Lesions of the basolateral amygdala complex block propofol-induced amnesia for inhibitory avoidance learning in rats.

BACKGROUND: As the unitary theory of anesthesia gives way to the "multiple sites, multiple mechanisms" concept, the sites involved in mediating the components of anesthesia must be identified. In the current study, we test the hypothesis that the basolateral amygdala complex (BLAC) is a brain site involved with mediating propofol-induced amnesia. METHODS: Male Sprague-Dawley rats were divided into two groups, sham-operated control animals and rats given bilateral excitotoxic N-methyl-D-aspartate lesions of the BLAC. For each group, animals were given intraperitoneal saline or propofol (25 mg/kg) 5 min before inhibitory avoidance learning. Rats were given a foot shock (0.4 mA) upon entering the dark side of a two-sided apparatus. Rats could escape additional shock by returning to and staying in the light side. Training ended after shock avoidance for greater than 60 s. Memory was tested at 24 h. Longer latencies to enter the dark side 24 h after training imply better memory. RESULTS: Sham-saline-treated animals had a robust memory latency (median latency [interquartile range] = 300 [163-567] s). Sham-propofo-treated animals exhibited a significant anterograde amnesia (latency = 63 [14-111] s) (P < 0.05 vs. sham-saline-treated animal). Both the saline-injected and propofol-injected animals with BLAC lesions showed robust memory (latency = 300 [264-485] and 323 [143480] s, respectively). These latencies did not differ from performance in the sham-saline-treated group and were significantly higher than the latency of the sham-propofol-treated group (both P < 0.05). CONCLUSIONS: Discrete BLAC lesions blocked the amnestic effect of propofol. BLAC activity appears to be a requirement for propofol-induced amnesia. This finding suggests that the BLAC is a key brain site mediating anesthetic-induced amnesia.

Generation and analysis of GluR5(Q636R) kainate receptor mutant mice.

The physiological significance of RNA editing of transcripts that code for kainate-preferring glutamate receptor subunits is unknown, despite the fact that the functional consequences of this molecular modification have been well characterized in cloned receptor subunits. RNA editing of the codon that encodes the glutamine/arginine (Q/R) site in the second membrane domain (MD2) of glutamate receptor 5 (GluR5) and GluR6 kainate receptor subunits produces receptors with reduced calcium permeabilities and single-channel conductances. Approximately 50% of the GluR5 subunit transcripts from adult rat brain are edited at the Q/R site in MD2. To address the role of glutamate receptor mRNA editing in the brain, we have made two strains of mice with mutations at amino acid 636, the Q/R-editing site in GluR5, using embryonic stem cell-mediated transgenesis. GluR5(RloxP/RloxP) mice encode an arginine at the Q/R site of the GluR5 subunit, whereas GluR5(wt(loxP)/wt(loxP)) mice encode a glutamine at this site, similar to wild-type mice. Mutant animals do not exhibit developmental abnormalities, nor do they show deficits in the behavioral paradigms tested in this study. Kainate receptor current densities were reduced by a factor of six in acutely isolated sensory neurons of dorsal root ganglia from GluR5(RloxP/RloxP) mice compared with neurons from wild-type mice. However, the editing mutant mice did not exhibit altered responses to thermal and chemical pain stimuli. Our investigations with the GluR5-editing mutant mice have therefore defined a set of physiological processes in which editing of the GluR5 subunit is unlikely to play an important role.

Central neuronal loss and behavioral impairment in mice lacking neurotrophin receptor p75.

The neurotrophin receptor p75 is a low-affinity receptor that binds neurotrophins. To investigate the role of p75 in the survival and function of central neurons, p75 null-mutant and wild type litter mate mice were tested on behavioral tasks. Null mutants showed significant performance deficits on water maze, inhibitory avoidance, motor activity, and habituation tasks that may be attributed to cognitive dysfunction or may represent a global sensorimotor impairment. The p75 null-mutant and wild type litter mate mice were assessed for central cholinergic deficit by using quantitative stereology to estimate the total neuronal number in basal forebrain and striatum and for subpopulations expressing the high-affinity tyrosine receptor kinase A (trkA) neurotrophin receptor and choline acetyltransferase (ChAT). In the adult brain, cholinergic neurons of the basal forebrain receive target-derived trophic support, whereas cholinergic striatal neurons do not. Adult p75 null-mutant mice had significant reduction of basal forebrain volume by 25% and had a corresponding significant loss of 37% of total basal forebrain neurons. The basal forebrain population of ChAT-positive neurons in p75-deficient mice declined significantly by 27%, whereas the trkA-positive population did not change significantly. There was no significant change in striatal volume or in striatal neuronal number either in total or by cholinergic subpopulation. These results demonstrate vulnerability to the lack of p75 in adult central neurons that are neurotrophin dependent. In addition, the loss of noncholinergic central neurons in mice lacking p75 suggests a role for p75 in cell survival by an as yet undetermined mechanism. Possible direct and indirect effects of p75 loss on neuronal survival are discussed.

Hippocampal grafts of acetylcholine-producing cells are sufficient to improve behavioural performance following a unilateral fimbria-fornix lesion.

Lesions of the septohippocampal pathway produce cognitive deficits that are partially attenuated by grafts of cholinergic-rich tissue into denervated target regions or by systemic administration of cholinomimetic drugs. In the present study, fibroblasts engineered to produce acetylcholine were used to test the hypothesis that restoration of hippocampal acetylcholine in rats with septohippocampal lesions is sufficient to improve cognitive processing post-damage. Rats received unilateral grafts of acetylcholine-producing or control fibroblasts into the hippocampus immediately prior to an aspirative lesion of the ipsilateral fimbria-fornix. Some rats with fimbria-fornix lesions were implanted with acetylcholine-producing or control fibroblasts into the neocortex, another major target of the basal forebrain cholinergic system, to determine if the site of acetylcholine delivery to the damaged brain is critical for functional recovery. Rats were tested in a hidden platform water maze task, a cued water maze task and activity chambers between one and three weeks post-grafting. Compared to unoperated controls, rats with fimbria fornix lesions only were significantly impaired in hidden platform water maze performance. Hippocampal grafts of acetylcholine-producing cells reduced lesion-induced deficits in the water maze, whereas hippocampal control grafts and cortical grafts of either cell type were without effect. Locomotor activity and cued water maze performance were unaffected by the lesion or the implants. Taken together, these data indicate that water maze deficits produced by fimbria fornix lesions, which disrupt a number of hippocampal neurotransmitter systems, can be attenuated by target specific replacement of acetylcholine in the hippocampus and that this recovery occurs in the absence of circuitry repair.

Altered synaptic physiology and reduced susceptibility to kainate-induced seizures in GluR6-deficient mice.

L-glutamate, the neurotransmitter of the majority of excitatory synapses in the brain, acts on three classes of ionotropic receptors: NMDA (N-methyl-D-aspartate), AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) and kainate receptors. Little is known about the physiological role of kainate receptors because in many experimental situations it is not possible to distinguish them from AMPA receptors. Mice with disrupted kainate receptor genes enable the study of the specific role of kainate receptors in synaptic transmission as well as in the neurotoxic effects of kainate. We have now generated mutant mice lacking the kainate-receptor subunit GluR6. The hippocampal neurons in the CA3 region of these mutant mice are much less sensitive to kainate. In addition, a postsynaptic kainate current evoked in CA3 neurons by a train of stimulation of the mossy fibre system is absent in the mutant. We find that GluR6-deficient mice are less susceptible to systemic administration of kainate, as judged by onset of seizures and by the activation of immediate early genes in the hippocampus. Our results indicate that kainate receptors containing the GluR6 subunit are important in synaptic transmission as well as in the epileptogenic effects of kainate.

Bicuculline administered into the amygdala after training blocks benzodiazepine-induced amnesia.

Male Sprague-Dawley rats were injected (i.p.) with either midazolam (MDZ, 2.0 mg/kg) or vehicle (1.0 ml/kg) 10 min before they were trained on a multiple-trial inhibitory avoidance task. Immediately following the training, bicuculline methiodide (BMI; 2.0, 5.6, 56.0 or 197.0 pmol/0.5 microl) or vehicle (0.5 microl) was infused bilaterally into the amygdala. On a 48 h retention test the performance of the MDZ-treated animals was significantly poorer than that of controls. The retention of MDZ-treated animals given intra-amygdala injections of the lowest dose of BMI (2.0 pmol) was comparable to that of controls, whereas higher doses of BMI impaired retention. The present results are consistent with other findings indicating that the amygdala mediates the amnestic effects of benzodiazepines on aversive learning. Furthermore, these data suggest that benzodiazepines impair memory by disrupting post-training processes underlying memory consolidation.

Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation.

The hippocampus is one of the few areas of the rodent brain that continues to produce neurons postnatally. Neurogenesis reportedly persists in rats up to 11 months of age. Using bromodeoxyuridine (BrdU) labeling, the present study confirms that in the adult rat brain, neuronal progenitor cells divide at the border between the hilus and the granule cell layer (GCL). In adult rats, the progeny of these cells migrate into the GCL and express the neuronal markers NeuN and calbindin-D28k. However, neurogenesis was drastically reduced in aged rats. Six-to 27-month-old Fischer rats were injected intraperitoneally with BrdU to detect newborn cells in vivo and to follow their fate in the dentate gyrus. When killed 4-6 weeks after BrdU labeling, 12- to 27-month-old rats exhibited a significant decline in the density of BrdU-positive cells in the granule cell layer compared with 6-month-old controls. Decreased neurogenesis in aging rats was accompanied by reduced immunoreactivity for poly-sialylated neural cell adhesion molecule, a molecule that is involved in migration and process elongation of developing neurons. When animals were killed immediately (12 hr) after BrdU injection, significantly fewer labeled cells were observed in the GCL and adjacent subgranular zone of aged rats, indicative of a decrease in mitotic activity of neuronal precursor cells. The reduced proliferation was not attributable to a general aged-related metabolic impairment, because the density of BrdU-positive cells was not altered in other brain regions with known mitotic activity (e.g., hilus and lateral ventricle wall). The decline in neurogenesis that occurs throughout the lifespan of an animal can thus be related to a decreasing proliferation of granule cell precursors.

Midazolam administered to rats induces anterograde amnesia for changes in reward magnitude.

L. P. Crespi (1942) showed that rats trained to run an alley for a large food reward slowed down when shifted to a small reward. This effect is usually interpreted as an aversive emotional response to reward reduction (A. Amsel, 1958). Benzodiazepines attenuate the behavioral effects of reward reduction (cf. C. F. Flaherty, 1990), but the emphasis has been on their anxiolytic, not memory-impairing, effects. Researchers trained rats (175-200 g) to run an alley for food until asymptote was reached. Reward magnitude was then either decreased (Experiment 1) or increased (Experiment 2). The benzodiazepine midazolam (1 mg/kg ip), injected immediately prior to a decrease or increase in reward magnitude, impaired the later retention of both changes in a manner consistent with anterograde amnesia. The findings suggest that the memory-impairing effects of benzodiazepines may, at least in part, influence the response to reward reduction.

Infusion of the GABAergic antagonist bicuculline into the medial septal area does not block the impairing effects of systemically administered midazolam on inhibitory avoidance retention.

This experiment investigated the effect of intraseptal administration of the GABAergic antagonist bicuculline methiodide on benzodiazepine-induced amnesia. Male Sprague-Dawley rats were implanted with cannula aimed at the medial septal area and allowed to recover for 1 week. Ten minutes prior to training in a continuous multiple trial inhibitory avoidance task, buffer solution or bicuculline methiodide (56 or 100 pmol/0.5 microliter) was injected into the medial septal area. This infusion was immediately followed by systemic (ip) administration of saline or midazolam (1.5 or 3.0 mg/kg). In comparison with saline controls, animals given the higher dose of midazolam (3.0 mg/kg), required more trials to reach acquisition criterion (remaining in the starting chamber for 100 s). This midazolam-induced acquisition deficit was blocked by an intraseptal infusion of bicuculline methiodide (100 pmol). On a 48-h retention test the performance of animals given either dose of midazolam was significantly impaired relative to vehicle controls. Furthermore, although intraseptal infusion of bicuculline methiodide prior to systemic injection of midazolam blocked the midazolam-induced acquisition impairment, bicuculline did not block the midazolam-induced retention impairment. These results suggest that although the medial septal area may be involved in midazolam-induced acquisition deficits, this area is not a critical site of action for benzodiazepine-induced effects on inhibitory avoidance retention.

Localization in the amygdala of the amnestic action of diazepam on emotional memory.

It is well known that systemically administered benzodiazepines (BZDs) induce anterograde amnesia in a variety of learning tasks. BZs effects are mediated through the GABAA complex by enhancing GABA-induced synaptic inhibition. As the GABAergic system in the amygdaloid complex (AC) is a site of action for the anxiolytic effects of BZs, such findings suggest that BZs may also influence memory through the amygdala. The present report summarizes a recent series of experiments designed to examine this implication. In a first experiment rats received either sham or bilateral AC lesion using N-methyl-D-aspartic acid (NMDA). One week later, animals were trained on an inhibitory avoidance task and tested 48 h later. Diazepam (DZP; 1.0 and 2.0 mg/kg, i.p.) or vehicle was injected 30 min prior to acquisition. The results demonstrate that DZP-induced retention deficits was blocked in rats with AC lesions. In a second experiment, in an attempt to localize the site of BZDs amnestic action in the AC, we tested the effects of DZP in rats with bilateral ibotenic acid-induced lesions of central (CE), lateral (LAT) or basolateral (BL) amygdala nuclei. The results shown that retention was impaired in animals with CE and LAT lesions but not in animals with BL lesions. In a third experiment we tested the effects of DZP microinjections in different nuclei of the AC on retention performance of rats trained in an avoidance task. The results demonstrate that DZP microinjection prior training in the BL/LAT, but not CE nuclei produce anterograde amnesia.(ABSTRACT TRUNCATED AT 250 WORDS)

Midazolam administered into the amygdala impairs retention of an inhibitory avoidance task.

This experiment investigated the amnestic effects of injections of the benzodiazepine midazolam administered into the amygdala prior to training on an inhibitory avoidance task. Male Sprague-Dawley rats were implanted bilaterally with cannulae aimed at the amygdala. After 1 week recovery a buffer solution or midazolam (3 or 10 micrograms/0.5 microliters) was injected bilaterally 5 min before a single training trail in a two-compartment inhibitory avoidance apparatus. The pretraining intra-amygdala injections of midazolam did not affect the training step-through latencies. However, on a 48-h retention test the step-through latencies of the midazolam-treated animals were significantly lower than those of the buffer controls. These findings are consistent with other recent evidence indicating that the amygdala is involved in mediating the amnestic effects of benzodiazepines.

Bicuculline administered into the amygdala blocks benzodiazepine-induced amnesia.

This experiment investigated the effect of intra-amygdala administration of the GABAergic antagonist bicuculline methiodide on benzodiazepine-induced amnesia. Male Sprague-Dawley rats were implanted bilaterally with cannulae aimed at the amygdala and allowed to recover for 1 week. Ten minutes before training in a continuous multiple trial inhibitory avoidance task a buffer solution or bicuculline methiodide (56 pmol/0.5 microliters) was injected bilaterally into the amygdala and this injection was immediately followed by a systemic injection of saline or midazolam (1.0 mg/kg). In comparison with saline controls, midazolam-treated animals required more trials to reach the acquisition criterion of remaining in the starting chamber for 100 s. The midazolam effect on acquisition was not attenuated by intra-amygdala infusion of bicuculline methiodide, suggesting that the midazolam-induced changes in acquisition behavior do not involve the amygdaloid GABAergic system. On a 48-h retention test the performance of the midazolam-treated animals was significantly poorer than that of the controls. However, the retention performance of animals given intra-amygdala injections of bicuculline methiodide prior to the systemic injection of midazolam was comparable to that of the saline controls. These results suggest that the amygdaloid GABAergic system mediates the impairing effects of midazolam on retention of inhibitory avoidance training.

Basolateral amygdala lesions block diazepam-induced anterograde amnesia in an inhibitory avoidance task.

This experiment examined the effects of diazepam (DZP) on acquisition and retention of an inhibitory avoidance response by rats with excitotoxic-induced lesions of central (CE), lateral (LAT), or basolateral (BL) amygdala nuclei. Sham-operated and lesioned rats received i.p. injections of DZP (2.0 mg per kg of body weight) 30 min before training in a continuous multiple-trial inhibitory avoidance task. Retention was tested 48 h later. Acquisition was not impaired by the lesions or the DZP. Retention was impaired in animals with CE and LAT lesions in comparison with sham-operated controls. DZP impaired retention in the sham-operated controls as well as CE- and LAT-lesioned animals but did not affect retention in animals with BL lesions. These findings indicate that the DZP-induced anterograde amnesia for inhibitory avoidance training is mediated through influences involving the BL amygdala nucleus.

Amygdala lesions block the amnestic effects of diazepam.

This experiment examined the effects of pre-training systemic injections of the benzodiazepine (BZ) diazepam (DZP) on learning and retention of an inhibitory avoidance response in rats with bilateral lesions of the amygdaloid complex (AC) induced by intra-amygdala injections of the excitotoxin N-methyl-D-aspartic acid (NMDA). Unoperated, sham-operated and AC-lesioned rats received i.p. injections of DZP (1.0 or 2.0 mg/kg) or vehicle 30 min prior to training in a continuous multiple-trial inhibitory avoidance task. Retention was tested 48 h later. The acquisition and retention of the AC-lesioned rats were impaired, relative to that of the unoperated and sham controls. In the unoperated and sham controls, DZP impaired retention but did not affect acquisition. In contrast, in animals with AC lesions, DZP did not affect either acquisition or retention. These findings suggest that the amnestic effects of DZP are mediated, at least in part, through influences involving the AC.