Long-term memory formation in the chick requires mobilization of ryanodine-sensitive intracellular calcium stores.

Training chicks (Gallus domesticus) on a one-trial passive avoidance task results in transient and time-dependent enhanced increases in N-methyl-d-aspartate- or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-stimulated intracellular calcium concentration in synaptoneurosomes isolated from a specific forebrain region, the intermediate medial hyperstriatum ventrale. This increase could result from either calcium entry from the extracellular medium or from mobilization of intracellular calcium stores. We have therefore examined the effects of dantrolene, an inhibitor of calcium release from the intracellular ryanodine-sensitive store, on these processes. Dantrolene, 50 nmol per hemisphere injected intracerebrally 30 min pre- or 30 min posttraining, blocked longer term memory for the passive avoidance task, whereas memory for the task was unaffected when dantrolene was injected at earlier or later times. Preincubation of synaptoneurosomes, isolated from the intermediate hyperstriatum ventrale 10 min after training, with 100 nM dantrolene abolished the enhanced training-induced increase in intracellular calcium concentration elicited by 0.5 mM N-methyl-d-aspartate. By contrast, the training-induced enhancement of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-stimulated increase in intracellular calcium concentration in synaptoneurosomes prepared 6 h posttraining was unaffected by preincubation with dantrolene, which was not amnestic at this time. Calcium release from ryanodine-sensitive intracellular stores may thus be a necessary stage in the early phase of the molecular cascade leading to the synaptic modulation required for long-term memory storage.

Passive avoidance training results in increased responsiveness of voltage- and ligand-gated calcium channels in chick brain synaptoneurosomes.

A temporal cascade of events has been described from a number of biochemical investigations of passive avoidance training in day-old chicks. Among these, within minutes of training, there is a transient, enhanced release of glutamate and increased agonist and antagonist binding to N-methyl-D-aspartate-sensitive glutamate receptors in the intermediate medial hyperstriatum ventrale of the forebrain. Some 6.5 h later, alpha-amino-3-hydroxy-5-methyl-4-isoxazo lepropionate binding to glutamate receptors is also increased in the same region. These processes might be predicted to affect the uptake of calcium via voltage-sensitive calcium channels or glutamate receptor-associated channels, thereby changing the intracellular calcium concentration. To test this possibility, we have measured the calcium concentration in synaptoneurosomes, containing both pre- and postsynaptic elements, prepared from left and right intermediate medial hyperstriatum ventrale at various times following training, using Fura 2-AM as the indicator of intracellular calcium concentration. Synaptoneurosomes, prepared immediately and 5 min after training, were stimulated with 70 mM potassium chloride in the presence of 2 mM calcium, resulting in a significantly enhanced increase in calcium concentration in synaptoneurosomes from the left hemisphere of trained chicks. This effect was absent in samples obtained at later times after training. N-Methyl-D-aspartate (0.5 mM) induced a significant enhancement in the increase in calcium concentration in intermediate medial hyperstriatum ventrale from both left and right hemispheres 10 min and 30 min after training. At 3 h and 6 h after training, alpha-amino-3-hydroxy-5-methyl-4-isoxazo lepropionate (0.5 mM) induced a significantly enhanced increase in calcium concentration in samples from either hemisphere. These results suggest that immediately after training there is an engagement of both pre- and postsynaptic voltage-sensitive calcium channels, followed by an increased reponse to N-methyl-D-aspartate receptor stimulation, and coinciding with the enhanced calcium-dependent glutamate release and an increase in N-methyl-D-aspartate-sensitive glutamate receptor binding that has been reported previously. The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-sensitive mechanisms are activated at a later stage of memory formation, when increased alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate binding to glutamate receptors has been reported. Thus, responsiveness of calcium channels to agonist stimulation is implicated in temporally diverse stages in the cascade of events involved in memory formation following passive avoidance training in the chick.

Changes in binding to muscarinic and nicotinic cholinergic receptors in the chick telencephalon, following passive avoidance learning.

Changes in nicotinic and muscarinic cholinergic receptors 30 min after one-trial passive avoidance training were studied in day-old chicks (Gallus domesticus), by quantitative receptor autoradiography. [3H]-alpha-bungarotoxin (alpha-BgT) and [3H]-quinuclidinyl benzilate (QNB) were used to monitor changes in 15 forebrain regions for nicotinic and muscarinic receptors, respectively. A significant increase occurred bilaterally in the quantity of bound alpha-BgT in the lobus parolfactorius, while the amount of bound QNB decreased significantly, and bilaterally, in the hippocampus, hyperstriatum ventrale, lobus parolfactorius and posterolateral telencephalon, pars dorsalis. The data support an involvement of cholinergic receptor types in the neural mechanisms underlying passive avoidance learning.

Exposure to light prior to hatching induces asymmetry of receptor binding in specific regions of the chick forebrain.

This paper describes neurochemical asymmetries present in forebrain regions of the newly hatched chick that result from environmental conditions; specifically from asymmetrical exposure of the chick embryo to light prior to hatching. Quantitative autoradiography was used to determine GABA and glutamate receptor subtype binding in a number of regions of the left and right forebrain hemispheres of chicks that had either the left (LES), or the right (RES), eye system exposed to light prior to hatching. On day 19 of incubation the embryo's head was withdrawn from the egg and the left or the right eye was occulded until hatching. [3H]MK-801, [3H]AMPA and [3H]muscimol binding assays were performed on frozen sections from 2 different coronal regions of the forebrain, sampled on day-1 posthatching. Significant [3H]MK-801, [3H]AMPA and [3H]muscimol binding asymmetries were determined in forebrain regions from chicks that had their RES exposed to light prior to hatching, particularly in forebrain regions which are known to receive afferent visual input. The reverse pattern of asymmetry was found for all 3 ligands in regions such as the ectostriatum of chicks that had their LES exposed to light, while asymmetry of muscimol and AMPA binding, present in many regions in right eye system chicks was not present in chicks that had the left eye system exposed to light during incubation. Thus, the presence and pattern of experience-dependent neurochemical asymmetries in the chick forebrain are specific to both region and receptor type.

Passive avoidance learning in the day-old chick is modulated by GABAergic agents.

Injection of drugs directly into the intermediate medial hyperstriatum ventrale (IMHV) of day-old chicks, prior to training on a chrome bead dipped in either the strong aversant methyl anthranilate (MeA), or the weak aversant quinine, allows investigation of the effects of potential amnestic and memory-enhancing agents on retention of a passive avoidance task. Chicks were injected into the left and right IMHV, with either saline or muscimol (GABA agonist), 30 minutes before training on an MeA-coated bead. On test, either 10 min, 30 min or 24 h after training, birds were presented with a dry chrome bead. Normally, trained birds will avoid the test bead; however, significantly more muscimol-injected birds pecked the dry bead than did saline-injected chicks, indicating amnesia in the muscimol-injected birds. In chicks injected bilaterally into the IMHV with bicuculline, a GABAA antagonist, 30 minutes prior to training on a quinine-coated bead, avoidance scores were significantly improved on testing at 24 h compared with saline-injected control chicks, indicating enhanced retention in bicuculline-treated birds. These results suggest a role for the GABAergic system in the acquisition and retention of passive avoidance learning in the day-old chick.

Quantitative autoradiographic demonstration of changes in binding to delta opioid, but not mu or kappa receptors, in chick forebrain 30 minutes after passive avoidance training.

Day-old domestic chicks (Gallus domesticus) were trained on a one-trial passive avoidance task in which the aversive stimulus was a bitter tasting substance, methylanthranilate. Thirty minutes later, localization of binding of highly specific ligands (([D-Ala2, Gly-ol]-enkephalin ([3H]DAGO) for mu (mu) receptor sites, [D-Pen2,D-Pen5]-enkephalin ([3H]-DPDPE) for delta (delta) sites, and [3H]-U- 69593 for kappa (kappa 1) sites) to opioid receptors in various regions of the forebrain of methyl-anthranilate trained (M-) and control (water trained (W-)) chicks was determined using quantitative receptor autoradiography. Significant differences in binding to delta ([3H]-DPDPE), but not mu or kappa receptors, were found in several regions of the forebrain, of trained compared to control chicks. There were decreases in binding in the hyperstriatum dorsale of the left hemisphere (14%) and a decrease in binding in the lateral hyperstriatum ventrale of the right hemisphere (14%). However, significant increases were observed in delta binding in the paleostriatum augmentatum of the right hemisphere (16%) and the lobus parolfactorius of both hemispheres (left, 20%; right, 21%). In a control experiment designed to determine whether the taste of methylanthranilate contributed to the increase in 3H-DPDPE binding, there was no significant difference in the level of binding between blindfolded birds in which methylanthranilate was placed in the beak, and blindfolded birds in which water was placed on the bead and inserted into the beak. These findings demonstrate that changes occur in an opioid receptor sub-type in specific regions of forebrain of the chick following passive avoidance training which may be related to events concerned with the process of memory formation.

Loss of gamma-aminobutyric acid (GABA) immunoreactivity from mouse first somatosensory (SI) cortex following neonatal, but not adult, denervation.

Whisker follicles were surgically ablated (lesioned) on two entire rows (B and C) of the left snout of two groups of Swiss mice, in the first 2 days after birth, and in the second group at 8 weeks of age (adults). Two months after surgery GABA-immunoreactivity (GABA-IR) was examined in tangential sections of the first somatosensory (SI) 'barrel' cortex of these two groups (at which time it was also confirmed that the follicles had not regrown). In the adult-lesioned mice all of the barrels appeared intact and visually similar in both the experimental (right) and control (left) hemispheres. The staining pattern of immunopositive cells and puncta was qualitatively similar to that which we have described previously. However, in the neonatally-lesioned mice barrel rows B and C were not visible in the right hemisphere sections and there was a marked reduction in GABA-IR, with fewer immunopositive cells. Many of those that did show GABA-IR stained only weakly and the puncta were also fewer in number than in the left (control) hemisphere where the GABA-IR pattern and staining intensity was normal.

Cerebral Glycoprotein Synthesis and Long-term Memory Formation in the Chick (Gallus domesticus) Following Passive Avoidance Training Depends on the Nature of the Aversive Stimulus.

Chicks that peck a small bright bead coated in a distateful substance can learn in a single trial to subsequently avoid a similar bead. The taste aversant commonly used is methyl anthranilate, which also has a strong pervasive odour. We have compared the efficacy of methyl anthranilate and the apparently odourless quinine as aversants. Methyl anthranilate-trained chicks learnt the task and the memory apparently persisted undiminished for at least 24 h. Quinine-trained chicks exhibited a memory for the task similar to that of methyl anthranilate-trained chicks 45 min after training, this thereafter declined until, at 24 h after training, they showed no recall. We investigated the incorporation of a radio-labelled synaptic membrane glycoprotein precursor, [3H]fucose, into three regions of the chick forebrain; two of these regions have previously been implicated in learning using methyl anthranilate as the aversant. There was a significant increase in [3H]fucose incorporation into the left lateral cerebral area and numerically similar, but non-significant, increases in the intermediate part of the medial hyperstriatum ventrale and lobus parolfactorius. There were no such increases in the right hemisphere of methyl anthranilate-trained chicks or any region of either hemisphere of quinine-trained chicks. Thus, the memory for methyl anthranilate is longer-lasting than that for quinine and is associated with increased fucosylation in the left cerebral hemisphere and although in the short-term, chicks can retain a memory of the one-trial passive avoidance task with quinine as the aversant, this does not result in a localized increase in cerebral [3H]fucose incorporation.

Distribution of mu, delta, and kappa opioid receptor binding sites in the brain of the one-day-old domestic chick (Gallus domesticus): an in vitro quantitative autoradiographic study.

Three highly specific opioid ligands--[D-Ala2,Gly-ol]-enkephalin (DAGO) for mu (mu) receptor sites, [D-Pen2,D-Pen5]-enkephalin (DPDPE) for delta (delta) sites, and U-69593 for kappa (kappa) sites--were used to determine the regional distribution of the three major subtypes of opioid receptor binding sites in the brains of 1-day-old domestic chicks by the technique of quantitative receptor autoradiography. Whilst there was a degree of heterogeneity in the binding levels of each of the ligands, some notable similarities existed in the binding of the mu and kappa ligands in several forebrain regions, and in the optic tectum of the midbrain where mu and delta binding was very high. In the forebrain there was a high level of binding of mu and kappa ligands in the hyperstriatum, and for the mu ligand there was a very distinct lamination of binding sites in hyperstriatum accessorium, intercalatum supremum, dorsale and ventrale. Levels of binding of the mu and kappa ligands were also high in nucleus basalis, and (for mu only) in the neostriatum. The distribution of binding of the delta specific ligand in the forebrain showed marked differences to that of mu and kappa, being particularly low in the hyperstriatum and neostriatum. Very high levels of labelling of delta binding sites were, however, found in the nucleus rotundus. Binding of the three ligands was generally low or absent in the cerebellum and medulla, apart from a distinct labelling of the granule cell layer by the mu-ligand. A kinetic analysis was made of the binding of the three ligands to whole forebrain sections using scintillation counting methods.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of GABA-like immunoreactivity in the ectostriatum of domestic chicks: GABA immunocytochemistry combined with Golgi impregnation.

The distribution of GABA-like immunoreactivity (GABA-LI) in the ectostriatal core (Ec) of domestic chicks (one to two days old) was investigated using (1) preembedding GABA immunocytochemistry and (2) Golgi impregnation and gold-toning combined with postembedding GABA immunocytochemistry. Two major classes of neurons which display GABA-LI were identified in chick Ec. Firstly, large GABA immunopositive cells which comprise at least two further subtypes: an ovoid or polygonal form of 14-18 microns diameter with no apparent polarity of dendrites and a smaller cell (10-14 microns) with ovoid or basket-shaped soma and often more polarized dendritic ramification. In both subtypes the dendritic surface is smooth or sparsely spiny. Secondly, a small GABA immunopositive cell which is characterized by a round cell body of 5-8 microns diameter and thin and sparsely ramifying dendrites of smooth surface or with irregular protrusions. Based upon comprehensive descriptions of ectostriatal cytoarchitectonics (Tömböl et al., 1988c), and synaptology (Watanabe et al., 1985), we argue that the GABA-immunopositive cell types of chick Ec are likely to represent inhibitory interneurons comparable with GABAergic inhibitory cell types described in mammalian visual cortex.

[3H]naloxone binding in the brain of the domestic chick (Gallus domesticus) determined by in vitro quantitative autoradiography.

The distribution of [3H]naloxone binding sites was investigated in the brains of one-day-old domestic chicks using quantitative receptor autoradiography. Among the forebrain regions, the greatest levels of opiate binding were found in hyperstriatum dorsale, hyperstriatum ventrale, hyperstriatum intercalatum supremum and neostriatum. Intermediate levels were found throughout the paleostriatal regions, septum, thalamus, archistriatum, hyperstriatum accessorium and area parahippocampalis whilst in hippocampus and ectostriatum the density of [3H]naloxone binding sites was low. In the hindbrain, high levels of opiate binding were found in optic tectum whereas in cerebellum the density of binding sites was barely above background. The greatest densities of opiate binding appear to coincide with regions found to be involved in sensory processing and memory storage.

Quantitative autoradiographic analysis of the distribution of [3H]muscimol binding to GABA receptors in chick brain.

Quantitative receptor autoradiography was used to investigate the distribution of high-affinity GABA receptors (GABAA) in left and right hemispheres of the brains of 3-week-old chicks. The receptors were labelled with the potent GABA agonist [3H]muscimol. High levels of [3H]muscimol labelling were found throughout the fore-, mid-, and hindbrain, though considerable variation was found in different regions. In the telencephalon the highest concentration of specific binding was found in the hyperstriatum ventrale followed by the neostriatum, and then the lobus parolfactorius of the paleostriatal complex, whilst in the diencephalon highest levels of labelling were present in the infundibulum. In the midbrain distinct lamination was observed in the high levels of [3H]muscimol binding in the optic tectum and in the hind brain the highest density of labelling occurred in the granular layers of the cerebellum. Levels of labelling were generally low in the brainstem regions. The distribution of [3H]muscimol binding in the optic tectum and in the hind brain the highest density of labelling occurred in the granular layers of the cerebellum. Levels of labelling were generally low in the brainstem regions. The distribution of [3H]muscimol binding sites is in good agreement with our previous work on the distribution of GABA-immunoreactivity in the chick brain.

gamma-Aminobutyric acid (GABA) immunoreactivity in mouse and rat first somatosensory (SI) cortex: description and comparison.

The location and morphological characteristics of gamma-aminobutyric acid (GABA)-immunopositive cells and their processes were studied in rat and mouse first somatosensory (SI) cortex (including 'barrels') in layer IV, and layers above (I-III), and below (V and VI). In coronal sections of SI cortex of both species GABA-immunopositive cells and punctate profiles were found in each of layers I-VI. The cells were of various sizes; the largest, located in layers III and V of each species, resemble the large basket cells seen in Golgi-impregnated material. Most of the immunopositive cells were multipolar and circular or ellipsoidal in shape, but occasionally bipolar cells with fusiform perikarya were also seen. In coronal sections, immunopositive cells did not form a characteristic pattern. GABA-immunopositive cells were observed to be most numerous in the supragranular layers whereas GABA-positive punctate profiles were more numerous in layer IV. In tangential sections from layer IV of SI cortex of both species, GABA-immunopositive cells, processes and punctate profiles were visible throughout the entire barrel field. The pattern of distribution of immunopositive cells was similar (a) in two different morphological groups--i.e. the posteromedial barrel subfield (PMBSF) and the anterolateral barrel subfield (ALBSF) in rat barrel field, and (b) in PMBSF barrels of both rat and mouse (excluding differences due to structural dissimilarities between rat and mouse barrels). GABA-immunopositive neurons were grouped mainly in the barrel side and septum and were visible frequently in small clusters. In barrels of both species GABA-immunopositive cells were of a variety of sizes and ranged in shape from ellipsoidal to circular.

Autoradiographic localization of gamma-aminobutyric acid receptors in mouse barrel field.

The distribution of high-affinity gamma-aminobutyric acid (GABA) receptors in mouse posteromedial barrel subfield (PMBSF) in layer IV of the somatosensory (SI) cortex was studied using [3H]muscimol autoradiography in vitro. A qualitative study revealed a heterogeneous distribution in the density of [3H]muscimol binding in the barrel field. In the barrel sides and septum between the barrels [3H]muscimol binding exhibits the lowest level of labelling in the entire tissue. In comparison, [3H]muscimol binding in the hollows is considerably higher than in the barrel sides although the level is not homogeneous. These findings suggest that the barrel hollows are much richer in GABA receptors than the surrounding barrel sides and septa.

Ontogeny of 3H-muscimol binding to membranes of chick forebrain.

A potent GABA agonist, 3H-muscimol, was used to investigate the development of GABA receptors in left and right hemispheres of chick forebrain from day 12 in ovo to day 21 post-hatch. Total specific 3H-muscimol binding (p mol mg-1 protein) increases rapidly in ovo, reaching a peak at around day one post-hatch and then showing a slow decline to approximately 50% of the maximal level at day 21 post-hatch. Despite the considerable evidence from previous studies of lateralization of avian brain function, no significant hemispheric differences were found in 3H-muscimol binding (either of p mol hemisphere-1 or p mol mg protein-1) at any of the developmental ages examined.

gamma-Aminobutyric acid immunoreactivity in mouse barrel field: a light microscopical study.

The barrel field of the mouse somatosensory cortex (SmI) was investigated immunocytochemically using an antiserum against the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). GABA-immunopositive cells and processes are grouped largely in the barrel side, whereas the barrel hollow is only weakly immunostained. The GABA-immunopositive cells have an ellipsoidal appearance similar to that of non-pyramidal class II barrel neurones described previously in Golgi impregnation studies of the mouse and rat barrel field.

Decreased levels of an astrocytic marker, glial fibrillary acidic protein, in the visual cortex of dark-reared rats: measurement by enzyme-linked immunosorbent assay.

An antibody to glial fibrillary acidic protein (GFAP) (an immunocytochemical marker for astrocytes) has been used in an enzyme-linked immunosorbent assay (ELISA) to determine the amount of GFAP in three visual regions, the dorsal lateral geniculate nucleus (dLGN), the superior colliculus (SC) and the visual cortex (VC) (area 17) of dark-reared (D), normal (N) and light-exposed (L) rats. In all experiments GFAP was also measured in a control non-visual region, the motor cortex (MC) (area 4). No significant differences were found in GFAP in dLGN, SC or MC between D, L or N rats. However, in the visual cortex, the amount of GFAP in N rats was significantly greater than that in D rats (by 32%).

Tritiated etorphine and naloxone binding to opioid receptors in caudate nucleus in schizophrenia.

Opioid receptor binding sites were assessed in membrane preparations of caudate nucleus from post-mortem brains of controls and of patients with schizophrenia. There was no difference between the two groups in the total specific binding of 3H-etorphine or in its 'mu' and ('delta + kappa') components. Similarly, the binding of 3H-naloxone did not differ between patients and controls. It is concluded that a previous report of reduced opioid receptors in caudate of schizophrenics is unlikely to prove a consistent finding and that the results of the present study offer no support to the claim that there is a general disturbance in opiate mechanisms in schizophrenia.

Adverse effects of anticholinergic medication on positive schizophrenic symptoms.

In a series of 36 patients with acute schizophrenia flupenthixol dosage was blindly adjusted to give a fixed level of sedation. Patients were than randomly allocated to procyclidine or placebo. The patients receiving procyclidine experienced more positive schizophrenic symptoms and less severe extrapyramidal features by comparison with placebo patients. Blood levels of prolactin and flupenthixol estimated by radioimmunoassay were not significantly changed by the addition of procyclidine. Flupenthixol dosage and levels and prolactin levels were significantly related. There was no significant association between clinical and laboratory measures, with the exception that a curvilinear (inverted U) relationship was demonstrated between flupenthixol levels and antipsychotic and extrapyramidal effects. This relationship may be due to the fact that, in a study of this design, patients resistant to the effects of neuroleptic medication are likely to be given the highest doses. The findings support earlier claims that anticholinergic medication has adverse effects on schizophrenic symptoms.

Spontaneous orofacial dyskinesia and dopaminergic function in rats after 6 months of neuroleptic treatment.

A syndrome of spontaneous orofacial dyskinesia was identified in groups of rats treated for 6 months with a wide range of neuroleptic drugs. Phenothiazines, thioxanthenes, and substituted benzamides were particularly likely to induce the syndrome. It was observed in the presence of a functional blockade of dopamine receptors and endured for at least 2.5 months after drug withdrawal. There was no relation between the syndrome and changes in striatal dopamine receptors, as indexed by the binding of tritiated spiperone and tritiated cis(Z)-flupenthixol. The syndrome parallels several of the features of clinical tardive dyskinesia, whose pathophysiology thus may not involve changes in the characteristics of striatal dopamine receptors.