Glutamic acid decarboxylase and somatostatin immunoreactivities in rat visual cortex

Antibodies to glutamic acid decarboxylase (GAD) and somatostatin (SS) were used to determine the laminar distribution and morphology of GAD- and SS-immunoreactive neurons and terminals in rat visual cortex. The present study demonstrates that GAD-immunoreactive neurons constitute several morphologically distinct subclasses of neurons in rat visual cortex. These subclasses of neurons can be distinguished by differences in soma size, soma shape, dendritic branching patterns, axonal arborizations, and location in the neuropil. GAD-immunoreactive neurons are found throughout all layers of visual cortex. They have nonpyramidal morphology and constitute roughly 15% of the total neuronal population. The laminar pattern of GAD-immunoreactive puncta is uneven, with a prominent band of terminals in layer IV. Numerous large GAD-positive puncta surround the somata and proximal dendrites of pyramidal cells in layers II, III, and V. SS-immunoreactive neurons constitute a less numerous and more restricted population of nonpyramidal neurons. Their somata are located mainly in layers II, III, V, and VI. Very few, if any, SS-immunoreactive neurons are found in layers I and IV. SS-immunoreactive terminals are arranged along vertical and diagonal collateral branches that have a beaded appearance. Finally, many neurons in the supra- and infragranular layers and in the white matter are immunoreactive to both glutamic acid decarboxylase and somatostatin. This coexistence of immunoreactivity to both GAD and SS may characterize a broad subclass of cortical nonpyramidal neurons.     

Glutamic acid decarboxylase immunoreactivity is present in perikarya of neurons in nucleus tractus solitarius of rat

Immunohistochemical procedures reveal perikarya containing glutamic acid decarboxylase immunoreactivity in the nucleus tractus solitarius of the rat. After colchicine pretreatment, neurons were observed in all subdivisions of the nucleus with a particular concentration in the ventrolateral and intermediate subdivisions.     

Morphological heterogeneity of layer VI neurons in mouse barrel cortex

Understanding the basic neuronal building blocks of the neocortex is a necessary first step toward comprehending the composition of cortical circuits. Neocortical layer VI is the most morphologically diverse layer and plays a pivotal role in gating information to the cortex via its feedback connection to the thalamus and other ipsilateral and callosal corticocortical connections. The heterogeneity of function within this layer is presumably linked to its varied morphological composition. However, so far, very few studies have attempted to define cell classes in this layer using unbiased quantitative methodologies. Utilizing the Golgi staining technique along with the Neurolucida software, we recontructed 222 cortical neurons from layer VI of mouse barrel cortex. Morphological analyses were performed by quantifying somatic and dendritic parameters, and, by using principal component and cluster analyses, we quantitatively categorized neurons into six distinct morphological groups. Additional systematic replication on a separate population of neurons yielded similar results, demonstrating the consistency and reliability of our categorization methodology. Subsequent post hoc analyses of dendritic parameters supported our neuronal classification scheme. Characterizing neuronal elements with unbiased quantitative techniques provides a framework for better understanding structure–function relationships within neocortical circuits in general. J. Comp. Neurol. 512:726–746, 2009. © 2008 Wiley‐Liss, Inc.     

Glutamic acid decarboxylase in the striate cortex of normal and monocularly deprived kittens

Degeneration of the thalamic fibers in the visual cortex of turtles leads to an increase in the numerical density of cortical synapses with flattened vesicles and symmetrical membrane differentiations (Smith, L. M., and F. F. Ebner (1980) Soc. Neurosci. Abstr. 6: 328). This change correlates with an increase in the cortical activity of glutamic acid decarboxylase (GAD), the synthetic enzyme for gamma-aminobutyric acid (GABA). These data are consistent with the hypothesis that removal of thalamic input activity is the stimulus for cortical GABAergic neurons to form new synapses. Pharmacological evidence suggests that even simple environmental deprivation may induce a similar increase in the numerical density of GABAergic synapses in kitten striate cortex (Duffy, F. H., S. R., Snodgrass, J. L. Burchfiel, and J. L. Conway (1976) Nature 260: 256-257). We have examined this possibility in monocularly deprived kittens using methods to localize and measure GAD. GAD in kitten striate cortex was localized using immunocytochemistry. GAD-positive cells were found in all layers and were uniformly distributed in layers II to VI. Immunoreactivity associated with axon terminals (puncta), in contrast, was laminated with a distinct band in layer IV. Monocular deprivation (MD), by either unilateral enucleation or lid closure, had no detectable effect on the distribution of GAD in striate cortex. The band of layer IV puncta remained uniform even under conditions that produced alterations in layer IV cytochrome oxidase activity. We measured GAD activity in homogenates of striate cortex to address the possibility that MD causes an absolute change in the density of GABAergic synapses. Again, however, GAD activity in the binocular and monocular segments of striate cortex was found to be unaffected by early enucleation. These data suggest two conclusions: first, that the numerical density of GABAergic synapses in visual cortex is not regulated directly by thalamic activity, and second, that changes in GABAergic synapse density do not account for the ocular dominance shift observed in kitten striate cortex after MD.     

Glutamic acid decarboxylase-like immunoreactivity in brainstem auditory nuclei of the rat

The distribution of GABA-producing neurons in the brainstem auditory nuclei of the rat was investigated immunohistochemically by using an antibody to glutamic acid decarboxylase (GAD). In the cochlear nuclei, GAD immunoreactive neurons are present only in the superficial granular and molecular layers, whereas terminals are found in all subdivisions of the nuclei and are particularly dense surrounding large spherical cells and one type of stellate cell. In the superior olivary complex, GAD immunoreactive neurons are located in the lateral olivary nucleus and throughout the periolivary region. Immunoreactive terminals are distributed along dendrites of principal cells of the medial and lateral olivary nuclei and are clustered around somata of globular neurons of the nucleus of the trapezoid body. An extremely dense band of immunoreactive somata and terminals is present along the ventral edge of the olivary complex. The ventral, intermediate, and dorsal nuclei of the lateral lemniscus contain small fusiform GAD-immunoreactive neurons and a moderately dense plexus of immunoreactive terminals. The inferior colliculus contains a large population of GAD-immunoreactive perikarya and an extremely dense accumulation of immunoreactive terminals in the central, dorsomedial, and external nuclei. These observations indicate that GABA systems are involved in function at all levels of the brainstem auditory pathway.     

Glutamic acid decarboxylase autoantibodies and neurological disorders

Glutamic acid decarboxylase (GAD) is the enzyme that catalyses the production of GABA, a major neurotransmitter of the central nervous system. Antibodies to GAD (GAD-Ab) were first recognised in a patient affected by stiff-person syndrome; subsequently they were reported in a large number of cases with type 1 diabetes. Recently GADAb have been described in a number of patients affected by chronic cerebellar ataxia, drug-resistant epilepsy and myoclonus. These cases usually harbour other autoantibodies or are affected by organ-specific autoimmune diseases. The role of GAD-Ab is still unclear; the lack of experimental models makes it difficult to investigate their potential pathogenetic role. However two mechanisms have been suggested: the reduction by GAD-Ab of GABA synthesis in nerve terminals or the interference with exocytosis of GABA. Received: 16 May 2002 / Accepted in revised form: 10 July 2002 Correspondence to B. Giometto     

Vibrissectomy induced changes in GAP-43 immunoreactivity in the adult rat barrel cortex.

Within the rat primary somatosensory cortex, neurons responding principally to movement of each individual mystacial vibrissa are grouped together in structures termed barrels. Previous studies have examined changes in the area of cortex showing increased 2-deoxyglucose uptake in response to vibrissal stimulation. These studies have shown that chronic removal of all but the central (C3) vibrissa in adult rats induces an enlarged representation of the remaining C3 barrel in the contralateral cortex. This increase is prevented by cortical norepinephrine depletion. The major question raised by such studies is whether such plasticity is due to structural rearrangement or unmasking of otherwise silent synapses. In this study, antibodies to GAP-43, a presynaptic protein whose synthesis is related to neuronal development and regeneration, were used to investigate this issue. In adult rat brain, tangential sections through layer IV of the barrel receptor field normally show moderate levels of GAP-43 immunoreactivity (GAP-IR) in the inter-barrel septa and low levels within the barrels themselves. The present study examined changes in the pattern of GAP-IR from 1 to 8 weeks after vibrissectomy with sparing of C3 as an index of possible physical reorganization of cortical circuits. Quantitative analysis of the cortices of animals with unilateral vibrissectomy with sparing of C3 showed that the area of low GAP-IR within the barrels surrounding C3 was decreased at 1 week (8.4% shrinkage; P less than 0.01) and 8 weeks (12.0% shrinkage; P less than 0.015), relative to the cortex ipsilateral to the surgery. Both bilateral vibrissectomy with sparing of C3 and ibotenic acid lesions of the ventrobasal thalamus produced similar results. Some evidence was also seen that the area of low GAP-IR in the C3 barrel shrank to a similar degree after such manipulations. Cortical norepinephrine depletion had no apparent effect on vibrissectomy-induced GAP-IR changes. These results suggest that removal of vibrissal input to the adult rat barrel cortex produces transynaptic induction of axonal sprouting within the barrel cortex.     

Elevated immunoreactivity for glutamic acid decarboxylase in the rat cerebral cortex following transient middle cerebral artery occlusion

We investigated the expression of glutamic acid decarboxylase (molecular weight 67,000; GAD67) immunohistochemically in the rat cerebral cortex following transient middle cereral artery occlusion (MCAO) capable of producing slowly progressive neuronal damage. An increase in GAD67 immunoreactivity was observed in the cerebral cortex ipsilateral to the ischemic insult, most prominent in lamina IV, 3 to 14 days after MCAO. At this stage, light microscopy showed GAD67-positive puncta to be larger and more strongly immunoreactive in the ipsilateral cortex than those in the contralateral side. The elevated expression of GAD67 in the insulted cortex may reflect part of the adaptive functional changes in GABA transmission with slowly progressive cortical ischemic damage.Supported in part by Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan     

Activity-dependent regulation of glutamic acid decarboxylase in the rat barrel cortex: effects of neonatal versus adult sensory deprivation.

Immunocytochemical techniques were used to study the effects of tactual deprivation on glutamic acid decarboxylase (GAD) containing neurons in rat somatosensory barrel cortex. In normal rats GAD immunoreactive neurons and puncta are present in all laminae, with dense patches of GAD immunoreactive puncta centered on the barrels in lamina IV. Trimming whiskers of adult rats leads to a reversible decrease of GAD immunoreactivity in barrels corresponding to trimmed hairs. Intensity of GAD staining also is reversibly altered in supragranular laminae of nondeprived barrel columns flanked by deprived barrels. This indicates that GAD levels in the barrel cortex ordinarily fluctuate with changes in sensory input. By contrast, animals whose whiskers are trimmed from birth have normal GAD staining in both deprived and nondeprived barrels. Moreover, if trimmed whiskers of neonatally deprived animals are allowed to grow to normal lengths and are retrimmed later in adulthood GAD staining is not affected. Thus early tactual deprivation disrupts mechanisms that permit modulation of transmitter enzyme levels in cortical neurons following changes in sensory experience.     

Corticothalamic projections from layer 5 of the vibrissal barrel cortex in the rat

This study bears on the projections of layer 5 cells of the vibrissal sensory cortex to the somatosensory thalamus in rats. Small groups of cells were labeled with biotinylated dextran amine (BDA), and their axonal arborizations were individually reconstructed from horizontal sections counterstained for cytochrome oxidase. Results show that the vast majority ( approximately 95%) of layer 5 axons that innervate the somatosensory thalamus are collaterals of corticofugal fibers that project to the brainstem. The anterior pretectal nucleus, the deep layers of the superior colliculus, and the pontine nuclei are among the structures most often coinnervated. In the thalamus, layer 5 axons terminate exclusively in the dorsal part of the posterior group (Po), where they form clusters of large terminations. Because dorsal Po projects to multiple cortical areas, we sought to determine whether all recipient areas return a layer 5 projection to this part of the thalamus. Additional experiments using fluoro-gold and BDA injections provided evidence that the primary somatosensory area is the sole source of layer 5 projections to dorsal Po but that this thalamic region receives convergent layer 6 projections from the primary and second somatosensory areas and from the motor and insular cortices. These results show that layer 5 projections do not overlap in associative thalamic nuclei, thus defining area-related subdivisions. Furthermore, the coinnervation of brainstem nuclei by layer 5 CT axons suggests that this pathway conveys to the thalamus a copy of the cortical output aimed at brainstem structures.     

Developmental distribution of calretinin in mouse barrel cortex

We describe the postnatal development of calretinin expression in the mouse barrel cortex by immunohistochemistry. A densely staining neuropil and numerous cell bodies appeared throughout layer V, but only within barrel septa of layer IV, at postnatal day 4. This staining pattern became most robust at postnatal day 8. Thereafter, calretinin expression became reduced until the third postnatal week when it attained its mature levels, and the barrel-specific staining was no longer apparent.     

Glutamic acid decarboxylase autoimmunity in Batten disease and other disorders

Degenerative diseases of the CNS, such as stiff-person syndrome (SPS), progressive cerebellar ataxia, and Rasmussen encephalitis, have been characterized by the presence of autoantibodies. Recent findings in individuals with Batten disease and in animal models for the disorder indicate that this condition may be associated with autoantibodies against glutamic acid decarboxylase (GAD), an enzyme that converts the excitatory neurotransmitter glutamate to the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). Anti-GAD autoantibodies could result in excess excitatory neurotransmitters, leading to the seizures and other symptoms observed in patients with Batten disease. The pathogenic potential of GAD autoantibodies is examined in light of what is known for other autoimmune disorders, such as multiple sclerosis, SPS, Rasmussen encephalitis, and type 1 diabetes, and may have radical implications for diagnosis and management of Batten disease.     

Local intra- and interlaminar connections in mouse barrel cortex

Focal injections of horseradish peroxidase (HRP) in dimethylsulfoxide (DMSO) were targeted into mouse somatosensory cortex, in vitro, with a template. Injections were made at different depths and in different locations in the whisker-barrel-defined somatosensory map in order to determine quantitative connectivity patterns within and between barrel-defined cortical columns. Cortices were sectioned in a plane parallel to the pia at 75 microns. Data were collected directly from microscope slides by computer. Data are presented as: 1) Plots of computer-mapped HRP reaction product density in neurons and cell locations for each section in relation to barrel boundaries; 2) histograms of label in cortical layers related to individual barrel-defined columns; 3) polar plots of relative amounts of label within individual barrel columns in sections through each barrel column; 4) vectors which represent HRP reaction product density as a function of direction and distance from the injection site; 5) statistical analysis of the shape of the label distribution pattern in the plane of the cortex as a function of injection site depth; and 6) probability of labeling of any other barrel column given a labeled barrel column. The principal findings are: 1) The pattern of label distribution, after an injection directly above or directly below an individual barrel, is hour-glass shaped with the waist of the hour-glass in layer IV. 2) Connections within barrel cortex are asymmetrical. Barrel-related columns within a row are more strongly interconnected than those in different rows. 3) Connections of the small barrels associated with whiskers on the upper lip are strongest with other small barrels, but strong connections also exist between these small barrels and the larger barrels. 4) The pattern of intracortical connections in SII is not asymmetrical; interlaminar connections in SII are fundamentally different from those in barrel cortex. 5) Quantitative intracortical projection patterns are highly consistent with functional data on intracortical processing of whisker information. As such, the quantitative data clearly indicate the spatial extent and relative magnitude of populations of neurons involved in intracortical processing of sensory information. The spatial arrangements of these intracortical connections, in conjunction with known developmental events, make it highly likely that the distribution of intracortical axons in mouse barrel cortex is sculpted in part by experience.     

Glutamic acid decarboxylase in cerebrospinal fluid in infancy and childhood Part II. Glutamic acid decarboxylase activity in cerebrospinal fluid of children with neurological diseases

Glutamic acid decarboxylase (GAD) activity in cerebrospinal fluid (CSF) was determined in 53 patients with neurological diseases as follows: Epilepsy (n:17), febrile convulsions (n:3), meningoencephalitis (n:17), encephalopathies (n:10), CNS leukemia (n:3), congenital hydrocephalus (n:2) and pseudoileus neonatorum (n:1). Compared with the mean normal value (5.2 +/- 2.5 pmol CO2 formed/hr/ml) reported in Part I, a significant increase of GAD activity in CSF was demonstrated in patients with uncontrolled epileptic seizures (11.4 +/- 3.9 pmol CO2 formed/hr/ml), febrile convulsions (13.5 +/- 8.7), viral meningitis with or without encephalitis (20.3 +/- 13.6), encephalopathies (30.0 +/- 25.9), CNS leukemia (11.1 +/- 5.0), congenital hydrocephalus (20.5 +/- 7.3) and pseudoileus neonatorum (28.6). Markedly high GAD activity was found in patients with CNS leukemia several days after intrathecal injection of methotrexate (39.8 +/- 18.0). On the other hand, significantly low GAD activity was shown in patients with bacterial meningitis or brain abscess (1.3 +/- 1.2). This suggests that some bacterial factors may be inhibitory toward GAD activity in CSF. High GAD activity in CSF may be useful as an indicator of aseptic brain dysfunction, although it was not always correlated with the severity of symptoms.     

Glutamic acid decarboxylase in cerebrospinal fluid in infancy and childhood. Part I. Glutamic acid decarboxylase activity in cerebrospinal fluid of normal infants and children

Glutamic acid decarboxylase (GAD) activity in the cerebrospinal fluid (CSF) of normal infants (n:14) and children (n:28) was determined by measuring the amount of 14CO2 released from L-[1-14C]-glutamic acid. The mean GAD activity in CSF of infants and children was 5.2 +/- 2.5 pmol CO2 formed/hr/ml. Dividing these subjects into 4 groups according to age, GAD activities in CSF were 5.4 +/- 1.6 pmol CO2 formed/hr/ml in neonates (0-1 m), 3.6 +/- 1.6 pmol CO2 formed/hr/ml in infants (2-12 m), 3.9 +/- 1.1 pmol CO2 formed/hr/ml in young children (2-6 yr) and 7.1 +/- 2.3 pmol CO2 formed/hr/ml in school children (7-16 yr), respectively. In neonates and school children, GAD activities were significantly higher (p less than 0.001) than those in the other age groups. In infants under 6 months of age, a significantly negative correlation between GAD activity in CSF and their ages was recognized (r = -0.52, p less than 0.001). In infants and children ranging from 6 months to 16 years of age, a significantly positive correlation between GAD activity in CSF and their ages was found (r = 0.67, p less than 0.001). These data suggest that high GAD activity in neonates may be due to hypoxia at birth and the activity gradually increases from 6 months to 15 years of age.