Tunnel crossing fibers and their synaptic connections within the inner hair cell region in the organ of corti in the maturing mouse.

Combined ultrastructural and immunocytochemical studies reveal that in the adolescent 12- to 17-day-old mouse the afferent tunnel crossing fibers that innervate outer hair cells receive synaptic contacts from three distinct sources: the GABAergic fibers (GABA = gamma-aminobutyric acid) of the lateral olivocochlear bundle, the non-GABAergic efferent tunnel crossing fibers, and the inner hair cells themselves. The GABAergic fibers give off collaterals that synapse with the afferent tunnel fibers as they cross the inner hair cell region. These collaterals also form synapses with afferent radial dendrites that are synaptically engaged with the inner hair cells. Vesiculated varicosities of non-GABAergic efferent tunnel fibers also synapse upon the outer spiral afferents. Most of this synaptic activity occurs within the inner pillar bundle. Distinctive for this region are synaptic aggregations in which several neuronal elements and inner hair cells are sequentially interconnected. Finally, most unexpected were the afferent ribbon synapses that inner hair cells-formed en passant on the shafts of the apparent afferent tunnel fibers. The findings indicate that: (1) the afferent tunnel (i.e., outer spiral) fibers may be postsynaptic to both the inner and the outer hair cells; (2) the non-GABAergic efferent and the afferent tunnel fibers form extensive synaptic connections before exiting the inner pillar bundle; (3) the GABAergic component of the lateral olivocochlear system modulates synaptically both radial and outer spiral afferents.

Compound synapses within the GABAergic innervation of the auditory inner hair cells in the adolescent mouse.

Ultrastructural investigation of the gamma-aminobutyric acid (GABA) component of the inner spiral bundle in adolescent mice revealed a pathway of glutamic acid decarboxylase (GAD)-positive and -negative fibers and vesiculated endings that contact inner hair cells and their afferents through a complex of axosomatic and axodendritic synapses. Ultrastructural details were investigated by using conventional electron microscopy. Several synaptic arrangements were observed: Main axosomatic synapses form between vesiculated endings and individual or adjoining inner hair cells (interreceptor synapses). Spinous synapses form on long, spinelike processes that protrude from inner hair cells to reach distant efferent endings. The efferent endings associate with inner hair cells and their synaptic afferents through compound synapses-serial, "converging," and triadic-otherwise characteristic of sensory relay nuclei. Serial synapses form by the sequential presynaptic alignment of the efferent-->receptor-->afferent components. Converging synapses result from the simultaneous apposition of a receptor ribbon synapse and a presynaptic efferent terminal on a recipient afferent dendrite. Triadic synapses comprise a vesiculated efferent ending in contact with an inner hair cell and with its synaptic afferent. Additionally, efferent endings may form simple axodendritic and axoaxonal synapses with GAD-negative vesiculated endings. The combination of different synaptic arrangements leads to short chains of compound synapses. It is assumed that these synaptic patterns seen in the adolescent mouse represent adult synaptology. The patterns of synaptic connectivity suggest an integrative role for the GABA/GAD lateral efferent system, and imply its involvement in the pre- and postsynaptic modulation of auditory signals.

The GABA/GAD innervation within the inner spiral bundle in the mouse cochlea.

Stains with antibodies to glutamic acid decarboxylase (GAD) and gamma-aminobutyric acid (GABA) in the cochlea of postnatal and adult mice reveal within the inner spiral bundle a distinctive neuronal plexus intimately associated with the inner hair cells. This innervation provides endings that cradle the receptor poles of the sensory cells and lateral end collaterals that wind between the cells, distributing endings alongside and around them. Some GAD-positive fibers enter the inner pillar bundle, from where they distribute tunnel fibers to the outer hair cells and recurrent collaterals to the inner hair cells. The GABAergic innervation within the inner spiral bundle is present along the entire cochlear axis, with the highest density in its basal half. Stainings against calcitonin gene-related peptide (CGRP), which localizes in the cholinergic counterpart of the inner spiral bundle, reveal that some of these fibers parallel the GABAergic circuit. The present data, together with our previous demonstration of compound (serial, converging, triadic) efferent synapses within this pathway (Sobkowicz et al. (1995) Abst. ARO 18, 171) evidences the presence of a distinctive innervation to the inner hair cells, hitherto unrecognized. The expression of growth-associated protein-43 (GAP-43) within the inner hair cell innervation in the adult cochlea provides evidence for a continuous synaptic turnover and plasticity, thus emphasizing its functional importance.

Global cerebral ischemia associated with cardiac arrest in the rat: I. Dynamics of early neuronal changes.

Light microscopic neuronal changes were studied in rats subjected to 10 min of global ischemia produced by compression of the major cardiac vessels. Observations of cresyl violet-stained sections revealed early changes involving predominantly GABAergic neurons in various locations. In rats killed 15 min after recirculation, the changes were characterized by the appearance of a clear peripheral zone with condensation of the remaining neuronal cytoplasm. After 1 h, these zones appeared to be compartmentalized into individual pearl-like vacuoles, especially prominent in the nucleus reticularis thalami. After 3 h, the cytoplasmic vacuoles disappeared and the neuronal changes, particularly in the cerebral cortex, striatum, hippocampus, and pars reticulata of the substantia nigra, consisted mainly of hyperchromasia or loss of Nissl substance. After 2 days, the cerebral cortex and thalamus contained occasional neurons with conspicuously large nucleoli. After 7 days, the hippocampus revealed an approximately 50% loss of CA1 pyramidal neurons, associated with intense microglial reactivity in the stratum radiatum, whereas the neuronal destruction was more complete in the nucleus reticularis thalami. Our observations suggest a possibility that early changes in GABAergic neurons may provide a period of neuronal disinhibition and thus contribute to an excitatory ischemic damage in regions connected by GABAergic circuitry.

Organization of the GABAergic system in the rat hippocampal formation: a quantitative immunocytochemical study.

Specific antibodies against gamma aminobutyric acid (GABA) and glutamic acid decarboxylase (GAD) were used to study the organization of the GABAergic system in the rat hippocampal formation. Both the number of GABA-like-immunoreactive (Li) somata and neuropil density were assessed in semithin sections. Cell counts revealed that approximately 11% of the hippocampal neuronal population showed GABA-Li within the various planes of section. Each layer in the hippocampal formation had a characteristic organization of GABA-Li elements. In Ammon's horn, 80-95% of the neuronal somata within the apical and basal dendritic regions were GABA-Li positive. Within the pyramidal cell layer 5-8% of the cells were GABA-Li in the CA1 to CA3 subfields of Ammon's horn and only 3% were GABA-Li within that portion of the pyramidal cell layer that inserts into the hilus. Only slight differences in the density of the GABA-Li neuropil were observed within the CA1-CA3 dendritic regions. Restricted to the stratum lucidum was a dense band of GABA-Li label. Counts of immunoreactive grains localized on the perimeter of pyramidal (CA1-CA3) and granule somata revealed more terminal boutons on the CA3 cells than on CA1 and granule neuronal somata. A topographical distribution of GABA-Li somata and neuropil was found in the fascia dentata: There the label particularly concerned its suprapyramidal and rostrolateral portions. Approximately 40% of neurons in the molecular layer, 60% in the polymorph layer, and 18% within the hilar region were GABA-Li. Within the granule cell layer only 2% of the neurons were GABA-Li positive. Distinct differences in the density of the GABA-Li neuropil were present in the molecular, pericellular granular, and hilar regions of the fascia dentata. While the morphology of GABA-Li neuronal somata varied according to their hippocampal layer, the most heterogeneous cell types were found in the regio inferior of the hippocampus. There we have identified neurons that are reminiscent of the inferior region interneuron described in Golgi material by Amaral and Woodward (Brain Res. 124:225-236, '77). Moreover, particularly in the sagittal plane, we have identified oval, triangular, and round cells and that have processes oriented in a parallel arrangement, appearing to be aligned along the granule cell mossy fibers.

Cholinergic-GABAergic synaptic interconnections in the rat amygdaloid complex: an electron microscopic double immunostaining study.

A correlated light and electron microscopic immunocytochemical study was performed to analyze 1) the distribution of cholinergic and GABAergic perikarya and terminals in the rat amygdala, and 2) the cholinergic innervation of GABAergic neurons in some amygdaloid nuclei. We will demonstrate here that cholinergic terminals establish synaptic contacts with GABAergic neurons in the basolateral amygdaloid region. These GABAergic neurons in turn are supposed to exert an inhibitory influence on the centromedial amygdaloid region. Our data suggest that the amygdaloid nuclei provide a useful model for studies of cholinergic-GABAergic synaptic interconnections in the CNS.

Organization and synaptic interconnections of GABAergic and cholinergic elements in the rat amygdaloid nuclei: single- and double-immunolabeling studies.

The aim of this study was to describe the localization of cholinergic and GABAergic neurons and terminals in the amygdaloid nuclei of the rat. Double immunolabeling was performed to study cholinergic-GABAergic synaptic interconnections. Cholinergic elements were labeled by using a monoclonal antibody to choline acetyltransferase (ChAT), the acetylcholine synthesizing enzyme. Antibodies against glutamate decarboxylase (GAD), the GABA- synthesizing enzyme, were employed to identify GABAergic perikarya and terminals. The tissue sites of the antibody bindings were detected by using either Sternberger's peroxidase-antiperoxidase (PAP) method or a biotinylated secondary antibody and avidinated ferritin. These two contrasting immunolabels allowed us to study GABAergic-cholinergic interconnections at the electron microscopic level. Our study revealed a characteristic distribution of GABAergic and cholinergic elements in the various amygdaloid nuclei: 1) Large, ChAT-immunopositive cells with heavily labeled dendrites were observed in the anterior amygdaloid area and in the lateral and medial zones of the central nucleus. These cells seem to constitute the intraamygdaloid extension of the magnocellular basal nucleus. Their dendrites invaded other amygdaloid nuclei, in particular the intercalated nuclei, the lateral olfactory tract nucleus, and the central zone of the central nucleus. These ChAT-immunoreactive dendrites formed synaptic contacts with GAD-positive terminals. GABAergic terminals probably thus exert an inhibitory amygdaloid influence onto cholinergic neurons of the magnocellular basal nucleus. 2) Two amygdaloid nuclei-the basal dorsal nucleus and the lateral olfactory tract nucleus-contained a dense network of ChAT-immunoreactive fibers and terminals, but they also contained numerous GAD-positive perikarya. Double-immunolabeling experiments revealed cholinergic terminals forming synaptic contacts on GAD-immunopositive cell bodies, dendritic shafts, and spines. 3) The central and medial nucleus seem to be the main target of GABAergic fibers to the amygdala. Both nuclei contained a dense plexus of GAD-immunoreactive terminals that may arise, at least in part, from the GABAergic neurons in the basal dorsal nucleus. Inhibition of the centromedial "excitatory" region through intraamygdaloid GABAergic connections may reduce excitatory amygdaloid influence onto hypothalamus and brainstem.

Distribution of GABA-like immunoreactivity in the rat amygdaloid complex.

The distribution of GABA-like (GABA-Li) immunoreactivity in the rat amygdaloid complex was studied by using an anti-GABA antibody. GABA-Li positive neurons and processes were present in every nucleus of the complex. Three patterns of immunoreactivity were revealed: (1) the intercalated masses and the lateral olfactory tract nucleus exhibited the most intense staining of the neuropil, and virtually every neuron was labeled, (2) the central and medial nuclei contained intensely labeled neuropil and moderately labeled neurons, and (3) in the remaining nuclei, the neuropil was weakly labeled, and relatively numerous GABA-Li neurons were present. Our results suggest that: (1) the intercalated masses and lateral olfactory tract nucleus consist of large aggregates of GABA-Li immunoreactive neurons, and (2) the lateral, basal dorsal, and the posterior cortical nuclei may constitute a significant source of GABAergic connections to other amygdaloid nuclei, in particular to the medial and central nuclei.

Maturation of kainic acid seizure-brain damage syndrome in the rat. I. Clinical, electrographic and metabolic observations.

The maturation of the seizure/brain damage syndrome produced by parenteral administration of kainate was studied in the rat. The motor, electrographic and metabolic alterations are described in the present report, the maturation of the pathological abnormalities and of the specific kainate binding sites are described in the two following companion papers. Parenteral kainate produces tonico-clonic seizures until the end of the third week of age when limbic motor signs (wet-dog shakes, facial myoclonia, paw tremor etc.) were first produced. Using the 2-deoxyglucose autoradiographic method, we found that in animals of 3 days of age and until the third week of age, kainate produced a rise in metabolism restricted to the hippocampus and lateral septum. This was paralleled by paroxysmal discharges which were recorded in the hippocampus. Starting from the end of the third week of age approximately--i.e. when the toxin produced limbic motor seizures--there was a rise of labelling in other structures which are part of or closely associated to the limbic system i.e. the amygdaloid complex, the mediodorsal and adjacent thalamic nuclei, piriform, entorhinal and rostral limbic cortices and areas of projection of the fornix. These metabolic maps are thus similar to those seen in adults. Two main conclusions can be drawn from these experiments: kainate activates the hippocampus from a very early age probably by means of specific receptors present in this structure and the limbic syndrome will only be produced by the toxin once the limbic circuitry--including in particular the amygdaloid complex--is activated by the procedure i.e. after the third week of age.

Maturation of kainic acid seizure-brain damage syndrome in the rat. III. Postnatal development of kainic acid binding sites in the limbic system.

The progressive appearance of [3H]kainic acid binding sites with age has been studied in membrane suspensions prepared from various regions of the rat limbic system, and by autoradiography. Binding sites with fast dissociation rate appeared earlier than binding sites with slow dissociation rate. Scatchard analysis demonstrated apparent receptor heterogeneity for both subclasses. High affinity components were detected in the hippocampus as early as 10 days after birth, but in the amygdala + piriform lobe were found only towards the end of the third week, when animals also respond to parenteral kainic acid, for the first time, with limbic seizures accompanied by metabolic activation of the amygdala. Slice autoradiography revealed distinct labelling of the hippocampal CA3 region by postnatal day 10. A comparison with the ontogenesis of the kainic acid-induced seizure-brain damage syndrome suggests a role of high affinity receptors as mediators of metabolic nerve cell activation by kainic acid. However, this receptor interaction per se does not result in neuronal damage to the vulnerable region of the Ammon's horn, which will only occur at an age when also the amygdala is activated by the neurotoxin.

Maturation of kainic acid seizure-brain damage syndrome in the rat. II. Histopathological sequelae.

The histopathological sequelae of parenteral administration of kainic acid were investigated in immature rats (3-35 days of age). The brains were fixed 1-14 days after the administration of kainate and the damage evaluated by means of argyrophylic (Fink-Heimer, Gallyas or Nauta-Gygax) and Nissl stains. In animals of less than 18 days of age there was no sign of damage even after 1-2 h of severe tonico-clonic convulsions. Between 18 and 35 days after birth, there was a progressive increase in the severity of the damage, the adult pattern being reached at the latter age. As in adult animals, brain damage was most severe in structures which are part of the limbic system, i.e. the hippocampal formation, lateral septum, amygdaloid complex, claustrum, piriform cortex, etc. In addition to neuronal abnormalities, the following reactions were observed: hypertrophy and swelling of satellite oligodendroglia, proliferation of hypertrophic microglia, proliferation of astroglia and hypertrophy of endothelial cells in the capillary wall. The latter type of change, together with local coagulative necrosis, was almost exclusively restricted to the granular and molecular layers of the fascia dentata. In the hippocampal formation we found a temporal gradient of vulnerability. The earliest and most consistent neuronal alterations were largely restricted to interneurons of the hilar region and to a lesser extent to non-pyramidal neurons of strata oriens and radiatum. The severe necrotic destruction of the pyramidal layer of CA3 is conspicuous at a later age (postnatal day 30-35) and with longer survival times. Our results suggest that: (1) the neurotoxin only induces brain damage once it also causes limbic motor seizures and its associated metabolic activations, notably in the amygdala; (2) the earliest pathological sequelae occur in interneurons of the hilar region and (3) sclerosis of the vulnerable region of the Ammon's horn--the CA3 region--is only obtained once the dentate granules and their mossy fibres are fully operational, thereby reflecting the crucial role of this axonal connection in eliciting hippocampal damage.

A multidisciplinary study of folic acid neurotoxicity: interactions with kainate binding sites and relevance to the aetiology of epilepsy.

Folic acid has been injected unilaterally into the amygdaloid complex of awake chronically implanted rats, or in rats under anaesthesia. Clinical, electrographic, and metabolic changes (estimated by means of the 2-deoxyglucose method) have been studied in relation to subsequently demonstrated neuropathology using Fink-Heimer and Nissl stains. The observations are compared to the corresponding effects of intra-amygdaloid application of kainic acid. Major differences were noted between the folate and the kainate induced seizure/brain damage syndrome. Thus: folate produced essentially stereotypies, alternating with myoclonic unilateral jerks of head and limbs. In contrast, limbic motor seizures which are characteristically produced by kainic acid, were extremely rare. Folate did not produce the preferential and sequential electrographic activation of limbic structures as observed after kainate. 2-Deoxyglucose autoradiography revealed an enhanced metabolic activity in the injected amygdala and in the overlying piriform and entorhinal cortices. The most conspicuous rise in labelling, however, occurred in the entire fronto-parietal cortex (ipsilaterally) up to the cingulate region, as well as in the ventral thalamic complex and the globus pallidus, i.e. in structures which are not labelled after kainate treatment. Some extent of local damage was observed 1-8 days after the injection; distant from the injection site, we found massive anoxic-ischemic type of damage in the superficial layers of the fronto-parietal cortex, a complete necrosis of the piriform lobe, and neuronal cell loss in the ventral thalamus and several extrapyramidal structures. The full range of limbic damage associated with kainate was never produced by folate. The CA3 region of the hippocampus, most susceptible to kainate, was only mildly affected by folate. These differences between kainate and folate prompted us to re-evaluate the recently reported high affinity of folates for kainic acid membrane binding sites. We found that folic acid competed only very weakly with [3H]kainic acid for binding sites on striatal, cortical, hippocampal, amygdaloid, and cerebellar membranes. It is thus concluded, that folate is not a good candidate for an endogenous kainate-like substance. We propose intracerebral injections of folic acid as a useful tool to study the vulnerability of brain structures to anoxic-ischemic conditions.

Kainic acid seizure syndrome and binding sites in developing rats.

In rats up to 16 days after birth, parenteral kainic acid (KA) produced tonico-clonic convulsions, metabolic activation limited to the hippocampus, and no brain damage. Starting with the 19th day after birth, KA produced limbic seizures associated with metabolic activation and subsequent damage in the hippocampus, the amygdala, and other limbic structures. Membranes prepared from hippocampi 10 days after birth bound [3H]KA with a high affinity component, which was localized in the mossy fiber region by slice autoradiography. In contrast, on amygdaloid membranes this component appeared only 17-19 days after birth. Our results further stress the crucial role of the amygdala in the KA seizure syndrome.

The different types of synapses in the thalamic nucleus ventralis anterior (VA) of Saimiri sciureus and their degeneration after pallidum coagulation.

In the squirrel monkey (Saimiri sciureus) the nucleus VA or lateropolaris (L. po), occupying the rostro-lateral pole of the thalamus, contains 7 principal types of synapses, two of which have axo-spinous variants, i.e., type I LR (spi) and type V SO (spi). The synapses of type I LR (Large boutons, Round to ovoid vesicles) and type II EF (Encapsulated bouton, elongated to Flat vesicles) contain large vesicles sometimes attached by neurofilaments. Because the type IV MR (Medium size, Round vesicles) synapses have virtually the same kind of axo-dendritic synaptic contacts as type I, either with or without aggregations of presynaptic vesicles, they belong to the same category of afferent boutons, differing only in size and in the number of mitochondria. The small axo-dendritic type III SR synapses (Small boutons, Round vesicles) are densely packed with vesicles having a relatively dense center, and undergo markedly asymmetric contacts with dendrites. The type V SO (Small bouton, Ovoid vesicles) synapses, the most frequent type of synapses, have larger pale ovoid vesicles in a bell-shaped bouton that is in asymmetric contact with a small dendrite. The subtype V St, which likewise contains pale ovoid vesicles has a stiletto-shaped rather than bell-shaped bouton which undergoes an asymmetric contact. In parallel to the other ventro-oral (or VL) nuclei, the nucleus VA possesses two types of presynaptic dendrites: type VI DE (Dendritic terminal with sparse, Evenly distributed vesicles) suspended among the neurofilaments; and type VII DC (Dendritic terminal in which many ovoid vesicles are Clustered). In the type VI junctions, the synaptic membrane specializations are difficult to detect. After circumscribed pallidum externum lesions in the squirrel monkey, many type I LR and type IV MR synapses, and a lesser number of type II EF synapses, undergo dark degeneration. In contrast, after such lesions the type VI DE and type VII DC dendritic terminals never show signs of degeneration. Also the bell-shaped type V SO terminal occasionally undergoes degeneration with darkening of the axoplasm; the subtype V St terminal, however, does not. Thus, it may be assumed that the type I LR, type IV MR, type II EF and type V SO boutons represent terminals of direct neuronal connections between the pallidum and thalamic nucleus VA.

Interamygdaloid connections in the rat studied by the horseradish peroxidase method.

Neurons of the rat amygdaloid body were labeled with horseradish peroxidase following its injection into contralateral nuclei of the amygdala. The results strongly suggest that there is a contralateral amygdaloid projection from the basal (dorsal and ventral) nuclei of amygdala; it terminates in the medial, central and lateral nucleus. True commissural connections were found only between posterior parts of the cortical nuclei of amygdala and between homonymous areas of the piriform cortex.