Electron microscopic study of GABA-immunoreactive neuronal processes in the superficial gray layer of the rat superior colliculus: their relationships with degenerating retinal nerve endings

Summary GABA-immunoreactive neuronal elements were detected in the stratum griseum superficiale or superficial gray layer of the rat superior colliculus in an electron microscopic study, using postembedding immunocytochemistry with protein A-gold as a marker. In addition to neuronal somata, two types of GABA-immunoreactive neuronal processes were observed. Numerous profiles of axon terminals (1 m in diameter) with clear round or pleomorphic synaptic vesicles and mitochondria were found to establish mostly symmetrical synaptic contacts with GABA-immunonegative dendrites of various diameters. Some axosomatic synapses could also be observed. The gold particle density in this axon terminal compartment was between seven and 13 times the background level. The stratum griseum superficiale also included GABA-immunoreactive dendrites, some of which contained clear synaptic vesicles. These dendritic profiles always formed the presynaptic component of dendrodendritic synaptic contacts. The density of the gold particles in the dendritic compartment, taken as a whole, was between three and 13 times the background level. Furthermore, the relationship between the GABA-immunoreactive neuronal elements and degenerating retinal nerve endings identified in the left stratum griseum superficiale following enucleation of the right eye was investigated after a 7-day survival period. The profiles of degenerating retinal nerve endings (0.7 m in diameter) were found to be devoid of any specific labelling. Most of the retinal boutons established axodendritic synapses of the asymmetrical type with an immunonegative dendrite, which was also contacted in some cases by a GABA-immunopositive axon terminal. Other retinal endings were presynaptic to GABA-immunopositive dendritic profiles with synaptic vesicles, some of which were found to contact in turn an unlabelled dendrite, thereby completing serial synaptic relationships. More rarely, retinal endings formed the presynaptic component of possible axoaxonic synapses with GABA-positive terminals presumed to be axonic in nature. It can be concluded that the retinal input to the superficial gray layer often converges with a GABAergic axonal input on a dendritic target, the neurotransmitter specificity of which is unknown. In other cases, retinal terminals synaptically contact GABA-immunolabelled conventional and presynaptic dendrites and probably also some axon terminals; this might provide an anatomical substrate for the control of GABA release from these GABAergic processes. These results indicate that transmitter GABA plays an important role in retinocollicular transmission.     

Changes in GABA-immunoreactive cell density during motor focal epilepsy induced by cobalt in the rat

Summary The distribution of GABA-immunoreactive cell bodies and terminals was studied using an anti-GABA serum during the development of chronic focal epilepsy induced by cobalt deposits onto the motor cortex of the rat. Cell counts of GABA-positive neurons were carried out in the epileptogenic area and correlated with the electrophysiological activity of the cobalt focus. In normal control rats, we identified GABA-immunoreactive somata and processes in the motor agranular cortex; they were multipolar or bipolar but never pyramidal and were present in all layers, especially in layer II. GABA-immunoreactive terminals were widely scattered in the neuropil and surrounded the unlabelled cell bodies. In the cobalt-treated animals, changes in the GABAergic innervation were observed during the development of the epileptic focus: decreases in the GABA-positive cell density and in the number of GABA-positive terminals were present before the onset of epileptic discharges and became more marked during the period of maximal spiking activity; a progressive return to normal values of GABA-positive cell density (except in the deep layers) as well as the reappearance of GABA positive terminals were associated with the extinction of the epileptic syndrome. Our observations suggest that the impaired inhibitory neurotransmission mediated by GABA plays a role in the development of the cobalt-induced epilepsy; moreover the recovery of GABAergic function which occurs during the extinction of the epileptic syndrome might imply a capacity for axonal regeneration of the GABAergic neurons.     

Effects of diffuse axonal injury on speed of information processing following severe traumatic brain injury

To test the hypothesis that slowed information processing in traumatic brain injury is related to diffuse axonal injury (DAI), the authors compared 10 patients with predominant DAI (diffuse group) and minimal DAI (mixed injury group) on the Symbol Digit Modalities Test, simple and choice reaction time, Trail Making Tests A and B, and the Stroop Neuropsychological Screening Test. The diffuse group was slower than the mixed injury and control groups on basic speed of processing tasks. This difference was not apparent on complex speeded tasks once basic speed of processing was controlled for. The diffuse group's slower speed of processing was not accounted for by differences in injury severity, age, or time postinjury. The diffuse group showed greater recovery over time.     

Role of GABA in the extraocular motor nuclei of the cat: a postembedding immunocytochemical study.

The GABAergic innervation of the extraocular motor nuclei in the cat was evaluated using postembedding immunocytochemical techniques. The characterization of GABA-immunoreactive terminals in the oculomotor nucleus was carried out at the light and electron microscopic levels. GABA-immunopositive puncta suggestive of boutons were abundant in semithin sections throughout the oculomotor nucleus, and were found in close apposition to somata and dendrites. Ultrathin sections revealed an extensive and dense distribution of GABA-immunoreactive synaptic endings that established contacts with the perikarya and proximal dendrites of motoneurons and were also abundant in the surrounding neuropil. GABAergic boutons were characterized by the presence of numerous mitochondria, pleiomorphic vesicles and multiple small symmetrical synaptic contacts. The trochlear nucleus exhibited the highest density of GABAergic terminations. In contrast, scarce GABA immunostaining was associated with the motoneurons and internuclear neurons of the abducens nucleus. In order to further elucidate the role of this neurotransmitter in the oculomotor system, retrograde tracing of horseradish peroxidase was used in combination with the GABA immunostaining. First, medial rectus motoneurons were identified following horseradish peroxidase injection into the corresponding muscle. This was carried out because of the peculiar afferent organization of medial rectus motoneurons that contrasts with the remaining extraocular motoneurons, especially their lack of direct vestibular inhibition. Semithin sections of the oculomotor nucleus containing retrogradely labeled medial rectus motoneurons and immunostained for GABA revealed numerous immunoreactive puncta in close apposition to horseradish peroxidase-labeled somata and in the surrounding neuropil. At the ultrastructural level, GABAergic terminals established synaptic contacts with the somata and proximal dendrites of medial rectus motoneurons. Their features and density were similar to those found in the remaining motoneuronal subgroups of the oculomotor nucleus. Second, oculomotor internuclear neurons were identified following the injection of horseradish peroxidase into the abducens nucleus to determine whether they could give rise to GABAergic terminations in the abducens nucleus. About 20% of the oculomotor internuclear neurons were doubly labeled by retrograde horseradish peroxidase and GABA immunostaining. A high percentage (80%) of the oculomotor internuclear neurons projecting to the abducens nucleus showed immunonegative perikarya. It was concluded that the oculomotor internuclear pathway to the abducens nucleus comprises both GABAergic and non-GABAergic neurons and, at least in part, the GABA input to the abducens nucleus originates from this source. It is suggested that this pathway might carry excitatory and inhibitory influences on abducens neurons arising bilaterally.     

Structure and function of gustatory neurons in the nucleus of the solitary tract. IV. The morphology and synaptology of GABA-immunoreactive terminals.

In the visual, auditory and somatosensory systems, insight into the synaptic arrangements of specific types of neurons has proven useful in understanding how sensory processing within that system occurs. The neurotransmitter GABA is present in the nucleus of the solitary tract and based on the fact that the vast majority of cells respond to GABA, its agonists and antagonists, and that over 45% of synaptic terminals in the rostral subdivision of the nucleus of the solitary tract are GABA-immunoreactive, GABA is thought to play an important role in gustatory processing. The following study was carried out to establish the distribution of GABA-immunoreactive terminals within the nucleus of the solitary tract. Specifically, the distribution on to physiologically-identified gustatory neurons was determined using post-embedding electron immuno-histochemistry. GABA-immunoreactive terminals synapse with gustatory neuronal somata and all portions of their dendrites, but non-GABAergic terminals synapse only with distal dendrites of the gustatory cells and on to correspondingly small unidentified dendritic profiles in the neuropil. There is a differential distribution of two subtypes of GABA-immunoreactive terminals on to proximal and distal portions of the gustatory neurons as well. Finally, a model for the synaptic arrangements involving gustatory and GABAergic neurons is proposed.     

Anatomical distribution and ultrastructural organization of the gabaergic system in the rat spinal cord. An immunocytochemical study using anti-GABA antibodies

γ-Aminobutyric acid (GABA)-containing elements have been studied by light and electron microscopy in the rat spinal cord, using immunocytochemistry with anti-GABA antibodies. Light microscopy showed immunoreactive somata localized principally in laminae I–III, and occasionally in the deeper laminae of the dorsal horn and in the ventral horn. Small somata were also observed around the central canal. Punctate GABA-immunoreactive profiles were particularly concentrated in laminae I–III, and moderately abundant in the deeper laminae and in the ventral horn where they were observed surrounding the unlabelled motoneurons.

At the ultrastructural level, the punctate profiles corresponded to GABA-containing axonal varicosities or small dendrites. GABA-immunoreactive varicosities were presynaptic to labelled or unlabelled dendrites and cell bodies. Some unlabelled terminals presynaptic to unlabelled dendrites received symmetrical synaptic contacts from GABA-immunoreactive terminals.

These results confirm data obtained withl-glutamate decar☐ylase immunocytochemistry, and support the role of GABA in pre- and postsynaptic inhibition in the spinal cord, respectively via axoaxonal and axosomatic or axodendritic synapses.     

Magnetic resonance identified ventricular dilation in traumatic brain injury: comparison of pre- and postin jury scan and postin jury results.

A case study is presented in which a patient received magnetic resonance (MR) imaging of the brain 3 months prior to a severe traumatic brain injury (TBI). The post-TBI MR findings are compared and contrasted with the pre-TBI MR images. The posttraumatic changes demonstrate a significant dilation of the ventricular system which reflects diffuse axonal injury and loss of brain substance. Correspondingly, the neuropsychological studies in this individual reflect global deficits which match the nonspecific, traumatically induced degenerative changes found in the postinjury MR scan. This case study is unique in that specific preinjury MR findings are available for direct comparison and quantitative analysis of TBI-associated changes in brain structure with neuropsychological outcome.     

Traumatically induced axonal injury: pathogenesis and pathobiological implications.

This work reviews the pathobiology of traumatically induced axonal injury. Drawing upon literature gleaned from the experimental and clinical setting, this review attempts to emphasize that, other than the most destructive insults, traumatic brain injury does not typically cause direct mechanical disruption of the axon. Rather, this review documents that with traumatic injury focal, subtle axonal change occurs, and that over time, such change leads to impaired axoplasmic transport, continued axonal swelling, and ultimate disconnection. The initial intra-axonal events that trigger the above described sequence of reactive axonal change are considered with focus on the possibility of either traumatically altered axolemmal permeability, direct cytoskeletal damage/perturbation, or more overt metabolic/functional disturbances. Not only does this review focus on the sequence of traumatically induced axonal change, but also, it considers its attendant consequences in terms of Wallerian degeneration and subsequent deafferentation. The concept that traumatically induced diffuse axonal injury leads to diffuse deafferentation is emphasized together with its pathobiological implications for morbidity and recovery. The potential for either adaptive or maladaptive neuroplasticity subsequent to such diffuse deafferentation is considered in the context of mild, moderate and severe traumatic brain injury.     

Axonal injury following mild traumatic brain injury is exacerbated by repetitive insult and is linked to the delayed attenuation of NeuN expression without concomitant neuronal death in the mouse

Mild traumatic brain injury (mTBI) affects brain structure and function and can lead to persistent abnormalities. Repetitive mTBI exacerbates the acute phase response to injury. Nonetheless, its long‐term implications remain poorly understood, particularly in the context of traumatic axonal injury (TAI), a player in TBI morbidity via axonal disconnection, synaptic loss and retrograde neuronal perturbation. In contrast to the examination of these processes in the acute phase of injury, the chronic‐phase burden of TAI and/or its implications for retrograde neuronal perturbation or death have received little consideration. To critically assess this issue, murine neocortical tissue was investigated at acute (24‐h postinjury, 24hpi) and chronic time points (28‐days postinjury, 28dpi) after singular or repetitive mTBI induced by central fluid percussion injury (cFPI). Neurons were immunofluorescently labeled for NeuroTrace and NeuN (all neurons), p‐c‐Jun (axotomized neurons) and DRAQ5 (cell nuclei), imaged in 3D and quantified in automated manner. Single mTBI produced axotomy in 10% of neurons at 24hpi and the percentage increased after repetitive injury. The fraction of p‐c‐Jun+ neurons decreased at 28dpi but without neuronal loss (NeuroTrace), suggesting their reorganization and/or repair following TAI. In contrast, NeuN+ neurons decreased with repetitive injury at 24hpi while the corresponding fraction of NeuroTrace+ neurons decreased over 28dpi. Attenuated NeuN expression was linked exclusively to non‐axotomized neurons at 24hpi which extended to the axotomized at 28dpi, revealing a delayed response of the axotomized neurons. Collectively, we demonstrate an increased burden of TAI after repetitive mTBI, which is most striking in the acute phase response to the injury. Our finding of widespread axotomy in large fields of intact neurons contradicts the notion that repetitive mTBI elicits progressive neuronal death, rather, emphasizing the importance of axotomy‐mediated change.     

Morphological heterogeneity of the GABAergic network in the suprachiasmatic nucleus, the brain's circadian pacemaker

GABA (gamma-amino-butyric acid) is the predominant neurotransmitter in the mammalian suprachiasmatic nucleus (SCN), with a central role in circadian time-keeping. We therefore undertook an ultrastructural analysis of the GABA-containing innervation in the SCN of mice and rats using immunoperoxidase and immunogold procedures. GABA-immunoreactive (GABA-ir) neurons were identified by use of anti-GABA and anti-GAD (glutamic acid decarboxylase) antisera. The relationship between GABA-ir elements and the most prominent peptidergic neurons in the SCN, containing vasopressin-neurophysin (VP-NP) or vasoactive intestinal polypeptide (VIP), was also studied. Within any given field in the SCN, approximately 40–70% of the neuronal profiles were GABA-ir. In GABA-ir somata, immunogold particles were prominent over mitochondria, sparse over cytoplasm, and scattered as aggregates over nucleoplasm. In axonal boutons, gold particles were concentrated over electron-lucent synaptic vesicles (diameter 40–60 nm) and mitochondria, and in some instances over dense-cored vesicles (DCVs, diameter 90–110 nm). GABA-ir boutons formed either symmetric or asymmetric synaptic contacts with somata, dendritic shafts and spines, and occasionally with other terminals (axo-axonic). Homologous or autaptic connections (GABA on GABA, or GAD on GAD) were common. Although GABA appeared to predominate in most neuronal profiles, colocalisation of GABA within neurons that were predominantly neuropeptide-containing was also evident. About 66% of the VIP-containing boutons and 32% of the vasopressinergic boutons contained GABA. The dense and complex GABAergic network that pervades the SCN is therefore comprised of multiple neuronal phenotypes containing GABA, including a wide variety of axonal boutons that impinge on heterologous and homologous postsynaptic sites.     

Alterations in excitatory and GABAergic inhibitory connections in hippocampal transplants

Solid pieces of embryonic hippocampal tissue were implanted in a cavity formed by aspiration of the fimbria-fornix and the overlying cingulate cortex in adult rats. Six to 8 months after the transplantation, chronic recording electrodes were implanted into the graft and the host hippocampi for the recording of electroencephalogram and unit activity in the freely moving animal. Irregularly occurring sharp waves or electroencephalogram spikes and concurrent synchronous discharge of large groups of neurons dominated the electrical activity of the grafts, in contrast to the situation in normal animals. Light microscopy and GABA immunocytochemistry in the grafts revealed that the three major cell types of the hippocampal formation, i.e. pyramidal neurons, dentate granule cells and GABA-immunoreactive interneurons were present in the hippocampal grafts. At the ultrastructural level, however, significant alterations in connectivity were observed. The most striking finding was the absence or sparse occurrence of synapses on the axon initial segments of pyramidal neurons. The axon initial segments are normally densely covered by GABAergic synapses derived from a specialized type of interneuron, the chandelier or axo-axonic cell. On the other hand, numerous GABA-immunoreactive terminals were found in synaptic contact with somata of pyramidal neurons, suggesting that other types of GABAergic interneurons and their efferent connections may have developed in a normal manner. The cell bodies of pyramidal neurons received, in addition, several asymmetric synapses from GABA-negative terminals. These presumably excitatory synapses are not present on the somata of pyramidal cells in the normally developing hippocampus. We hypothesize that the somatic excitatory synapses originate, at least in part, from the axon collaterals of the neighbouring pyramidal cells in the graft. We suggest that the hyperexcitability of the neuronal circuitry within the graft is due to reduced inhibition (lack of axo-axonic synapses) coupled with increased collateral excitation of the pyramidal neurons.     

Ultrastructural evidence for enkephalin mediated disinhibition in the ventromedial nucleus of the hypothalamus

The ventromedial nucleus of the hypothalamus (VMN) regulates the estrogen-dependent appearance of female mating behavior, lordosis. Accumulating evidence suggests that estrogen might exert its control over lordosis by acting, in part, on neurons that contain enkephalin in the VMN. The expression of the enkephalin precursor gene is robustly stimulated by estrogen and is correlated with the later appearance of lordosis. GABA has also been implicated as an important neurotransmitter for the appearance of lordosis. Because enkephalin is thought to act in several brain areas to modulate the activity of GABAergic neurons, we studied the ultrastructural morphology and relationship between neurons containing these neurochemicals using dual-labeling immunocytochemistry in ovariectornized rats, half of which received estrogen replacement. Immunolabeling for enkephalin was almost always detected within axon terminals (695 axonal profiles sampled), while GABA immunoreactivity was more often localized to cell bodies and dendrites (191 profiles), than to axons (63 profiles). Axon terminals containing enkephalin immunolabeling provided a major innervation to soma or dendrites containing GABA. That is, over one third (94/245) of the axon terminals in contact with GABA-immunoreactive dendrites contained enkephalin. Furthermore, these GABA-immunoreactive dendrites accounted for a fifth of the somatodendritic processes associated with enkephalin-containing axon terminals. These findings support the hypothesis that enkephalin may act in the VMN by inhibiting GABAergic neurons, which could result in the disinhibition of neural circuits relevant for lordosis.     

Ultrastructural features of non-commissural GABAergic neurons in the medial vestibular nucleus of the monkey.

The ultrastructural characteristics of non-degenerating GABAergic neurons in rostrolateral medial vestibular nucleus were identified in monkeys following midline transection of vestibular commissural fibers. In the previous papers, we reported that most degenerated cells and terminals in this tissue were located in rostrolateral medial vestibular nucleus, and that many of these neurons were GABA-immunoreactive. In the present study, we examined the ultrastructural features of the remaining neuronal elements in this medial vestibular nucleus region, in order to identify and characterize the GABAergic cells that are not directly involved in the vestibular commissural pathway related to the velocity storage mechanism. Such cells are primarily small, with centrally-placed nuclei. Axosomatic synapses are concentrated on polar regions of the somata. The proximal dendrites of GABAergic cells are surrounded by boutons, although distal dendrites receive only occasional synaptic contacts. Two types of non-degenerated GABAergic boutons are distinguished. Type A terminals are large, with very densely-packed spherical synaptic vesicles and clusters of large, irregularly-shaped mitochondria with wide matrix spaces. Such boutons form symmetric synapses, primarily with small GABAergic and non-GABAergic dendrites. Type B terminals are smaller and contain a moderate density of round/pleomorphic vesicles, numerous small round or tubular mitochondria, cisterns and vacuoles. These boutons serve both pre- and postsynaptic roles in symmetric contacts with non-GABAergic axon terminals. On the basis of ultrastructural observations of immunostained tissue, we conclude that at least two types of GABAergic neurons are present in the rostrolateral portion of the monkey medial vestibular nucleus: neurons related to the velocity storage pathway, and a class of vestibular interneurons. A multiplicity of GABAergic bouton types are also observed, and categorized on the basis of subcellular morphology. We hypothesize that "Type A" boutons correspond to Purkinje cell afferents in rostrolateral medial vestibular nucleus, "Type B" terminals represent the axons of GABAergic medial vestibular nucleus interneurons, and "Type C" boutons take origin from vestibular commissural neurons of the velocity storage pathway.     

GABAergic structures in the ventral part of the oral pontine reticular nucleus: An ultrastructural immunogold analysis

GABA mediates inhibitory effects in neurons of the ventral part of the oral pontine reticular nucleus (vRPO). Evidence increasingly suggests that GABA plays an important role in the modulation of rapid eye movement (REM) sleep generation in the cat vRPO. Here, we investigate the anatomical substrate of this modulation using GABA immunocytochemistry. Immunoperoxidase labeling revealed a few small GABA-immunoreactive cell bodies scattered throughout the vRPO. The numerical densities of all vRPO synapses and the GABA-immunoreactive synapses were estimated, at the electron microscopical level, by using a combination of the physical disector and the post-embedding immunogold techniques. We estimated that 30% of all vRPO synaptic terminals were immunoreactive to GABA. Our findings support the hypothesis that vRPO neuron activity is significantly controlled by inhibitory GABAergic terminals that directly target somata and the different parts of the dendritic tree, including distal regions. GABAergic input could inhibit vRPO REM sleep-inducing neurons during other states of the sleep-wakefulness cycle such as wakefulness or non-REM sleep.     

Role of Excitatory Amino Acid-Mediated Ionic Fluxes in Traumatic Brain Injury

One major event taking place at the moment of traumatic brain injury in neuronal cells is the occurrence of massive ionic fluxes across the plasma membrane, which can be referred to as traumatic depolarization (TD). Unlike spreading depression, TD can occur over wide brain areas simultaneously. Furthermore, recovery from TD often takes far longer than recovery from ionic perturbation elicited by the passage of a single wave of spreading depression. Neuronal cell damage caused by ischemic brain injury is also initiated by massive ionic fluxes, termed anoxic depolarization. The occurrence of similar ionic events in these two forms of brain injury may account for the genesis of diffuse ischemia-like damage without actual episodes of hypoxia or ischemia in traumatic brain injury. We review the data indicating that excitatory amino acids (EAA) may play a vital role in producing TD, and that such EAA-mediated ionic perturbation is responsible for a number of posttraumatic events including subcellular metabolic dysfunction and cellular responses such as microglial activation and astrocytic transformation. TD may represent one of the most important mechanisms of diffuse neuronal cell dysfunction and damage associated with traumatic brain injury.     

Perisomatic changes in the maturing hypoglossal nucleus after axon injury

Summary The perisomatic retrograde reactions to peripheral nerve injury during postnatal maturation were investigated by quantitative light and electron microscopy. The hypoglossal nerve was crushed, ligated or transected in 10 and 21 day postnatal (dpn) rats. Only crush injury was made in 7 dpn rats. Survival periods ranged from 3 to 40 days postoperative (dpo). Normal and sham operated animals of corresponding ages served as controls. A remarkable, transient response of neuronal somata in the young (7–10 dpn) rats, following all three types of axon injury, was the formation of excrescences which engulfed neuronal processes: boutons, dendrites, small neurites and a few myelinated axons in adjacent neuropil. The somal engulfment was rarely evident after nerve injury to 21 dpn rats. Bouton displacement from the somal surface, accompanied by glial incursion, followed each type of nerve injury but was less extensive and occurred later in the young rats. There seemed to be no association in the amount of boutons displaced from neuronal somata with the type of nerve injury for any of the three experimental age groups. However, the rate and intensity of the perineuronal glial reaction were related to the severity of the nerve injury in the older (21 dpn) but not in the younger (7–10 dpn) rats. Substantial loss of neurons occurred in the affected nucleus after each type of trauma to the young neurons. The degree of neuronal loss was related to the age and the volume of axoplasm disrupted. Only nerve transection in 21 dpn rats resulted in appreciable neuronal loss. The responses of axotomized neurons, their afferent axon terminals and the surrounding glia are quantitatively and qualitatively different in the hypoglossal nucleus of rats of increasing postnatal ages.     

GABAergic neurons in the cerebral cortex of the brain of a lizard (Podarcis hispanica)

To check the occurrence of GABAergic neurons in a reptilian species, sections from brains of the lizard Podarcis hispanica were immunoreacted with an antibody to GABA. GABA-immunoreactive somata were found predominantly in the external and internal plexiform layers, some also in the granular layer of the medial, dorsomedial and dorsal cortices. GABAergic boutons were also found in the 3 layers; however, their densest distribution occurred in the granular layer and in the outer zone of the external plexiform layer. The distribution of GABAergic somata and boutons supports the suggestion that some areas of the cerebral cortex of reptiles may be homologous to parts of the hippocampus of the mammalian brain.