Stratum Moleculare Research Articles

Histophotometric evaluation of glutamate dehydrogenase activity of the rat hippocampal formation during postnatal development, with special reference to the glutamate transmitter metabolism.

Transmitter glutamate/aspartate synthesis is known to proceed along different metabolic pathways. In this light, the functional relevance of glutamate dehydrogenase in postnatally maturing glutamatergic/aspartatergic structures was studied by means of quantitative enzyme histochemistry. The basic requirements concerning the kinetics and calibration of the histochemical glutamate dehydrogenase reaction used were proved to be met in order to obtain valid quantitative data. The histochemically demonstrable activity of glutamate dehydrogenase (EC 1.4.1.3) in the hippocampal formation of the rat increased markedly during postnatal development. On day 30, the distribution pattern observed was similar to that in adult animals. While the enzyme activity rose within cell body layers from day 0 to day 30 by 240-285%, the increase in neuropil layers was found to be up to 830%. Maximum values were seen in the stratum lacunosum-moleculare of CA1 and CA3 and the stratum moleculare of the dentate fascia on day 30. Since the hippocampal neuropil is supposed to be copiously provided with glutamatergic (and aspartatergic?) structures which become functional in rats during the first weeks of postnatal life, the increase in enzyme activity is discussed to be primarily a consequence of maturing synaptic systems using glutamate and/or aspartate as transmitters.

Neuropeptide Y innervation of the hippocampal region in the rat and monkey brain.

Using antibodies to neuropeptide Y (NPY) in combination with immunohistochemical techniques we have studied the distribution of cell bodies and nerve terminals containing NPY immunoreactivity (-i) in the hippocampal region of rats and monkeys (cynomolgus). In colchicine-pretreated rats a large number of NPY-positive cells are present in all areas of the hippocampal region. The NPY-i cells range in size from small (diameter across soma: 10-15 micron) to large (approximately 20 micron). Most of the NPY-i cells are situated in the hilus, in the subgranular zone of the area dentata, and in the stratum oriens of Ammon's horn. A majority of these are polymorphic cells but cells of different morphology are present in these layers as well. These include small spheroid cells and dentate pyramidal basket cells that are distinct from the polymorphic cells in the subgranular zone. The subicular complex (e.g., the subiculum, pre-, and parasubiculum) and the entorhinal area contain fewer NPY-i cells than the rest of the hippocampal region. In the dorsal parts of the pre- and parasubiculum numerous small cells are scattered throughout all layers, while in the entorhinal area the NPY-stained cells are situated primarily in the deep layers (V and VI). In the ventral part of the lateral entorhinal area large multipolar and bitufted cells are found in layers II-VI. In the untreated monkey brain NPY-positive cells are found in the hilus of the area dentata and in the deep (IV through VI) layers of both the medial and lateral entorhinal area. Fewer NPY-stained cells are present in the subicular complex and in the entorhinal area. In the monkey as well as in the rat, NPY-stained cells are present in the angular bundle and in the alveus. A dense network of NPY-i fibers innervates the entire hippocampal region in both the rat and the monkey. The hippocampal NPY-i preterminal processes are present primarily in stratum moleculare of Ammon's horn and in the outer one-third of this layer in the area dentata. The NPY-positive innervation of the dentate molecular layer is far more prominent in the monkey than in the rat brain. Numerous NPY-stained fibers are scattered in other areas as well. In all retrohippocampal structures, and in particular the entorhinal area, the NPY-i fibers form a massive network that innervates all layers to about the same extent, with the exception of the molecular layer, which is more densely innervated than the other layers.

An analysis of cholinoceptive neurons in the hippocampal formation by direct microinfusion

Microinfusion of cholinergic agents were made in various sites in the dorsal hippocampal formation of urethane anaesthetized rats. Infusions of eserine or carbachol elicited hippocampal theta activity when made in areas containing high levels of cholinergic markers: the stratum oriens and radiatum of the CA1 and CA3, the stratum moleculare and stratum granulosum of the dentate gyrus and the infragnular region of the hilus. Subsequent infusions of atropine sulfate antagonized the theta activity. Control infusions of equal volumes of saline in active sites were without effect. Infusions of eserine or carbachol in the vicinity of the hippocampal fissure, the stratum lacunosum/moleculare of the CA1 or CA3, in the deep regions of the hilus, and in the lateral ventricle and overlying neocortex, were also without effect. Furthermore, in active sites, the latency to onset of theta and subsequent theta frequency, were both directly related to the total amount of carbachol infused. Thus, areas in which theta could be elicited with a cholinergic agonist (carbachol), or an anticholinesterase (eserine) and antagonized with atropine, were found to correspond well to areas previously found to contain well to areas previously found to contain a high density of cholinoceptive neurons, using autoradiographic and immunohistochemical techniques. These results provide further support for the involvement of acetylcholine as a neurotransmitter in the generation of type 2 theta in the hippocampal formation.

The cholinergic innervation of the rat fascia dentata: Identification of target structures on granule cells by combining choline acetyltransferase immunocytochemistry and Golgi impregnation

A monoclonal antibody against choline acetyltransferase (ChAT), the acetylcholine-synthesizing enzyme, was used to study cholinergic synapses on identified (Golgi stained) granule cells in the rat fascia dentata. Choline acetyltransferase immunocytochemistry was applied to 40-microns Vibratome sections cut perpendicular to the longitudinal axis of the hippocampus. Light microscopy revealed fine varicose ChAT-immunoreactive axons in all layers of the fascia dentata, i.e., in the stratum moleculare, the stratum granulosum, and the subgranular polymorph zone. Most fibers were observed in the vicinity of granule cell bodies where they ran mainly parallel to the granular layer. Next, the immunostained Vibratome sections were sandwiched between small pieces of Parafilm and piled to form a block that was covered with agar and Golgi stained. After that, the sections were separated by cutting away the agar and removing the Parafilm. Sections containing well-impregnated granule cells were gold-toned (Fairén et al., '77), embedded in Araldite, and subjected to ultrathin sectioning for electron microscopy. A total of 14 gold-toned granule cells were examined in the electron microscope for synaptic contacts with cholinergic afferents. Choline acetyltransferase-immunoreactive axon terminals were observed that established symmetric synaptic contacts with the cell bodies and dendritic shafts of the gold-toned identified granule cells. Two types of contact were observed on spines arising from gold-toned granule cell dendrites. Immunoreactive terminals established asymmetric synaptic contacts with the head of small spines and symmetric contacts with the stalk of large, complex spines. The boutons forming asymmetric synaptic contacts with the cup-shaped spine head of the complex spines were not found to be immunoreactive. Our results demonstrate that cholinergic fibers to the rat fascia dentata establish characteristic types of synaptic contact with different postsynaptic elements of granule cells, suggesting a complex function of this afferent system.

Specific patterns of fibre outgrowth from transplants to host mice hippocampi, shown immunohistochemically by the use of allelic forms of Thy-1

Foetal mouse hippocampal primordia from mice homozygous for the Thy-1.1 allele were transplanted into the hippocampal region of adult histocompatible mice, homozygous for the Thy-1.2 allele. After survival periods of two months to one year the transplants consisted of a discrete tissue mass, well fused with the host, and distinguished from it by an intense Thy-1.1 immunoreactivity of the neuropil. The host hippocampus was entirely negative for Thy-1.1 immunoreactivity except for well defined projections arising from the transplant. These projections had three different patterns of distribution in the host: (1) a mossy-fibre-like distribution (to the stratum lucidum of field CA3); (2) a fimbria-like distribution (to the stratum oriens and stratum radiatum of fields CA1 and CA3), and (3) a commissural/association-like distribution (to the inner stratum moleculare of the dentate gyrus). Analysis of the position of the transplants and their component cell types indicated that, for each of the three distribution patterns, three conditions had to be fulfilled: (1) the transplant had to contain the appropriate type of cell (granule cells for mossy fibre projections, and larger pyramid-like cells for the other two); (2) the transplant neurons giving rise to the projection had to lie in direct contact with the host field to be innervated, and (3) the terminal field had to be specifically denervated. Thus, for three fibre systems the patterns of transplant-to-host projections observe rules of organization comparable to those of the normal hippocampal circuitry. This implies that in the adult host brain there remain (or there can be elicited) the molecular cues capable of establishing patterns of neuronal connectivity comparable to those formed during normal development.

Neuron density in the molecular layer of the frontal cortex in primary generalized epilepsy.

Developmental disturbance of the brain as a factor in the causation of epilepsies has been suspected for a long time. An increase of nerve cells in the stratum moleculare, which is qualitatively observed in primary generalized epilepsy, is thought to be an indicator for slight maldevelopment. In this morphometric study, the neuron density of layer I in the frontal lobe in primary generalized epilepsy is measured. Compared with age-matched controls, the neuron density in primary generalized epilepsy is significantly increased. Thus, neuron density could be a significant index for the disturbance of regional maturation of the cortex.

Gamma-Aminobutyric acid and benzodiazepine receptors in the kindling model of epilepsy: a quantitative radiohistochemical study

Quantitative radiohistochemistry was utilized to study alterations of gamma-aminobutyric acid (GABA) and benzodiazepine receptors in the kindling model of epilepsy. The radioligands used for GABA and benzodiazepine receptors were [3H] muscimol and [3H]flunitrazepam, respectively. GABA receptor binding was increased by 22% in fascia dentata of the hippocampal formation but not in neocortex or substantia nigra of kindled rats. Within fascia dentata, GABA receptor binding was increased to an equivalent extent in stratum granulosum and throughout stratum moleculare; no increase was found in dentate hilus or stratum lacunosummoleculare or stratum radiatum of CA1. The increased binding was present at 24 hr but not at 28 days after the last kindled seizure. The direction, anatomic distribution, and time course of the increased GABA receptor binding were paralleled by increased benzodiazepine receptor binding. Unexpectedly, GABA receptor-mediated enhancement of benzodiazepine receptor binding was slightly attenuated in fascia dentata of kindled compared to control rats. The anatomic distribution of the increased GABA receptor binding is consistent with a localization to somata and dendritic trees of dentate granule cells. We suggest that increased GABA and benzodiazepine receptor binding may contribute to enhanced inhibition of dentate granule cells demonstrated electrophysiologically in kindled animals.

Spontaneous epileptiform discharges in hippocampal slices induced by 4-aminopyridine

4-Aminopyridine (4-AP) induced 2 types of spontaneous field potentials (SFPs) in the hippocampal slice. Type I resembled spontaneous activity induced by other convulsants. They occurred at a rate of approximately 1 Hz, started in the CA2/CA3 region and spread at a velocity of 0.3 m/s to area CA1. Transsection experiments and laminar profiles indicated that they spread synaptically along the Schaffer collateral pathway. Synaptic blockade by low Ca 2+/high Mg 2+ or kynurenic acid reversibly abolished type I SFPs. Increasing [Ca 2+] o lowered the rate and slightly increased the amplitude. Possibly, increased spontaneous transmitter release, and not disinhibition, is responsible for the generation of type I SFPs. Type II occurred at a rate of about 0.15 Hz and travelled in the same direction, but a factor 10 slower. They could not be blocked by separation of the CA1 and CA3 region; coupling remained until stratum moleculare was severed. Type II could not be suppressed by blockade of synaptic transmission. The laminar profile is similar in shape to that of type I but not identical. Increasing [Ca 2+] o had the same but stronger effect as on type I. Type II SFPs depressed evoked population spikes up to a second and delayed the next type I SFP. The mechanisms involved remain largely speculative; further analysis is needed to help understand the epileptogenic action of 4-AP.

Intravesicular localization of zinc in rat telencephalic boutons. A histochemical study

A transition metal (presumably zinc) has been localized inside the synaptic vesicles of rat telencephalic boutons which make asymmetric synaptic contacts (Gray type I). A modification of Timm's sulphide-silver method for electron microscopy was applied to different areas of the rat forebrain: olfactory bulb, septum, caudate-putamen, amygdaloid complex, neocortex, entorhinal cortex and different parts of the hippocampal formation, viz. subiculum, stratum radiatum and oriens of both regio superior (CA1) and regio inferior hippocampi (CA3), the mossy fibre zone and the hilus fascia dentata and stratum moleculare in the gyrus dentatus. It was observed that the Timm staining in all these areas was located in synaptic boutons. Labelled boutons displayed a population of round clear vesicles and few dense core vesicles and made asymmetric synaptic contacts on dendritic spines. Silver granules in these boutons were confined to only a minor fraction of the round clear vesicles (approximately 10%). Labelled boutons occasionally showed silver granules in the synaptic clefts. The possible involvement of vesicular zinc in synaptic transmission is discussed.

The dentate gyrus in hypoglycemia: Pathology implicating excititoxin-mediated neuronal necrosis

A detailed light- and electron-microscopic study of the damage to the rat dentate gyrus in hypoglycemia was undertaken, in view of the previously advanced hypothesis that hypoglycemic nerve cell injury is mediated by a released neurotoxin. The distribution of neuronal necrosis showed a relationship to the subarachnoid cisterns. Electron microscopy of the dentate granule cells and their apical dendrites revealed dendrosomal, axon-sparing neuronal pathology. Dentate granule cells were affected first in the dendrites in the outer layer of the stratum moleculare, sparing axons of passage and terminal boutons. Subsequently, the neuronal perikarya were affected, and Wallerian degeneration of axons followed. Cell membrane abnormalities preceded the appearance of mitochondrial flocculent densities and degradation of the cytoskeleton, and are suggested to be early lethal changes. The observed early dendrotoxic changes, and the dendrosomal, axon-sparing nature of the lesion implicate an excitotoxin-mediated neuronal necrosis in hypoglycemia.

Ectopic parvalbumin-positive cells in the cerebellum of the adult mutant mouse 'nervous'.

The basket and stellate cells of the 'nervous' mouse cerebellum lose 50% of their presynaptic parallel fibres and more than 90% of Purkinje cells, their major postsynaptic targets, after postnatal day 23. To study the fate of these molecular layer cells we examined the cerebellum of the mutant mouse 'nervous' with antisera against the 'marker protein' parvalbumin, which exclusively tags Purkinje, basket, and stellate cells. Ten homozygous 'nervous' mice, 12-14 weeks old, and 30 control animals of several inbred strains were examined. The number of basket and stellate cells decreased in affected areas of the molecular layer. In addition, parvalbumin-positive cells were detected in the granular layer, in the white matter of the cerebellum and in the area of the cerebellar peduncles of all 'nervous' mice, but not in those of the control animals. These cells mainly occurred in areas which still displayed degenerating Purkinje cell axons. Ectopic parvalbumin-positive cells could either represent a class of interneurons, which have changed their antigenic properties, and therefore happen to stain with antisera against parvalbumin, or stratum moleculare cells which have failed to recognise their proper position during ontogenesis. Alternatively these cells could represent postdevelopmental basket cells, which have acquired the ability to migrate.

Mossy fiber sprouting in the fascia dentata after unilateral entorhinal lesions: Quantitative analysis using computer-assisted image processing

Axon sprouting typically occurs in a brain region that has been partially denervated. The present study demonstrates, quantitatively, evidence for sprouting outside the region of deafferentation. A modification of the Timm sulfide silver histochemical method was used to monitor an increase in the mossy fiber terminal field in the fascia dentata of adult rats following severe deafferentation of the outer three-fourths of stratum moleculare by unilateral entorhinal lesions. Computer-assisted image processing techniques were used to quantify mossy fiber sprouting. In stratum granulosum, and to a lesser extent in the deep (supragranular) portion of stratum moleculare (areas separated from the zone of deafferentation), there was a three-fold increase in the area of mossy fiber staining on the side of the lesion compared to the non-operated side (and unoperated animals). Much of the increased staining was located near the tip of the infrapyramidal (ventral) blade of the fascia dentata. Since mossy fiber sprouting apparently occurs in the absence of degeneration-produced synaptic dilution in that region, it may represent an example of post-lesion growth initiated by conditions fundamentally different from those normally believed to induce sprouting.

Medial septal projections to the dentate gyrus of the rat: electrophysiological analysis of distribution and plasticity.

Previous electrophysiological experiments in rabbits have suggested that medial septal stimulation activates dentate granule cells and evokes an associated negative field response at the granule cell layer, without an associated "dendritic" response. Anatomical studies have suggested that septal input to the granule cells may be to stratum moleculare, or close to the cell layer, or may not exist at all. The present experiments confirmed in rats anaesthetised with urethane that medial septal stimulation elicits single action potentials from cells in the granule layer. The associated negative field potential was maximal in the granule cell layer and there was no sign of a separate dendritic potential. The fibres responsible for this potential travel to the dorsal hippocampus in the fornix superior rather than the fimbria, taking the same course as the fibres which contribute to the dense cholinesterase staining just above the granule cell layer. Stimulation at 100 Hz for 1 s of either medial septal, or perforant path, input to the dentate granule cell layer produced long term potentiation of the subsequent evoked field responses to the stimulated pathway. The responses to the non-stimulated pathway were unchanged. Paired pulse stimulation produced both homosynaptic and heterosynaptic potentiation. These data suggest that medial septal input synapses close to granule cell bodies and produces a negative field potential which is a combination of dendritic and population spike potentials. Medial septal input also appeared to produce direct activation of hilar neurones, some of which may be basket cells or other interneurones. The data also show that long term potentiation is specific to this input, perhaps dependent on presynaptic mechanisms. Paired pulse potentiation, at least in the heterosynaptic case appears to depend on postsynaptic mechanisms.

Distribution of neuropeptides in the limbic system of the rat: The hippocampus

The distribution of several neuropeptides (vasoactive intestinal polypeptide, cholecystokinin octapeptide, substance P, neurotensin, methionine-enkephalin and somatostatin) in the hippocampal formation has been studied with immunocytochemical techniques. Numerous vasoactive intestinal polypeptide, cholecystokinin-octapeptide and somatostatin-positive cell bodies were found within the hippocampus and subiculum. Neurotensin-positive cell bodies were found within the subiculum, but no substance P or methionine-enkephalin-containing cell bodies were seen in either hippocampus proper or subiculum. Vasoactive intestinal polypeptide and cholecystokinin-octapeptide-positive cell bodies were predominantly located in the stratum moleculare and stratum radiatum of CA 1–2 regions and dentate gyrus, whilst somatostatin-containing cell bodies were found mainly in the stratum oriens. Nerve fibres containing each of the six peptides were found within the hippocampus. Large numbers of vasoactive intestinal polypeptide, cholecystokinin-octapeptide and somatostatin fibres innervated the pyramidal and granule cell layers, with smaller numbers in the stratum radiatum and fewer still in the stratum moleculare and stratum oriens. Other than a moderately dense neurotensin-positive fibre plexus in the dorsal subiculum, fewer neurotensin, substance P and methionine-enkephalin fibres were present. However, when present, these three peptides had a distribution restricted to a region close to the pyramidal layer in the CA 2/3 region and to the stratum moleculare of the CA 1 region. Peptide-containing fibres were identified entering or leaving the hippocampus in three ways, via (i) the fornix (all six peptides), (ii) the dorsal subiculum (neurotensin-positive fibres projecting to the cingulate cortex: somatostatin, vasoactive intestinal polypeptide, and cholecystokinin-octapeptide present in fibres running between the dorsal subiculum and occipito-parietal cortex) and (iii) the ventral subiculum (vasoactive intestinal polypeptide, cholecystokinin-octapeptide and somatostatin in fibres running between entorhinal cortex and hippocampus, and all six peptides in fibres crossing the amygdalo-hippocampal border). These findings indicate a major distinction between those peptides (vasoactive intestinal polypeptide, cholecystokinin-octapeptide, somatostatin, neurotensin) which are found in cell bodies intrinsic to the hippocampal formation and those peptides (substance P, methionine-enkephalin) which are found only in hippocampal afferents. Differences were also noted between the connections of the dorsal hippocampus, which are predominantly by way of the fornix and cingulate bundle, and the ventral hippocampus, which are with the entorhinal cortex and amygdala.

Functional anatomy of limbic seizures: Focal discharges from medial entorhinal cortex in rat

Focal seizure discharges were induced in the ventral aspect of the medial entorhinal cortex of awake, freely moving rats, either with cannula injections of penicillin or picrotoxin (0.02 μl every 10–15 min) or by repetitive tetanic electrical stimulation.[ 14C]Deoxyglucose autoradiography (DG) was performed when animals were in a ‘steady-state’ with respect to electrographic discharges and/or behavioral changes. During simple interictal spikes behavior remained normal and DG labeling was increased only in the entorhinal focus and stratum moleculare of the ventral dentate gyrus. With complex spikes and short seizures animals exhibited staring, decreased responsiveness, and occasional wer dog shakes. DG labeling was inceased in all layers of the dentate gyrus, Ammon's horn (ipsilateral > contralateral) and, to a lesser degree, in ipsilateral anygdala, and the accumbens-ventral pallidum area. During stong seizures rearing and forelimb clonus occured and metabolism was strongly activated bilaterally in the hippocampal formation, amygdala, accumbens, substantia nigra, and the anterior and periventricular thalamic nuclei. These studies indicate that the dentate gyrus initially restricts the entry of seizures from entorhinal cortex into the rest of hippocampus. As this is overcome there is rapid bilateral spread through the hippocampal formation with passive interruption of normal behavior. With prolonged seizure discharges there is further capture of amygdala and subcortical extrapyramidal and thalamic nuclei associated with behavioral convulsions.

Regional Metabolic Activity in the Rat Brain During Sleep-Wake Activity

[14C]2-Deoxyglucose autoradiography was used to demonstrate regional metabolic activity in the rat brain during wake, slow-wave sleep (SWS), and REM sleep. Data are expressed as values of relative metabolic activity (RMA, regional optic density/mean brain optic density), which show the level of metabolic activity in a region, relative to the mean level of the brain. Regions exhibiting obviously decreased RMA during SWS include the cerebellum, cortical layer IV, and most of the thalamus, including the massa intermedia and lateral geniculate nuclei. Low levels of RMA in sensory relay regions during SWS reflect functional deafference of the brain. In contrast, some limbic regions, including the hypothalamic core and hippocampal dentate fascia, exhibit increased RMA during SWS. Regions exhibiting increased RMA may have functions which are carried on at high levels during sleep. The metabolic appearance of the brain in REM is very different from that in SWS. The reticular core, substantia nigra, hippocampal dentate fascia, and stratum moleculare, and many regions associated with the extrapyramidal motor system, exhibit increased RMA. The cerebellum and ventral thalamic nucleus show decreased RMA, while the lateral thalamic nuclei and cortical layer IV show no metabolic state dependency. In sum, SWS is associated with a general decrease in RMA of sensory and motor areas, and with increased RMA in some limbic regions. RMA is similar in cerebellar and hippocampal regions during REM and SWS. In contrast, RMA in many sensory relay regions does not decrease during REM, and there is REM-specific increase of RMA in some pontomesencephalic regions associated with motor function.

Comparative localization of enkephalin and cholecystokinin immunoreactivities and heavy metals in the hippocampus

The present study is concerned with the cellular origins and identities of the hippocampal enkephalin and CCK-immunoreactive fibers and terminals. In the hippocampus of the rat, the guinea pig and the European hedgehog a system of enkephalin immunoreactive nerves emerges in the hilus of area dentata and can be followed to the apical dendrites of the hippocampal regio inferior pyramidal cells. This pattern of immunoreactive nerves corresponds to the hippocampal mossy fiber system as visualized by the Timm staining. Cholecystokinin immunoreactive nerve fibers and terminals reveal the same distribution in the guinea pig and the European hedgehog whereas in the rat the mossy fiber zone contains little or no cholecystokinin immunoreactivity. In the guinea pig degeneration of the mossy fibers after stereotactic lesions of the mossy fibers causes a complete loss of both enkephalin and cholecystokinin immunoreactivity in the mossy fiber zone. Only a few enkephalin immunoreactive cell bodies were scattered throughout the granular cell layer of area dentata, but inhibition of the axoplasmic transport by colchicine dramatically increased the number of enkephalin immunoreactive granule cell bodies. Enkephalin immunoreactive cell bodies were also detected in the hilus, throughout the pyramidal cell layer as well as in the stratum radiatum and stratum moleculare. Cholecystokinin immunoreactive cell bodies were seen in the hilus of the area dentata and in the stratum oriens, stratum pyramidale and stratum radiatum and the cell-rich layer of subiculum. No cholecystokinin immunoreactive cell bodies were observed in the granular cell layer of area dentata. Even after colchicine treatment the granule cells were devoid of cholecystokinin immunoreactivity. In the rat a system of nerves displaying enkephalin immunoreactivity was also observed in the superficial one-third of the molecular layer of area dentata, a zone which corresponds to the termination of the lateral perforant path. Another observation was that in the rat, but not in the guinea pig and the hedgehog, the terminal zone of both the medial perforant path and the zone of commissural and associational fibers of area dentata contained cholecystokinin immunoreactive molecules. In summary, our data show: (1) that the hippocampal mossy fibers contain enkephalin immunoreactive molecules; (2) that the cholecystokinin immunoreactivity in the mossy fiber zone is most likely also localized in the mossy fibers per se, although the granule cells seem devoid of cholecystokinin immunoreactivity; (3) zinc, here visualized as a Timm-positive substance, is also localized in the mossy fiber terminals; further, (4) other intrinsic cell bodies than the granule cells may contribute to both the enkephalin and cholecystokinin immunoreactive terminals within the hippocampus; (5) in the rat the lateral perforant path may be enkephalinergic; and (6) both the terminal zone of the medial perforant path and the associational and commissural fibers of the rat contain cholecystokinin immunoreactivity.

Changes in [3H]muscimol binding in substantia nigra, entopeduncular nucleus, globus pallidus, and thalamus after striatal lesions as demonstrated by quantitative receptor autoradiography

A quantitative autoradiographic technique for measuring the binding of [3H]muscimol to central nervous system GABA receptors is described using tritium-sensitive film. [3H]Muscimol binding was studied in primary and secondary striatal projection areas of rat brain following kainic acid lesions of the striatum. Seven days after the lesion, binding affinities in the striatum and its projection areas were not altered significantly. There was a loss of [3H]muscimol receptors in the striatum. Receptors increased in numbers in the ipsilateral globus pallidus (19%), entopeduncular nucleus (22%), and substantia nigra pars reticulata (38%). [3H]Muscimol binding was decreased in the ipsilateral anteroventrolateral and ventromedial (8%) thalamic nuclei. [3H]Muscimol binding in other brain areas (layer IV of the cerebral cortex, central gray, superior colliculus, and stratum moleculare of hippocampus) was not affected. The findings suggest that a loss of striatal innervation resulted in increased numbers of GABA receptors in striatal projection sites. It is further suggested that loss of inhibitory striatal inputs to neurons in the entopeduncular nucleus and substantia nigra pars reticulata may activate GABAergic projections to thalamus and thus result in decreased numbers of thalamic GABA receptors.

Quantitative autoradiography of L-[ 3H]glutamate binding to rat brain

A technique has been developed to investigate sodium-independent L-[ 3H]glutamate binding in rat brain sections using quantitative autoradiography and tritium-sensitive film. Binding is rapid (reaching equilibrium in 5 min) and reversible (having a t 1 2 of dissociation of 0.38 min). Glutamate apparently bound to a single population of sites with a K d of about 1.0 μM. The pharmacology of this binding site is similar to that observed in homogenate studies. There is marked regional variation in the amount of glutamate bound. Of the areas analyzed in detail, the density of sites is greatest in stratum moleculare of hippocampus, followed by striatum and cortex.

Ontogeny of opiate binding sites in the hippocampus, olfactory bulb and other regions of the rat forebrain by autoradiographic methods

The postnatal development of the distribution of opiate binding sites in the rat forebrain was studied by light microscopic autoradiography. [ 3H] dihydromorphine ([ 3H]DHM) was used to label receptors in the forebrain of neonates days 2, 6, 8, 12, 16 and 21 postpartum. The hippocampus of the 2-day-old rat was diffusely labeled by [ 3H]DHM. By day 8, autoradiographic grains had a different distribution and were now clustered around the pyramidal cells. By day 16, the adult distribution of opiate binding sites was established. These changes correlate with the development of this structure and suggests that these binding sites may be localized to inhibitory interneurons of the pyramidal cell layer. The appearance of specific binding sites in the stratum moleculare of the dentate gyrus at day 8 further correlates with the development of the granule cells and their dendritic processes. In the olfactory bulb, high levels of specific binding were seen in the external plexiform layer at day 2. This pattern gradually changed, so that in the adult, the binding sites were more enriched in the glomeruli and granule cell layer. These changes in the olfactory bulb may be related to the maturation of the tufted cells and granule cells. Other regions of the brain were also examined. Thus, the analysis of receptor binding in development can aid in the localization of receptors to specific neuronal elements.