Loss of glutamic acid decarboxylase (Gad67) in Gpr88-expressing neurons induces learning and social behavior deficits in mice.

GABA is the neurotransmitter of striatal projection neurons, however the contribution of the striatal GABAergic output to behavior is not well understood. We assessed motor function, spatial learning, social behavior, olfactory and object recognition preferences in mice lacking the GABA-synthesizing enzyme glutamic acid decarboxylase, Gad67, in neurons expressing the protein Gpr88, an orphan G-protein-coupled receptor primarily expressed in the striatum. Gad67-deficient mice show no impairments in motor coordination and balance, but exhibit enhanced locomotor activity and stereotypic grooming behavior. Furthermore, Gad67-deficient mice show impairments in spatial learning, social behavior, olfactory preferences, and they prefer a familiar compared to a novel object in the object recognition test. These findings provide original evidence that striatal Gad67 expression is involved in the modulation of learning and social behavior. Some of the behavioral abnormalities observed in Gad67-deficient mice are reminiscent of Autism-spectrum-disorder (ASD) deficits, suggesting that abnormal striatal GABAergic output may contribute to behavioral deficits in ASD.

Parvalbumin-, calbindin-, and calretinin-immunoreactive hippocampal interneuron density in autism.

BACKGROUND: There has been a long-standing interest in the possible role of the hippocampus in autism and both postmortem brain and neuroimaging studies have documented varying abnormalities in this limbic system structure. AIMS: This study investigates the density of subsets of hippocampal interneurons, immunostained with the calcium binding proteins, calbindin (CB), calretinin (CR) and parvalbumin (PV) to determine whether specific subpopulations of interneurons are impacted in autism. MATERIALS AND METHODS: Unbiased stereological techniques were used to quantify the neuronal density of these immunoreactive subpopulations of gamma-aminobutyric acid-ergic (GABAergic) interneurons analyzed in the CA and subicular fields in postmortem brain material obtained from five autistic and five age-, gender- and postmortem interval-matched control cases. RESULTS: Results indicate a selective increase in the density of CB-immunoreactive interneurons in the dentate gyrus, an increase in CR-immunoreactive interneurons in area CA1, and an increase in PV-immunoreactive interneurons in areas CA1 and CA3 in the hippocampus of individuals with autism when compared with controls. DISCUSSION/CONCLUSIONS: Although our sample size is small, these findings suggest that GABAergic interneurons may represent a vulnerable target in the brains of individuals with autism, potentially impacting upon their key role in learning and information processing. These preliminary findings further suggest the need for future more expanded studies in a larger number of postmortem brain samples from cases of autism and controls.

Decreased GABAA receptors and benzodiazepine binding sites in the anterior cingulate cortex in autism.

The anterior cingulate cortex (ACC; BA 24) via its extensive limbic and high order association cortical connectivity to prefrontal cortex is a key part of an important circuitry participating in executive function, affect, and socio-emotional behavior. Multiple lines of evidence, including genetic and imaging studies, suggest that the ACC and gamma-amino-butyric acid (GABA) system may be affected in autism. The benzodiazepine binding site on the GABA(A) receptor complex is an important target for pharmacotherapy and has important clinical implications. The present multiple-concentration ligand-binding study utilized (3)H-muscimol and (3)H-flunitrazepam to determine the number (B(max)), binding affinity (K(d)), and distribution of GABA(A) receptors and benzodiazepine binding sites, respectively, in the ACC in adult autistic and control cases. Compared to controls, the autistic group had significant decreases in the mean density of GABA(A) receptors in the supragranular (46.8%) and infragranular (20.2%) layers of the ACC and in the density of benzodiazepine binding sites in the supragranular (28.9%) and infragranular (16.4%) lamina [corrected]. These findings suggest that in the autistic group this downregulation of both benzodiazepine sites and GABA(A) receptors in the ACC may be the result of increased GABA innervation and/or release disturbing the delicate excitation/inhibition balance of principal neurons as well as their output to key limbic cortical targets. Such disturbances likely underlie the core alterations in socio-emotional behaviors in autism.

Birthdates and number of neurons in the serotonergic raphe nuclei in the rat with prenatal protein malnutrition.

The effect of prenatal protein deprivation on timing of neurogenesis and on number of neurons generated in the serotonergic dorsal (DR) and median raphe (MR) nuclei of the rat was studied. These neurons are of interest because their neurogenesis occurs during the period of malnutrition and their axonal projections participate in the earliest stages of brain development. In this study, dams were maintained on a 25% casein diet or a 6% casein diet 5 weeks prior to mating and throughout pregnancy. At birth, all pups were cross-fostered to dams on a 25% casein diet. Bromodeoxyuridine, a thymidine analog that is incorporated into nuclear deoxyribonucleic acid during the cell cycle synthetic phase, was used as a marker of neurogenesis. Bromodeoxyuridine was administered on either embryonic day 11, 12, 13 or 14. On postnatal day 30, serial sections of raphe nuclei were processed with bromodeoxyuridine immunocytochemistry to determine the number of raphe cells generated on each day and with Nissl stain to determine the total number of cells generated. There were no significant differences between the two diet groups in timing of generation or in total number of cells generated, indicating that neurogenesis of these early generated neurons appears unaffected by concomitant protein deprivation.

Neuropathology of progressive cognitive decline in chronically hypertensive rhesus monkeys.

Hypertension is an identified major risk factor for cerebrovascular disease, which is second only to Alzheimer's disease as a cause of dementia in the elderly. In addition, hypertension has been associated with a more subtle, progressive decline in cognitive function for which the neuropathology is not well understood. The present study was undertaken to explore this relationship in an experimental, nonhuman primate model, with hypertension produced by a coarctation of the thoracic aorta. Since prior studies with this model have shown a progressive decline in memory function, similar to that seen in human hypertension, as well as scattered microinfarcts in the cerebral white and gray matter, this study was designed to explore the relationship between these two. In addition to microinfarcts, the hypertensive monkeys with the highest arterial blood pressure also showed minute areas of focal gliosis without infarction. The number of these focal lesions showed a significant correlation with the severity of the hypertension, but not with the behavioral deficit. For four of these hypertensive monkeys, immunostaining demonstrated a pervasive, widespread activation of microglial cells and astroglial cells in the white matter as well as evidence of leaks in the blood-brain barrier, providing a more logical substrate for the cognitive decline.

Density and distribution of hippocampal neurotransmitter receptors in autism: an autoradiographic study.

Neuropathological studies in autistic brains have shown small neuronal size and increased cell packing density in a variety of limbic system structures including the hippocampus, a change consistent with curtailment of normal development. Based on these observations in the hippocampus, a series of quantitative receptor autoradiographic studies were undertaken to determine the density and distribution of eight types of neurotransmitter receptors from four neurotransmitter systems (GABAergic, serotoninergic [5-HT], cholinergic, and glutamatergic). Data from these single concentration ligand binding studies indicate that the GABAergic receptor system (3[H]-flunitrazepam labeled benzodiazepine binding sites and 3[H]-muscimol labeled GABA(A) receptors) is significantly reduced in high binding regions, marking for the first time an abnormality in the GABA system in autism. In contrast, the density and distribution of the other six receptors studied (3[H]-80H-DPAT labeled 5-HT1A receptors, 3[H]-ketanserin labeled 5-HT2 receptors, 3[H]-pirenzepine labled M1 receptors, 3[H]-hemicholinium labeled high affinity choline uptake sites, 3[H]-MK801 labeled NMDA receptors, and 3[H]-kainate labeled kainate receptors) in the hippocampus did not demonstrate any statistically significant differences in binding.

Effect of Prenatal Protein Malnutrition on Birthdates and Number of Neurons in the Rat Locus Coeruleus.

The effect of prenatal protein deprivation on the timing of neurogenesis and on the number of neurons generated in the locus coeruleus of the rat was studied. These neurons are of interest as their axon projections are involved in the earliest stages of cerebral cortical development. Dams were maintained on a 25% casein diet or a 6% casein diet five weeks prior to mating and the diets continued throughout the pregnancy. At birth, all pups were cross-fostered to dams on a 25% casein diet. BrDU, a thymidine analog that is incorporated into the nuclear DNA during the synthetic phase of the cell cycle, was used as a marker of the generation period. It was administered intraperitoneally (25 mg/kg body weight) on embryonic day 10, 11, 12, 13, or 14. On postnatal day 30, the brain stems were processed with BrDU immunocytochemistry to determine the relative number of neurons generated on each day, and with Nissl stain to determine the total number of neurons generated in the two groups. There were no significant differences between the two diet groups in the timing of their generation or in the total number of neurons generated, indicating a preservation of neurogenesis of these early generated neurons in these malnourished rats.

Organization of direct hippocampal efferent projections to the cerebral cortex of the rhesus monkey: projections from CA1, prosubiculum, and subiculum to the temporal lobe.

This study investigates direct hippocampal efferent projections to the temporal lobe of the rhesus monkey. Tritiated amino acid injections were placed into the hippocampal formation to identify terminal fields, and complementary fluorescent retrograde tracer injections were placed into the cortex to identify the cells of origin. Tritiated amino acid injections into CA1, prosubicular, or subicular subfields produced anterograde label over parts of the parahippocampal gyrus and temporal pole. Injections of fluorescent retrograde tracers demonstrated that these projections originate from longitudinal strips of neurons that occupy part of the CA1 subfield as well as from strips of neurons in adjacent prosubicular and subicular subfields. Thus, an injection into area TH of the posterior parahippocampal gyrus labeled neurons in a longitudinal strip of proximal CA1 (i.e., near CA2) as well as a strip in the subiculum; injections into areas TF, TL, 35, or Pro labeled neurons in a longitudinal strip of distal CA1 (i.e., near the prosubiculum) as well as one in the prosubiculum; and an injection into area TFO labeled neurons in a longitudinal strip in the middle of CA1. These strips of neurons extended longitudinally throughout the entire rostrocaudal length of the hippocampus. These results demonstrate that, in the monkey, CA1 projections to cortex arise topographically from longitudinally oriented strips of neurons that occupy only a part of the transverse extent of CA1 but that cover most of the anteroposterior extent of the hippocampus. Thus, in the monkey, CA1 is not a single uniform entity and may have a unique role as a source of direct hippocampal projections to the cerebral cortex.

Topographically specific hippocampal projections target functionally distinct prefrontal areas in the rhesus monkey.

The sources of ipsilateral projections from the hippocampal formation, the presubiculum, area 29a-c, and parasubiculum to medial, orbital, and lateral prefrontal cortices were studied with retrograde tracers in 27 rhesus monkeys. Labeled neurons within the hippocampal formation (CA1, CA1', prosubiculum, and subiculum) were found rostrally, although some were noted throughout the entire rostrocaudal extent of the hippocampal formation. Most labeled neurons in the hippocampal formation projected to medial prefrontal cortices, followed by orbital areas. In addition, there were differences in the topography of afferent neurons projecting to medial when compared with orbital cortices. Labeled neurons innervating medial cortices were found mainly in the CA1' and CA1 fields rostrally, but originated in the subicular fields caudally. In contrast, labeled neurons which innervated orbital cortices were considerably more focal, emanating from the same relative position within a field throughout the rostrocaudal extent of the hippocampal formation. In marked contrast to the pattern of projection to medial and orbital prefrontal cortices, lateral prefrontal areas received projections from only a few labeled neurons found mostly in the subicular fields. Lateral prefrontal cortices received the most robust projections from the presubiculum and the supracallosal area 29a-c. Orbital, and to a lesser extent medial, prefrontal areas received projections from a smaller but significant number of neurons from the presubiculum and area 29a-c. Only a few labeled neurons were found in the parasubiculum, and most projected to medial prefrontal areas. The results suggest that functionally distinct prefrontal cortices receive projections from different components of the hippocampal region. Medial and orbital prefrontal cortices may have a role in long-term mnemonic processes similar to those associated with the hippocampal formation with which they are linked. Moreover, the preponderance of projection neurons from the hippocampal formation innervating medial when compared with orbital prefrontal areas followed the opposite trend from what we had observed previously for the amygdala (Barbas and De Olmos [1990] (J Comp Neurol 301:1-23). Thus, the hippocampal formation, associated with mnemonic processes, targets predominantly medial prefrontal cortices, whereas the amygdala, associated with emotional aspects of memory, issues robust projections to orbital limbic cortices. Lateral prefrontal cortices receive robust projections from the presubiculum and area 29a-c and sparse projections from the hippocampal formation. These findings are consistent with the idea that the role of lateral prefrontal cortices in memory is distinct from that of either medial or orbital cortices. The results suggest that signals from functionally distinct limbic structures to some extent follow parallel pathways to functionally distinct prefrontal cortices.

Prenatal protein malnutrition effects on the serotonergic system in the hippocampal formation: an immunocytochemical, ligand binding, and neurochemical study.

Prenatally protein malnourished rats born to dams maintained on a 6% casein diet during pregnancy and then fostered at birth to females on a 25% casein diet show adult alterations in hippocampal kindling and long-term potentiation and behavioral changes that all suggest dysfunction of hippocampal formation (HF). In the present investigation, compared to well-nourished controls, 220 day malnourished rats exhibited a decrease in the 5-HT fiber density in the dentate gyrus (DG) and CA3 subfield and, a 15-25% decrease 5-HT uptake sites assayed with [3H]-citalopram in CA3 and CA1. In malnourished rats, 5-HT1A receptors assayed with [3H]8-OH-DPAT were decreased by 20% in CA3. Because most hippocampal subfields showed no 5-HT changes, hippocampal 5-HT levels determined via HPLC methods were similar in adult malnourished and control rats. These results suggest that there are localized changes in the 5-HT afferent system in the hippocampal formation of the 220 day prenatally protein malnourished rat. Considering the 5-HT afferent input to inhibitory intrahippocampal neurons, the decreased 5-HT plexus may result in increased inhibition within specific hippocampal subfields despite overall normal levels of 5-HT in the total HF.

The olivocerebellar projection in normal (+/+), heterozygous weaver (wv/+), and homozygous weaver (wv/wv) mutant mice: comparison of terminal pattern and topographic organization.

Olivocerebellar organization and topography were analyzed in adult normal (+/+), heterozygous weaver (wv/+), and homozygous weaver (wv/wv) mutant mice. The two genotypes (wv/+ and wv/wv) of the weaver mutant present a gradation of abnormal cerebellar morphology. Purkinje cell (PC) ectopia ranges from mild (wv/+) to moderate (wv/wv), and regional PC loss is also graded in the two types. To determine olivocerebellar organization and topography, tritiated amino acids were placed into different regions of the inferior olivary complex (IO) in normal, heterozygous, and homozygous weaver mice. Despite some PC loss and ectopia, olivocerebellar fiber (OCF) terminals in both homozygous and heterozygous weaver mice have an orthogonal distribution and topography similar to that seen in normal mice. Differences in OCF termination, such as an increased density of OCF terminal label in the lower portion of the molecular layer, the PC, and granule cell layers, are seen in homozygous weaver mice. In some heterozygous weaver and normal cases, multiple injections labeling most IO cells on one side of the IO resulted in continuous OCF terminal labeling in many regions of the contralateral cerebellar cortex, suggesting that all PCs receive OCF input. Retrograde analysis involving injections of horseradish peroxidase conjugated to wheat germ agglutinin into different mediolateral cerebellar regions in homozygous weaver mice further demonstrates a generally normal olivocerebellar topography.

Visual receptive field organization and cortico-cortical connections of the lateral intraparietal area (area LIP) in the macaque.

The visual receptive field physiology and anatomical connections of the lateral intraparietal area (area LIP), a visuomotor area in the lateral bank of the inferior parietal lobule, were investigated in the cynomolgus monkey (Macaca fascicularis). Afferent input and physiological properties of area 5 neurons in the medial bank of the intraparietal sulcus (i.e., area PEa) were also determined. Area LIP is composed of two myeloarchitectonic zones: a ventral zone (LIPv), which is densely myelinated, and a lightly myelinated dorsal zone (LIPd) adjacent to visual area 7a. Previous single-unit recording studies in our laboratory have characterized visuomotor properties of area LIP neurons, including many neurons with powerful saccade-related activity. In the first part of the present study, single-unit recordings were used to map visual receptive fields from neurons in the two myeloarchitectonic zones of LIP. Receptive field size and eccentricity were compared to those in adjacent area 7a. The second part of the study investigated the cortico-cortical connections of area LIP neurons using tritiated amino acid injections and fluorescent retrograde tracers placed directly into different rostrocaudal and dorsoventral parts of area LIP. The approach to area LIP was through somatosensory area 5, which eliminated the possibility of diffusion of tracers into area 7a. Unlike many area 7a receptive fields, which are large and bilateral, area LIP receptive fields were much smaller and exclusively confined to the contralateral visual field. In area LIP, an orderly progression in visual receptive fields was evident as the recording electrode moved tangentially to the cortical surface and through the depths of area LIP. The overall visual receptive field organization, however, yielded only a rough topography with some duplications in receptive field representation within a given rostrocaudal or dorsoventral part of LIP. The central visual field representation was generally located more dorsally and the peripheral visual field more ventrally within the sulcus. The lower visual field was represented more anteriorly and the upper visual field more posteriorly. In LIP, receptive field size increased with eccentricity but with much variability with in the sample. Area LIPv was found to have reciprocal cortico-cortical connections with many extrastriate visual areas, including the parieto-occipital visual area PO; areas V3, V3A, and V4: the middle temporal area (MT); the middle superior temporal area (MST); dorsal prelunate area (DP); and area TEO (the occipital division of the intratemporal cortex). Area LIPv is also connected to area TF in the lateral posterior parahippocampal gyrus.(ABSTRACT TRUNCATED AT 400 WORDS)

Regional and topographic organization of the olivocerebellar projection in homozygous staggerer (sg/sg) mutant mice: an anterograde and retrograde tracing study.

The purpose of this investigation was to examine the organization of the olivocerebellar projection in the homozygous staggerer mouse. The adult stagger cerebellum is devoid of most Purkinje cells which are the primary target neurons for olivocerebellar fibers. Many of the remaining Purkinje cells are ectopic. In the first experiments, a small injection of a tritiated amino acid was placed into the staggerer inferior olivary complex. In coronal sections, longitudinal strips of labeled axons and olivocerebellar fiber terminals were separated by similarly oriented regions that contained little or no olivocerebellar fiber label. Within a given orthogonal band, olivocerebellar fiber terminal label was visualized around Purkinje cell soma and primary dendrites. The labeled olivocerebellar fiber axons were usually located just ventral to these labeled olivocerebellar fiber terminals. Analysis of the distribution of olivocerebellar fiber terminals in the staggerer cases indicated the presence of 10-11 distinct zones, which is slightly less than that reported in normal mice. In the next set of experiments, a small injection of horseradish peroxidase conjugated with wheat germ agglutinin was made into different mediolateral cerebellar regions. These results demonstrated that staggerer olivocerebellar fibers are entirely contralateral and are also organized topographically in a manner similar to the pattern seen in the normal animal. Thus, severe depletion and ectopia of staggerer Purkinje cells does not greatly alter olivocerebellar fiber organization.

Topographic and zonal organization of the olivocerebellar projection in the reeler mutant mouse.

The organization of the olivocerebellar projection in the homozygous reeler mouse (rl/rl) was studied with the use of microinjections of 3H-leucine in different regions of the inferior olivary complex (IO) or horseradish peroxidase conjugated with wheat germ agglutinin (WGA-HRP) into medial, intermediate, or lateral regions of the reeler cerebellum. The purpose of this investigation was to determine the pattern of termination of olivocerebellar climbing fibers (CFs) in the cerebellum via an anterograde tracing technique, and to determine the topographic organization of the olivocerebellar projection via both anterograde and retrograde methods. The inferior olive injections were made via the ventral (i.e., retropharygeal) approach to the IO to minimize diffusion into other brainstem precerebellar nuclei and thus to ensure accurate well-restricted, injection sites. Labeled CF terminals were seen in both the superficial Purkinje cell (PC) layer (normally positioned PCs) and around PCs in the granular layer and central masses (ectopic PCs). The pattern of labeling is suggestive of orthogonal organization, in that vertical columns of cells are labeled. This is especially apparent in the medial PC group, where at least three bands are identified. Within an orthogonal band, CF terminals are seen around both superficial and deep Purkinje cells. Our data indicate that olivocerebellar topography is generally similar in reeler and normal mice despite severe abnormalities in target cell position in the reeler. The medial cerebellar region receives input from the caudal two-fifths of the medial accessory olive (MAO). The intermediate PC cluster receives input from more rostral portions of all three olivary divisions (MAO, principal olive [PO] and dorsal accessory olive [DAO] ), while rostral portions of MAO and PO project to the lateral cerebellum. These results indicate that the zonal organization of the olivocerebellar projection in the adult reeler exhibits a pattern generally similar to that seen in normal mice. This suggests that an afferent system can develop a normal organization despite having ectopic targets.

A qualitative and quantitative light microscopic study of the inferior olivary complex of normal, reeler, and weaver mutant mice.

In the normal mouse (+/+; +/rl) cerebellar Purkinje cells (PCs) are aligned in a monolayer and provide the main targets for incoming olivocerebellar climbing fibers (CF). In the neurological mutants, homozygous reeler (rl/rl), homozygous weaver (wv/wv) and heterozygous weaver (wv/+), cerebellar abnormalities exist in which many PCs are either missing or displaced. Therefore, it is of interest of determine if the inferior olivary complex (IO) in these mutants is also abnormal. This report concerns results obtained from a light microscopic study of the inferior olivary complex. Counts of IO cells revealed apparent differences in the IO in homozygous reeler when compared to normal littermates. Whereas in the normal mouse there are approximately 37,000 IO cells and clearly defined olivary subdivisions, the IO of the homozygous reeler has a 22.6% reduction in IO cells (mean = 28,770) and indistinct borders between the major olivary subdivisions. With regard to the heterozygous and homozygous weaver, surprisingly the IO morphology and cell numbers are similar to that of the wildtype mouse even though the animals have only 86% (wv/+, mean = 158,155) and 72% (wv/wv, mean = 131,882), respectively, of the normal numbers of PCs (+/+, mean = 183,857). Purkinje cell counts revealed that the midline vermal region is the most affected area in the cerebellum in wv/+ and wv/wv whereas counts in the lateral hemisphere are near normal. The PC/IO ratio in the homozygous weaver is approximately 3:1 as compared to 5:1 in the wildtype mouse. Recent electrophysiological findings in wv/wv indicate that PCs are multiply innervated by CFs. Since a transient phase of multiple innervation is normal in the immature rat, the situation in the adult homozygous weaver may represent a retention of this immature state. A factor which may play a role in this is the loss of parallel fiber (PF)-PC synapses resulting from massive postnatal granule cell death. An hypothesis suggesting an intrinsic PC time-dependent mutant gene effect is presented to account for the differences in the loss of Purkinje cells between wv/wv and wv/+ and between different regions of the cerebellum.

A qualitative and quantitative light microscopic study of the inferior olivary complex in the adult staggerer mutant mouse.

In the homozygous staggerer (sg/sg) mutant mouse, most of the Purkinje cells (the primary targets for olivocerebellar climbing fibers) are missing or ectopic. In this study, the organization and cell number of the inferior olivary complex in sg/sg were determined and compared to the inferior olive of the wildtype (+/+) mouse. Our results indicate that there is a marked disorganization and loss of almost 60% of the cells within the inferior olivary complex. This suggests that the inferior olive may be secondarily affected by the staggerer gene due to the defects in its main target cell, the Purkinje cell.

The olivocerebellar projection to the uvula in the mouse.

The organization of the olivocerebellar projection in the mouse was studied with the use of microinjections of horseradish peroxidase (HRP) or HRP conjugated to wheat germ agglutinin (WGA-HRP). Injections were made in medial, intermediate, and lateral sites along the width of the uvula. The purpose of this investigation was to determine the subnuclear origin of olivary afferents to different mediolateral regions of the uvula. Injections made in or adjacent to the midline of the uvula resulted in the retrograde labeling of cells, bilaterally in the caudal portion of the medial accessory olive (MAO). These labeled cells were located primarily in subnucleus C and nucleus beta of the MAO. Injections into the intermediate part of the uvula resulted in the labeling of cells in the caudal MAO (primarily nucleus beta), the dorsomedial cell column (dmcc), and a few cells in the ventral lamella of the principal olive (vPO). Laterally placed injections produced labeling of cells in dmcc and the vPO. These results are discussed in reference to the parasagittal organization of olivary afferents to the cerebellar cortex in the mouse (Beyerl et al., '82) and the organization of afferents to the involved regions of the inferior olivary (IO) complex. It is suggested that these parasagittal zones in the uvula may play different roles in the control of eye movements.