Regional Levels of Neurotransmitter Markers in the Pigeon Telencephalon: A Comparison with Possibly Homologous Areas of the Rat Telencephalon

The levels of cholinergic, gamma-aminobutyric acidergic (GABAergic), and excitatory amino acid neurotransmitter markers have been measured in 18 regions of the pigeon telencephalon as well as in supposedly homologous areas of the rat telencephalon. Among the basal telencephalic areas, some similar patterns of regional distribution were observed, with the noticeable exception of the ratio of levels of cholinergic markers between the striatum and globus pallidus, which was much larger in the rat than in the pigeon. In the rat cortical areas, some interesting differences were noticed among the archicortex, the paleocortex, and various parts of the neocortex. In particular, the area identified as prefrontal cortex by previous studies was significantly richer in cholinergic and excitatory amino acid markers and poorer in GABAergic activity than other neocortical regions. In the pigeon, presumedly neocortical equivalent areas--in particular, those constituting the dorsal ventricular ridge--were quite variable in levels of cholinergic markers, and some apparently well-established areas homologous to mammalian neocortex showed exceptionally low levels of cholinergic markers. The higher variability in levels of neurotransmitter-related markers shown by cortically equivalent areas of the avian dorsal ventricular ridge, as compared with the more uniform pattern present in basal telencephalic regions, may be the result of a greater plasticity of these structures during evolution, in response to different selective pressures.     

Differential Effects of Insulin on Choline Acetyltransferase and Glutamic Acid Decarboxylase Activities in Neuron-Rich Striatal Cultures

We studied the effects of insulin, nerve growth factor (NGF), and tetrodotoxin (TTX) on cellular metabolism and the activity of glutamic acid decarboxylase (GAD) and choline acetyltransferase (ChAT) in neuron-rich cultures prepared from embryonic day 15 rat striatum. Insulin (5 micrograms/ml) increased glucose utilization, protein synthesis, and GAD activity in cultures plated over a range of cell densities (2,800-8,400 cells/mm2). TTX reduced GAD activity; NGF had no effect on GAD activity. Insulin treatment reversibly reduced ChAT activity in cultures plated at densities of greater than 4,000 cells/mm2, and the extent of this reduction increased with increasing cell density. The number of acetylcholinesterase-positive neurons was not reduced by insulin, suggesting that insulin acts by down-regulating ChAT rather than by killing cholinergic neurons. Insulin-like growth factor-1 (IGF-1) reduced ChAT activity at concentrations 10-fold lower than insulin, suggesting that insulin's effect on ChAT may involve the IGF-1 receptor. NGF increased ChAT activity; TTX had no effect on ChAT activity. These results suggest that striatal cholinergic and GABAergic neurons are subject to differential trophic control.     

Whole-Brain Mapping of Inputs to Projection Neurons and Cholinergic Interneurons in the Dorsal Striatum

The dorsal striatum integrates inputs from multiple brain areas to coordinate voluntary movements, associative plasticity, and reinforcement learning. Its projection neurons consist of the GABAergic medium spiny neurons (MSNs) that express dopamine receptor type 1 (D1) or dopamine receptor type 2 (D2). Cholinergic interneurons account for a small portion of striatal neuron populations, but they play important roles in striatal functions by synapsing onto the MSNs and other local interneurons. By combining the modified rabies virus with specific Cre- mouse lines, a recent study mapped the monosynaptic input patterns to MSNs. Because only a small number of extrastriatal neurons were labeled in the prior study, it is important to reexamine the input patterns of MSNs with higher labeling efficiency. Additionally, the whole-brain innervation pattern of cholinergic interneurons remains unknown. Using the rabies virus-based transsynaptic tracing method in this study, we comprehensively charted the brain areas that provide direct inputs to D1-MSNs, D2-MSNs, and cholinergic interneurons in the dorsal striatum. We found that both types of projection neurons and the cholinergic interneurons receive extensive inputs from discrete brain areas in the cortex, thalamus, amygdala, and other subcortical areas, several of which were not reported in the previous study. The MSNs and cholinergic interneurons share largely common inputs from areas outside the striatum. However, innervations within the dorsal striatum represent a significantly larger proportion of total inputs for cholinergic interneurons than for the MSNs. The comprehensive maps of direct inputs to striatal MSNs and cholinergic interneurons shall assist future functional dissection of the striatal circuits.     

Topographic distribution of dopamine uptake,choline uptake,choline acetyltransferase,and GABA uptake in the striata of weaver mutant mice

The topographic distribution of dopamine (DA) uptake, choline uptake, choline acetyltransferase (ChAT) activity and GABA uptake within the striata of weaver mutant mice and control mice was determined. Uptake of [³H]dopamine, [³H]choline and [¹⁴C]GABA, as well as ChAT activity were determined in samples prepared from the dorsolateral, dorsomedial, ventrolateral and ventromedial portions of the striatum. In 45–60 day old control mice, dopamine uptake was homogeneously distributed throughout the striatum. On the other hand, striata from weaver mice exhibited an uneven distribution with the ventral aspects having greater uptake activity than the dorsal regions. Thus, although the ventral portion of the striatum is less severely affected than the dorsal portion, all areas of the striatum exhibited significantly reduced uptake rates. In 9 and 12 month old mice, choline uptake was higher in lateral than medial zones of the striatum of both genotypes and no differences were observed between genotypes. GABA uptake was higher in the ventral striatum than in the dorsal striatum but again no differences were found between weaver and control mice. The results of this study indicate that the entire weaver striatum is severely deficient in its ability to recapture dopamine and thus is functionally compromised. The results also indicate that the striatal cholinergic and GABAergic interneurons are not directly or indirectly affected by the weaver gene.Special ïssue dedicated to Dr. Morris H. Aprison     

Disease-related regressive alterations of forebrain cholinergic system in SOD1 mutant transgenic mice

Transgenic mice carrying the human mutated SOD1 gene with a glycine/alanine substitution at codon 93 (G93A) are a widely used model for the fatal human disease amyotrophic lateral sclerosis (ALS). In these transgenic mice, we carried out a neurochemical study not only restricted to the primarily affected regions, the cervical and lumbar segments of the spinal cord, but also to several other brain regions. At symptomatic (110 and 125 days of age), but not at pre-symptomatic (55 days of age) stages, we found significant decreases in catalytic activity of the cholinergic enzyme, choline acetyltransferase (ChAT) in the hippocampus, olfactory cortex and fronto-parietal cortex. In parallel, we observed a decreased number of basal forebrain cholinergic neurons projecting to these areas. No alterations of the cholinergic markers were noticed in the striatum and the cerebellum. A widespread marker for GABAergic neurons, glutamate decarboxylase (GAD), was unaffected in all the areas examined. Alteration of cholinergic markers in forebrain areas was paralleled by concomitant alterations in the spinal cord and brainstem, as a consequence of progressive apoptotic elimination of cholinergic motor neuron. Gestational supplementation of choline, while able to result in long-term enhancement of cholinergic activity, did not improve transgenic mice lifespan nor counteracted cholinergic impairment in brain regions and spinal cord.     

Regional Mapping of Neostriatal Neurotransmitter Systems as a Function of Aging

The purpose of the present investigation was to map chemically the distribution of certain neurotransmitter systems in the neostriatum of rats aged 6, 16, and 26 months. This mapping was carried out by microdissection of discrete striatal regions coupled with radiometric assays for choline acetyltransferase (ChAT), glutamate decarboxylase (GAD), dopamine (DA), and norepinephrine (NA). In all age groups, ChAT, DA, and NA were highest in the rostral relative to the caudal neostriatum. Additionally, ChAT was higher in the lateral than in the medial region, whereas GAD was more homogeneously distributed within the striatum. ChAT activity was decreased significantly primarily in the caudal regions in rats aged 16 and 26 months. DA levels were decreased in the caudal striatum in rats aged 26 months. NA levels were found to be significantly decreased primarily in the rostral neostriatal regions of the oldest rats. GAD activity remained unchanged in all age groups. These regional changes in selected neurotransmitter systems may underlie specific motor and cognitive deficits that often occur during aging.     

Parallel Decrease of Glutamic Acid Decarboxylase and Preproenkephalin mRNA in the Rat Striatum Following Chronic Treatment with a Dopaminergic D1 Antagonist and D2 Agonist

The levels of mRNA encoding glutamic acid decarboxylase (GAD) and preproenkephalin (PPE) were measured by Northern blot analysis, in the dorsal and the ventral part of the striatum, following long-term treatments with drugs acting selectively on D1 or D2 dopaminergic receptors. Chronic injection of the selective D1 antagonist SCH 23390 elicited a significant decrease in level of both GAD and PPE mRNA (-30%) in the dorsal striatum, whereas no significant change was observed in the ventral striatum. Chronic administration of both SCH 23390 and RU 24926, a D2 agonist, decreased the GAD and PPE mRNA levels in the dorsal (-38 and -57%, respectively) as well as in the ventral (-70 and -60%, respectively) striatum. In the ventral striatum the marked reduction of GAD mRNA levels was paralleled by a significant decrease of Vmax values of GAD enzymatic activity (-41%). These results suggest that the decrease in content of both GAD and PPE mRNA, promoted by the chronic blockade of D1 receptors, is mainly due to the action of dopamine acting on unaffected D2 receptors. Indeed, this decrease is further amplified when the D2 agonist and the D1 antagonist are administered together. Our results substantiate further the molecular mechanisms by which dopamine acts on different populations of GABAergic and enkephalinergic neurons in the two striatal regions examined.     

Differential sensitivity of cholinergic and GABAergic neurons in chick embryos treated intracerebrally with ethanol at 8 days of embryonic age

We have shown that in embryos treated with ethanol in ovo during days 1–3, a critical period of neuroembryogenesis, cholinergic neuronal phenotypic expression is decreased whereas GABAergic and catecholaminergic neuronal populations are increased as assessed by neuronal markers choline acetyltransferse (ChAT), glutamic acid decarboxylase (GAD) and tyrosine hydroxylase (TH) respectively. In this study, ethanol was administered intracerebrally to embryos at embryonic day 8, embryos were sacrificed at day 9 and ChAT and GAD activities assayed separately in cerebral hemispheres and remaining brain (diencephalon-midbrain and optic lobes). We found that ChAT activity was enhanced in the cerebral hemispheres only, whereas GAD activity was decreased in both cerebral hemispheres and remaining brain. We have concluded that the differential responses of neuronal phenotypes to ethanol may reflect compensatory mechanisms to ethanol insult. Moreover, these findings emphasize the vulnerability of the GABAergic neuronal phenotypes to ethanol neurotoxicity during early brain development in the chick.     

Morphological Diversity of GABAergic and Cholinergic Interneurons in the Striatal Dorsolateral and Ventromedial Regions of Rats

The striatum plays a fundamental role in sensorimotor and cognitive functions of the body, and different sub-regions control different physiological functions. The striatal interneurons play important roles in the striatal function, yet their specific functions are not clearly elucidated so far. The present study aimed to investigate the morphological properties of the GABAergic interneurons expressing neuropeptide Y (NPY), calretinin (Cr), and parvalbumin (Parv) as well as the cholinergic interneurons expressing choline acetyltransferase (ChAT) in the striatal dorsolateral (DL) and ventromedial (VM) regions of rats using immunohistochemistry and Western blot. The present results showed that the somatic size of Cr+ was the smallest, while ChAT+ was the largest among the four types of interneurons. There was no regional difference in neuronal somatic size of all types of interneurons. Cr+ and Parv+ neurons were differentially distributed in the striatum. Moreover, Parv+ had the longest primary dendrites in the DL region, while NPY+ had the longest ones in the VM region of striatum. But there was regional difference in the length of primary dendrites of Parv. The numbers of primary dendrites of Parv+ were the largest in both DL and VM regions of striatum. Both Cr+ and Parv+ primary dendrites displayed regional difference in the striatum. Western blot further confirmed the regional differences in the protein expression level of Cr and Parv. Hence, the present study indicates that GABAergic and cholinergic interneurons might be involved in different physiological functions based on their morphological and distributional diversity in different regions of the rat striatum.     

Comparison of the mammalian and avian telencephalon from the perspective of gene expression data.

Pallial and subpallial morphological subdivisions of the mouse and chicken telencephalon were examined from the new perspective given by gene markers expressed in these territories during development. The rationale of this approach is that common gene expression patterns may underlie similar histogenetic specification and, consequently, comparable morphological nature. The nested expression domains of the genes Dlx-2 and Nkx-2.1 are characteristic for the subpallium (lateral and medial ganglionic eminences). Similar expression of these markers in parts of the mouse septum and amygdala suggests that such parts may be considered subpallial. The genes Pax-6, Tbr-1 and Emx-1 are expressed in the pallium. Complementary areas of the septum and amygdala shared expression of these genes, suggesting these are the pallial parts of these units. Differences in the relative topography of pallial marker genes also define different regions of the pallium, which can be partially traced into the amygdala. Importantly, there is evidence of a novel "ventral pallium" subdivision, which is a molecularly distinct pallial territory intercalated between the striatum and the lateral pallium. Its derivatives in the mouse apparently belong to the claustroamygdaloid complex. Chicken genes homologous sequence-wise to these mouse developmental genes are expressed in topologically comparable patterns during development. The avian subpallium -the paleostriatum- expresses Dlx-2 and Nkx-2.1; expression extends as well into the septum and anterior and medial parts of the archistriatum. The avian pallium expresses Pax-6, Tbr-1 and Emx-1 and also contains a distinct ventral pallium, formed by the neostriatum and ventral intermediate parts of the archistriatum. The lateral pallium comprises the hyperstriatum ventrale, overlying temporo-parieto-occipital corticoid layer and piriform cortex, plus dorsal intermediate and posterior archistriatum. The dorsal pallium includes the dorsal, intercalated and accessory hyperstriatum, plus the dorsolateral corticoid area. The medial pallium contains the hippocampus and parahippocampal area. A dorsal part of the septum shares pallial molecular markers. Gene markers thus suggest common sets of molecular developmental determinants in either pallial or subpallial domains of the mouse and chicken telencephalon, extending all the way from the posterior pole (amygdala) to the septum. Ventral pallial derivatives identified as claustroamygdaloid in the mouse correlate with avian neostriatum and parts of the archistriatum.     

Thoughts on the development, structure and evolution of the mammalian and avian telencephalic pallium

Various lines of evidence suggest that the development and evolution of the mammalian isocortex cannot be easily explained without an understanding of correlative changes in surrounding areas of the telencephalic pallium and subpallium. These are close neighbours in a common morphogenetic field and are postulated as sources of some cortical neuron types (and even of whole cortical areas). There is equal need to explain relevant developmental evolutionary changes in the dorsal thalamus, the major source of afferent inputs to the telencephalon (to both the pallium and subpallium). The mammalian isocortex evolved within an initially small dorsal part of the pallium of vertebrates, surrounded by other pallial parts, including some with a non-cortical, nuclear structure. Nuclear pallial elements are markedly voluminous in reptiles and birds, where they build the dorsal ventricular ridge, or hypopallium, which has been recently divided molecularly and structurally into a lateral pallium and a ventral pallium. Afferent pallial connections are often simplified as consisting of thalamic fibres that project either to focal cell aggregates in the ventral pallium (predominant in reptiles and birds) or to corticoid areas in the dorsal pallium (predominant in mammals). Karten's hypothesis, put forward in 1969, on the formation of some isocortical areas postulates an embryonic translocation into the nascent isocortex of the ventropallial thalamorecipient foci and respective downstream ventropallial target populations, as specific layer IV, layers II- III, or layers V-VI neuron populations. This view is considered critically in the light of various recent data, contrasting with the alternative possibility of a parallel, separate evolution of the different pallial parts. The new scenario reveals as well a separately evolving tiered structure of the dorsal thalamus, some of whose parts receive input from midbrain sensory centres (collothalamic nuclei), whereas other parts receive oligosynaptic 'lemniscal' connections bypassing the midbrain (lemnothalamic nuclei). An ampler look into known hodological patterns from this viewpoint suggests that ancient collothalamic pathways, which target ventropallial foci, are largely conserved in mammals, while some emergent cortical connections can be established by means of new collaterals in some of these pathways. The lemnothalamic pathways, which typically target ancestrally the dorsopallial isocortex, show parallel increments of relative size and structural diversification of both the thalamic cell populations and the cortical recipient areas. The evolving lemnothalamic pathways may interact developmentally with collothalamic corticopetal collaterals in the modality-specific invasion of the emergent new areas of isocortex.     

Neurons with different neurotransmitters in embryonic neocortical allografts in the rat sciatic nerve

Different subsets of interneurons in the Wistar rat neocortex and in neocortical transplants developing in a damaged nerve were identified by the following immunohistochemical markers: glutamate decarboxylase (GAD 67) for GABAergic nerve cells, NO-synthase (NOS) for NO-ergic neurons, choline acetyltransferase (ChAT) for cholinergic cells, and tyrosine hydroxylase for catecholaminergic structures. Twentyeight days after surgery, individual GAD 67-ir, NO-ir, ChAT-ir, and very rarely TH-ir cells were detected in the graft. It was shown that the number of GAD 67-ir neurons per unit area in the grafts was less than in the rat neocortex P20.     

Specific lysis of GABAergic synaptosomes by an antiserum to glutamate decarboxylase

An antiserum to pure glutamate decarboxylase (GAD) when incubated with rat cortical synaptosomes in the presence of complement caused release of 33-53% of lactate dehydrogenase (LDH) and 22-41% of total GAD. In addition most of the gamma-aminobutyrate (GABA) present was released. Anti-GAD antiserum alone, or complement alone, were without action. The antiserum plus complement had no effect on noradrenaline or choline uptake, and did not release choline acetylase (ChAT). Anti-ChAT serum plus complement released 30-37% of ChAT and 10-13% of LDH. It prevented choline uptake. This serum did not produce GAD release or prevent GABA, choline or noradrenaline uptake. When cortical synaptosomes were exposed to both antisera plus complement, their actions were strictly additive. The data indicate specific lysis of GABAergic and cholinergic synaptosomal sub-populations.     

Choline acetyltransferase, glutamic acid decarboxylase and somatostatin in the kainic acid model for chronic temporal lobe epilepsy

The aim of the study was to investigate neurochemical changes in a kainic acid (KA; 10 mg/kg, s.c.)-induced spontaneous recurrent seizure model of epilepsy, 6 months after the initial KA-induced seizures. The neuronal markers of cholinergic and gamma-aminobutyric acid (GABA)ergic systems, i.e. choline acetyltransferase (ChAT) and glutamic acid decarboxylase (GAD) activities, and a marker for neuropeptide, i.e. level of somatostatin, have been investigated. The brain regions investigated were the hippocampus, amygdala/piriform cortex, caudate nucleus, substantia nigra and the frontal, parietal, temporal and occipital cortices. Six months after KA injection, reduced ChAT activity was observed in the amygdala/piriform cortex (47% of control; p<0.001), increased ChAT activity in the hippocampus (119% of control; p<0.01) and normal ChAT activity in the other brain regions. The activity of GAD was significantly increased in all analysed cortical regions (between 146 and 171% of control), in the caudate nucleus (144% of control; p<0.01) and in the substantia nigra (126% of control; p<0.01), whereas in the amygdala/piriform cortex, the GAD activity was moderately lowered. The somatostatin level was significantly increased in all cortical regions (between 162 and 221% of control) as well as in the hippocampus (119% of control), but reduced in the amygdala/piriform cortex (45% of control; p<0.01). Six months after KA injection, the somatostatin:GAD ratio was lowered in the amygdala/piriform cortex (49% of control) and in the caudate nucleus (41% of control), whereas it was normal in the hippocampus and moderately increased in the cortical brain regions. A positive correlation was found between seizure severity and the reduction of both ChAT activities and somatostatin levels in the amygdala/piriform cortex. The results show a specific pattern of changes for cholinergic, GABAergic and somatostatinergic activities in the chronic KA model for epilepsy. The revealed data suggest a functional role for them in the new network that follows spontaneous repetitive seizures.     

Effects of canonical Wnt signaling on dorso-ventral specification of the mouse telencephalon

Wnt signaling is involved in numerous processes during vertebrate CNS development. In this study, we used conditional Cre/loxP system in mouse to ablate or activate beta-catenin in the telencephalon in two time windows: before and after the onset of neurogenesis. We show that beta-catenin mediated Wnt signals are required to maintain the molecular identity of the pallium. Inactivation of beta-catenin in the telencephalon before neurogenesis results in downregulated expression of dorsal markers Emx1, Emx2 and Ngn2, and in ectopic up-regulation of ventral markers Gsh2, Mash1 and Dlx2 in the pallium. In contrast, ablation of ss-catenin after the onset of cortical neurogenesis (E11.5) does not result in a dorso-ventral fate shift. In addition, activation of canonical Wnt signaling in the subpallium leads to a repression of ventral telencephalic cell identities as shown by the down-regulation of subpallial markers Dlx2, Nkx2.1, Gsh2, Olig2 and Mash1. This was accompanied with an expansion of dorsal identities ventrally as shown by the expanded expression domains of pallial markers Pax6 and Ngn2. Thus, our data suggest that canonical Wnt signals are involved in maintaining the identity of the pallium by controlling expression of dorsal markers and by suppressing ventral programs from being activated in pallial progenitor cells.     

Distribution of glycogen phosphorylase and cytochrome oxidase in the central nervous system of the turtle Trachemys dorbigni

Glycogen phosphorylase (GP) and cytochrome oxidase (CO) activities were mapped histochemically in the brain of the turtle Trachemys dorbigni. In the telencephalon, both activities occurred in the olfactory bulb, in all cortical areas, in the dorsal ventricular ridge, striatum, primordium hippocampi and olfactory tubercle. In the diencephalon, they were identified in some areas of the hypothalamus, and in rotundus and geniculate nuclei. Both reactions were detected in the oculomotor, trochlear, mesencephalic trigeminal nuclei, the nucleus of the posterior commissure, torus semicircularis, substantia nigra and ruber and isthmic nuclei of the mesencephalon. In all layers of the optic tectum GP activity was found, but CO only labelled the stratum griseum centrale. In the medulla oblonga both enzymes appear in the reticular, raphe and vestibular nuclei, locus coeruleus and nuclei of cranial nerves. In the cerebellum, the granular and molecular layers, and the deep cerebellar nuclei were positive for both enzymes. The Purkinje cells were only reactive for CO. In the spinal cord, motor and commissural neurones exhibited a positive reaction for the two enzymes. However, CO also occurred in the marginal nucleus and in the lateral funiculus. These results may be useful as a basis for subsequent studies on turtle brain metabolism.     

Specific Inhibition of Dopamine D-1-Mediated Cyclic AMP Formation by Dopamine D-2, Muscarinic Cholinergic, and Opiate Receptor Stimulation in Rat Striatal Slices

The ability of different receptors to mediate inhibition of cyclic AMP accumulation due to a variety of agonists was examined in rat striatal slices. In the presence of 1 mM 3-isobutyl-1-methylxanthine, dopamine D-2, muscarinic cholinergic, and opiate receptor stimulation by RU 24926, carbachol, and morphine (all at 10(-8)-10(-5) M), respectively, inhibited the increase in cyclic AMP accumulation in slices of rat striatum due to dopamine D-1 receptor stimulation by 1 microM SKF 38393. In contrast, these inhibitory agents were unable to reduce the ability of a number of other agonists, including isoprenaline, prostaglandin E1, 2-chloroadenosine, vasoactive intestinal polypeptide, and cholera toxin, to increase cyclic AMP levels in striatal slices. These results suggest that in rat striatum either dopamine D-2, muscarinic cholinergic, and opiate receptors are only functionally linked to dopamine D-1 receptors or that the D-1 and D-2 receptors linked to adenylate cyclase lie on the cells, distinct from other receptors capable of elevating striatal cyclic AMP levels.     

Loss of Nkx2.1 homeobox gene function results in a ventral to dorsal molecular respecification within the basal telencephalon: evidence for a transformation of the pallidum into the striatum.

The telencephalon is organized into distinct longitudinal domains: the cerebral cortex and the basal ganglia. The basal ganglia primarily consists of a dorsal region (striatum) and a ventral region (pallidum). Within the telencephalon, the anlage of the pallidum expresses the Nkx2.1 homeobox gene. A mouse deficient in Nkx2.1 function does not form pallidal structures, lacks basal forebrain TrkA-positive neurons (probable cholinergic neurons) and has reduced numbers of cortical cells expressing GABA, DLX2 and calbindin that migrate from the pallidum through the striatum and into the cortex. We present evidence that these phenotypes result from a ventral-to-dorsal transformation of the pallidal primordium into a striatal-like anlage.