Rachel C. Bourne

Hemispheric asymmetry of synapses in chick medial hyperstriatum ventrale following passive avoidance training: a stereological investigation

A stereological analysis was made of synapses in the left and right hemispheres of chick medial hyperstriatum ventrale (MHV) 24h after passive avoidance training (PAL) and in water trained controls (W-control). The synaptic parameters examined were (D), the mean length of the postsynaptic thickening; (NV.syn), the number of synapses per unit volume of neuropil; (VV.syn), the volume density of the pre-synaptic bouton; (NV.ves), the number of synaptic vesicles per unit volume of neuropil and (ves.syn), the mean number of synaptic vesicles per pre-synaptic bouton. No significant differences exist in NV.syn between the left or right hemispheres of W-control and trained chicks, nor is NV.syn influenced by training. However, in W-control chicks D in the right MHV is significantly greater (12%) than in the left MHV and this difference disappears on training. There are no differences in VV.syn between left and right hemispheres of W-control chicks but following training VV.syn is 22.7% greater in the left MHV than in the right MHV. Training als influences the number of synaptic vesicles; in W-control chicks NV.ves in the right MHV is 12.25% greater than in the left MHV but following training these differences are reversed. When the data are expressed as numbers of vesicles per synapse (ves.syn), values for the left hemisphere of trained chicks exceed those in the right hemisphere by a staggering 61.38%. These results are discussed in the context of biochemical and electrophysiological studies which suggest that there is lateralization of the memory trace.

Quantitative autoradiographic demonstration of changes in binding to NMDA-sensitive [3H]glutamate and [3H]MK801, but not [3H]AMPA receptors in chick forebrain 30 min 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 an unpleasant tasting substance, methyl anthranilate. Thirty minutes later, localization of N‐methyl‐d‐aspartic acid (NMDA)‐sensitive [3H]glutamate receptor binding sites, [3H]MK801 and [3H]AMPA binding sites in 17 regions of the forebrain of methylanthranilate‐trained and control (water‐trained) chicks was determined using quantitative receptor autoradiography. Significant differences in binding to both MK801‐ and NMDA‐sensitive glutamate receptors, but not α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptors, were found in three regions of the forebrain of trained compared to control chicks; two of these regions have been implicated from previous lesion, biochemical and morphological studies as playing a key role in the process of memory formation and storage following passive avoidance training. For NMDA‐sensitive [3H]glutamate receptors, significant elevations in binding were observed in two regions, the left intermediate and medial hyperstriatum ventrale (IMHV) (39%) and the lobus parolfactorius (LPO) (34%), at 30 min post‐training, but a decrease (44%) occurred in binding to the lateral neostriatum. Significant increases in binding to MK801 receptors were observed in the left IMHV (19%) and right IMHV (13%), and left LPO (22%) at 30 min post‐training, though there was a decrease in the right LPO (15%). These findings, coupled with those described in a previous paper from our group (Burchuladze and Rose, Eur. J. Neurosci., 4, 533–538, 1992), demonstrate that a glutamate receptor subtype is involved in learning and memory formation in the chick.

Reminder effects: the molecular cascade following a reminder in young chicks does not recapitulate that following training on a passive avoidance task

Memory traces, once established, are no longer sensitive to disruption by metabolic inhibitors. However, memories reactivated by reminder are once again vulnerable, in a time‐dependent manner, to amnestic treatment. To determine whether the metabolic events following a reminder recapitulate those following initial training we examined the temporal dynamics of amnesia induced by the protein synthesis inhibitor anisomycin and the glycosylation inhibitor 2‐deoxygalactose. The effects of both were transient and dependent on time of reminder post‐training and time of injection relative to reminder, and differed from those following initial training. 2‐[14C]‐deoxyglucose uptake increased in two brain regions, the intermediate medial hyperstriatum ventrale (IMHV) and lobus parolfactorius (LPO) following reminder as it did following training, but the increase was bilateral rather than confined to the left hemisphere and was more marked in LPO than IMHV. C‐fos expression after reminder was increased only in the LPO, the chick brain region associated with a late phase of memory processing and recall. Thus although, like initial consolidation, memory processing after reminder is sensitive to inhibitors of protein synthesis and glycosylation, the temporal and pharmacological dynamics indicate differences between these two processes.

γ-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.

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.

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.

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 distasteful 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 nonsignificant, 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.

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%).

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.

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.