience 43:323–329.[ISI][Medline]

Reynolds GP, Czudek C, Andrews H (1990) Deficit and hemispheric asymmetry of GABA uptake sites in the hippocampus in schizophrenia. Biol Psychiat 27:1038–1044.[ISI][Medline]

Roth RH, Tam SY, Ida Y, Yang JX, Deutch AY (1988) Stress and the meso-corticolimbic dopamine systems. Ann NY Acad Sci 537:138–147.[ISI][Medline]

Schwartz R, Wess M, Labarca R, Skolnick P, Paul S (1987) Acute stress enhances the activity of the GABA receptor-gated ion channel in brain. Brain Res 411:151–155.[ISI][Medline]

Seeman P, Niznik HB (1990) Dopamine receptors and transporters in Parkinson's disease and schizophrenia. FASEB J 4:2737–2744.[Abstract/Free Full Text]

Seeman P, Guan H-C, Van Tol HHM (1993a) Dopamine D4 receptors elevated in schizophrenia. Nature 365:441–445.[ISI][Medline]

Seeman P, Ohara K, Ulpian C, Seeman MV, Jellinger K, Van Tol HHM, Niznik HB (1993b) Schizophrenia: normal sequence in the dopamine D2 receptor region that couples to G-proteins. DNA polymorphisms in D2. Neuropsychopharmacology 8:137–142.[ISI][Medline]

Seguela P, Watkins KC, Descarries L (1988) Ultrastructural features of dopamine axon terminals in the anteromedial and suprarhinal cortex of rat. J Comp Neurol 289:11–22.[ISI]

Sheldon PW, Aghajanian GK (1991) Excitatory responses to serotonin (5-HT) in neurons of the rat piriform cortex: evidence for mediation by 5-HT1C receptors in pyramidal cells and 5-HT2 receptors in interneurons. Synapse 9:208–218.[ISI][Medline]

Sheldon PW, Aghajanian GK (1990) Serotonin (5-HT) induces IPSPs in pyramidal layer cells of rat piriform cortex: evidence for the involvement of a 5-HT2-activated interneuron. Brain Res 506:62–69.[ISI][Medline]

Simpson MD, Slater P, Deakin JF, Royston MC, Skan WJ (1989) Reduced GABA uptake sites in the temporal lobe in schizophrenia. Neurosci Lett 107:211–215.[ISI][Medline]

Smiley JF, Goldman-Rakic PS (1993) Heterogenous targets of dopamine synapses in monkey prefrontal cortex demonstrated by serial section electron microscopy: a laminar analysis using the silver-enhanced diaminobenzidine sulfide (SEDS) immunolabeling technique. Cereb Cortex 3:223–238.[Abstract]

Smiley JF, Levey AI, Ciliax BJ, Goldman-Rakic PS (1994) D1 dopamine receptor immunoreactivity in human and monkey cerebral cortex: predominant and extrasynaptic localization in dendritic spines. Proc Natl Acad Sci USA 91:5720–5724.[Abstract]

Smiley JF, Goldman-Rakic PS (1996) Serotonergic axons in monkey prefrontal cerebral cortex synapse predominantly on interneurons as demonstrated by serial section electron microscopy. J Comp Neurol 367:431–443.[ISI][Medline]

Spoerri PE (1988) Neurotrophic effects of GABA in cultures of embryonic chick brain and retina. Synapse 2:11–22.[ISI][Medline]

Stanford SC (1993) Monoamines in response and adaptation to stress. In: Stress: from synapse to syndrome (Stanford SC, Salmon P, eds), pp. 282–332. London: Academic Press.

Tam S, Roth R (1990) Modulation of mesoprefrontal dopamine neurons by central benzodiazepine receptors. I. Pharmacological characterization. J Pharmacol Exp Ther 252:989–996.[Abstract]

Taylor JB, Benes FM (1996) Colocalization of glutamate decarboxylase, tyrosine hydroxylase and serotonin immunoreactivity in rat medial prefrontal cortex. Neuroscience-Net 1:10001.

Taylor JB, Cunningham M, Benes FM (1998) Neonatal raphe lesions increase dopamine fibers in prefrontal cortex of adult rats. Neuro-Report 9:1811–1815.[ISI][Medline]

Thierry AM, Tassin JP, Blanc G, Glowinski J (1976) Selective activation of the mesocortical DA system by stress. Nature 263:242–244.[ISI][Medline]

Thierry AM, Le Douarin C, Penit J, Ferron A, Glowinski J (1986) Variation in the ability of neuroleptics to block the inhibitory influence of dopaminergic neurons on the activity of cells in the rat prefrontal cortex. Brain Res Bull 16:155–160.[ISI][Medline]

Todtenkopf MS, Benes FM (1998) Distribution of glutamate decarboxylase65 immunoreactive puncta on pyramidal and non-pyramidal neurons in hippocampus of schizophrenic brain. Synapse 29:323–332.[ISI][Medline]

Van Bockstaele EJ, Cestari DM, Pickel VM (1994) Synaptic structure and connectivity of serotonin terminals in the ventral tegmental area: potential sites for modulation of mesolimbic dopamine neurons. Brain Res 647:307–322.[ISI][Medline]

Verney C, Berger B, Adrien J, Vigny A, Gay M (1982) Development of the dopaminergic innervation of the rat cerebral cortex. A light microscopic immunocytochemical study using anti-tyrosine hydroxylase antibodies. Devl Brain Res 5:41–52.[ISI]

Verney C, Alvarez C, Gerrard M, Berger B (1990) Ultrastructural double-labelling study of dopamine terminals and GABA-containing neurons in rat anteromedial cortex. Eur J Neurosci 2:960–972.[ISI][Medline]

Verney C, Alvarez C, Gerrard M, Berger B (1991) Ultrastructural double-labelling study of dopamine terminals and GABA-containing neurons in rat anteromedial cortex. Eur J Neurosci 2:960–972.[ISI]

Vincent SL, Khan Y, Benes FM (1993) Cellular distribution of dopamine D1 and D2 receptors in rat medial prefrontal cortex. J Neurosci 13: 2551–2561.[Abstract]

Vincent SL, Benes FM (1995) Postnatal maturation of GABA- immunoreactive neurons of rat medial prefrontal cortex. J Comp Neurol 355:81–92.[ISI][Medline]

Vincent SL, Khan Y, Wickramasinghe R, Benes FM (1995a) Differential regulation of GABAA and benzodiazepine receptor binding in the hippocampal formation of schizophrenics. Soc Neurosci Abstr 21:835.

Vincent SL, Khan Y, Benes FM (1995b) Cellular colocalization of dopamine D1 and D2 receptors in rat medial prefrontal cortex. Synapse 19:112–120.[ISI][Medline]

Walker EF, Diforio D (1997) Schizophrenia: a neural diathesis–stress model. Psychol Rev 104:667–685.[ISI][Medline]

Weibel ER (1979) Elementary introduction to stereological principles. In: Stereological methods, Vol. 1. Practical methods for biological morphometry, pp 46–57. New York: Academic Press.

West AR, Galloway MP (1996) Regulation of serotonin-facilitated dopamine release in vivo: the role of protein kinase A activating transduction mechanisms. Synapse 23:20–27.[ISI][Medline]

Weinberger DR (1987) Implications of normal brain development for the pathogenesis of schizophrenia. Arch Gen Psychiat 44:660–669.[Abstract]

Whitaker PM, Crow TJ, Ferrier IN (1981) Tritiated LSD binding in frontal cortex in schizophrenia. Arch Gen Psychiat 38:278–280.[Abstract]

Williams GV, Goldman-Rakic PS (1995) Modulation of memory fields by dopamine D1 receptors in prefrontal cortex. Nature 376:572–575.[ISI][Medline]

Woo T-U, Whitehead RE, Melchitzky DS, Lewis DA (1998) A subclass of prefrontal g-minobutyric acid axon terminals are selectively altered in schizophrenia. Proc Natl Acad Sci USA 95:5341–5346.[Abstract/Free Full Text]

Wu C, Yoder EJ, Shih J, Chen K, Dias P, Shi L, Ji SD, Wei J, Conner JM, Kumar S, Ellisman MH, Singh SK (1998) Development and characterization of monoclonal antibodies specific to the serotonin 5-HT2A receptor. J Histochem Cytochem 46:811–824.[Abstract/Free Full Text]

Yakovlev P, Lecours A (1967) The myelinogenetic cycles of regional maturation of the brain. In: Regional development of the brain early in life (Minkowski A, ed.), pp. 3–70. Oxford: Blackwell.

Yang CR, Seamans, JK (1996) Dopamine D1 receptor actions in layers V–VI rat prefrontal cortex neurons in vitro: modulation of dendritic– somatic signal integration. J Neurosci 16:1922–1935.[Abstract]

Zhou FM, Hablitz JJ, (1999) Dopamine modulation of membrane and synaptic properties of interneurons in rat cortex. J Neurophysiol 81:967–976.[Abstract/Free Full Text]