Patricia Shinnick-Gallagher

Fear conditioning induces a lasting potentiation of synaptic currents in vitro.

The amygdala plays a critical role in the mediation of emotional responses, particularly fear, in both humans and animals. Fear conditioning, a conditioned learning paradigm, has served as a model for emotional learning in animals, and the neuroanatomical circuitry underlying the auditory fear-conditioning paradigm is well characterized. Synaptic transmission in the medial geniculate nucleus (MGN) to lateral nucleus of the amygdala (LA) pathway, a key segment of the auditory fear conditioning circuit, is mediated largely through N-methyl-D-aspartate (NMDA) and non-NMDA (such as α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)) glutamate receptors; the potential for neural plasticity in this pathway is suggested by its capacity to support long-term potentiation (LTP),. Here we report a long-lasting increase in the synaptic efficacy of the MGN–LA pathway attributable to fear-conditioning itself, rather than an electrically induced model of learning. Fear-conditioned animals show a presynaptic facilitation of AMPA-receptor-mediated transmission, directly measured in vitro with whole-cell recordings in lateral amygdala neurons. These findings represent one of the first in vitro measures of synaptic plasticity resulting from emotional learning by whole animals.

LONG-TERM BEHAVIORAL AND NEURODEGENERATIVE EFFECTS OF PERINATAL PHENCYCLIDINE ADMINISTRATION: IMPLICATIONS FOR SCHIZOPHRENIA

Both acute and chronic administration of N-methyl-D-aspartate (NMDA) receptor antagonists such as phencyclidine and dizocilpine have been proposed to mimic some of the symptoms of schizophrenia. The purposes of the present study were first, to characterize the long-term behavioral and neurodegenerative effects of subchronic administration of phencyclidine to perinatal rats and second, to determine whether pretreatment with olanzapine could attenuate these effects. On postnatal days 7, 9 and 11 rat pups were pretreated with either vehicle or olanzapine prior to administration of either saline or phencyclidine (10 mg/kg). Some pups were killed on postnatal day 12 for biochemical determinations and others were tested on postnatal days 24-28 for prepulse inhibition of acoustic startle, on postnatal day 42 for phencyclidine-induced locomotor activity and between postnatal days 33 and 70 for acquisition of a delayed spatial learning task. Phencyclidine treatment resulted in a substantial increase in fragmented DNA in the frontal and olfactory cortices consistent with neurodegeneration by an apoptotic mechanism. An increase in the NMDA receptor NR1 subunit mRNA was also observed in the cortex. Gel shift assays showed that phencyclidine also increased the nuclear translocation of nuclear factor-kappaB proteins in the prefrontal cortex. In tissue from the frontal cortex, western blot analysis revealed that phencyclidine treatment increased Bax and decreased Bcl-X(L) proteins. Later in development, it was observed that perinatal phencyclidine treatment significantly retarded baseline prepulse inhibition of acoustic startle measured shortly after weaning. In 42-day-old rats, it was found that challenge with 2 mg/kg phencyclidine increased locomotor activity to a significantly greater extent in the rats that had been pretreated with phencyclidine. Similarly, perinatal phencyclidine treatment significantly delayed the acquisition of a delayed spatial alternation task. Each of the aforementioned changes (except for the spatial learning task, which was not tested) was significantly inhibited by olanzapine pretreatment, an antipsychotic drug known to be effective against both positive and negative symptoms of schizophrenia. Further, olanzapine treatment for 12 days following the administration of phencyclidine was also able to reverse the phencyclidine-induced deficit in baseline prepulse inhibition. Together these data suggest that perinatal administration of phencyclidine results in long-term behavioral changes that may be mechanistically related to the apoptotic neurodegeneration observed in the frontal cortex. It is postulated that these deficits may model the hypofrontality observed in schizophrenia and that this model may be helpful in designing appropriate pharmacotherapy.

Glucocorticoids alter calcium conductances and calcium channel subunit expression in basolateral amygdala neurons

Glucocorticoid hormones, which are released in high amounts after stress, enter the brain where they bind to intracellular receptors that are abundant in limbic areas, in particular the hippocampus and amygdala nuclei. Behavioural studies indicate that glucocorticoids modulate learning and memory processes via receptors in the hippocampus and amygdala. So far, the effects of glucocorticoids on amygdala neurons have not been investigated at the cellular and molecular level. We report here that in vitro application of glucocorticoids for 20 min increases 1–4 h later the amplitude of sustained, high‐voltage‐activated calcium currents in principal neurons of the basolateral amygdala. In contrast, the transient, low‐voltage‐activated currents were decreased. We examined whether these functional changes in calcium conductance were accompanied by transcriptional regulation of calcium channel subunits. Analysis of the RNA – collected after recording and then linearly amplified – revealed that glucocorticoid‐mediated increases in sustained calcium currents are associated with a parallel shift in the relative expression of the α1 subunit constituting the pore of the sustained, high‐voltage‐activated (L‐type) calcium channel. These data indicate that glucocorticoids, probably by selectively targeting genes encoding calcium channel subunits, largely alter the calcium influx into basolateral amygdala neurons. These actions could modify amygdala network function and thus contribute to the behavioural effects exerted by the stress hormones via the basolateral amygdala.

Corticotropin-Releasing Factor and Urocortin I Modulate Excitatory Glutamatergic Synaptic Transmission

Corticotropin-releasing factor (CRF)-related peptides serve as hormones and neuromodulators of the stress response and play a role in affective disorders. These peptides are known to alter complex behaviors and neuronal properties, but their receptor-mediated effects at CNS synapses are not well described. Here we show that excitatory glutamatergic transmission is modulated by two endogenous CRF-related peptide ligands, corticotropin-releasing factor [CRF rat/human (r/h)] and Urocortin I (Ucn I), within the central nucleus of the amygdala (CeA) and the lateral septum mediolateral nucleus (LSMLN). These limbic nuclei are reciprocally innervated, are involved in stress and affective disorders, and have high densities of the CRF receptors CRF1 and CRF2. Activation of these receptors exerts diametrically opposed actions on glutamatergic transmission in these nuclei. In the CeA, CRF(r/h) depressed excitatory glutamatergic transmission through a CRF1-mediated postsynaptic action, whereas Ucn I facilitated synaptic responses through presynaptic and postsynaptic CRF2-mediated mechanisms. Conversely, in the LSMLN, CRF caused a CRF1-mediated facilitation of glutamatergic transmission via postsynaptic mechanisms, whereas Ucn I depressed EPSCs by postsynaptic and presynaptic CRF2-mediated actions. Furthermore, antagonists of these receptors also affected glutamatergic neurotransmission, indicating that endogenous ligands tonically modulated synoptic activity at these synapses. These data show that CRF receptors in CeA and LSMLN synapses exert and maintain a significant synaptic tone and thereby regulate excitatory glutamatergic transmission. The results also suggest that CRF receptors may provide novel targets in affective disorders and stress.

Trans-ACPD and l-APB presynaptically inhibit excitatory glutamatergic transmission in the basolateral amygdala (BLA)

Intracellular recordings were obtained from neurones of the basolateral nucleus of the amygdala (BLA) and glutamate-mediated EPSPs evoked by stimulation of the stria terminalis (ST). The conformationally restricted analogue of glutamate trans-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD) caused a dose-dependent reduction in EPSP amplitude, EC50 approximately 50 microM. This effect was mimicked by the glutamate autoreceptor agonist, L-aminophosphonobutyric acid (L-APB, 50 microM). Furthermore, the effects of submaximal concentrations (50 microM) of trans-ACPD and L-APB were additive. The reduction in EPSP amplitude is observed with concentrations of both drugs that have no effect on either the resting membrane potential or the input resistance of BLA neurones. In addition, these compounds can reduce EPSP amplitude but not the response to exogenous application of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-proprionate (AMPA) suggesting activation of presynaptic receptors. These findings suggest that both trans-ACPD and L-APB act at presynaptic glutamate receptors on glutamatergic afferents to reduce excitatory transmission in the BLA.

Cocaine withdrawal enhances long‐term potentiation induced by corticotropin‐releasing factor at central amygdala glutamatergic synapses via CRF1, NMDA receptors and PKA

Cocaine addiction is an enduring, relapsing, behavioural disorder in which stressors reinstate cocaine‐seeking even after prolonged abstinence. Evidence suggests that the ‘anxiety‐like’ behaviour and stress associated with protracted withdrawal may be mediated by increased corticotropin‐releasing factor (CRF) in the central nucleus of the amygdala (CeA), a part of the limbic circuitry engaged in the coding and transmission of stimulus–reward associations. In the present study we describe a long‐lasting potentiation of glutamatergic transmission induced at lateral amygdala (LA)‐to‐CeA synapses by rat/human CRF. After 2 weeks of withdrawal from repeated intermittent exposure to cocaine, CRF‐induced long‐term potentiation (LTP) was greatly enhanced compared to the respective saline control group while, after short‐term withdrawal (24 h), there was no significant difference between the two treatment groups, indicating alterations in CRF systems during protracted withdrawal from chronic cocaine. After prolonged withdrawal, CRF‐induced LTP was dependent on activation of CRF2, CaV2.3 (R‐type) calcium channels and intracellular signalling through protein kinase C in both saline‐ and cocaine‐treated groups. The enhanced CRF‐induced LTP after 2 weeks of withdrawal was mediated through augmented CRF1 receptor function, associated with an increased signalling through protein kinase A, and required N‐methyl‐d‐aspartate (NMDA) receptors. Accordingly, single‐cell recordings revealed a significantly increased NMDA/AMPA ratio after prolonged withdrawal from the cocaine treatment. These results support a role for CRF1 receptor antagonists as plausible treatment options during withdrawal from chronic cocaine and suggest CaV2.3 blockers as potential candidates for pharmaceutical modulation of CRF systems.

Intracellular recordings from rat dorsolateral septal neurons, in vitro

We have made intracellular recordings from the dorsolateral septal nucleus (DLSN) in a rat brain slice. DLSN neurons fire short tetrodotoxin (TTX) sensitive action potentials followed by an after-hyperpolarization. The action potential is followed by either a Ca2+-dependent depolarization or a Ca2+-dependent after-hyperpolarization. Stimulation of medial septum results in antidromic invasion of DLSN neurons while stimulation of the fimbria/fornix results in orthodromic EPSPs or action potentials.

The effects of temperature, pH and Cl-pump inhibitors on GABA responses recorded from cat dorsal root ganglia

GABA applied by iontophoresis produced GABA-induced currents (GCs) and GABA-induced depolarizations (GDs) which were recorded intracellularly from cat dorsal root ganglia (DRG). Lowering the temperature (37 to 27 degrees C) of the preparation depressed the amplitude of GCs while prolonging their rise-time and decay time. This depressant action was mainly due to a hyperpolarizing shift in the GABA equilibrium potential (EGABA). GABA responses could also be depressed by alkalinization of the superfusion solution or addition of putative chloride pump inhibitors, e.g. SITS, furosemide or bumetanide. However, the mechanism by which these latter procedures depressed GABA responses was not due to a shift in EGABA as occurred with lowered temperature. Instead we suggest that alkalinization or the putative chloride pump inhibitors affect the chloride channel or some other site associated with the GABA receptor complex and cause the depression we observed. GABA responses could be facilitated by lowering the pH of the superfusion solution or by injecting ammonium ion into a DRG. These results suggest that a temperature-sensitive, inwardly directed chloride pump that is resistant to SITS, furosemide or bumetanide, operates in cat DRG.

Fear learning induces persistent facilitation of amygdala synaptic transmission

In the maintenance phase of fear memory, synaptic transmission is potentiated and the stimulus requirements and signalling mechanisms are altered for long‐term potentiation (LTP) in the cortico‐lateral amygdala (LA) pathway. These findings link amygdala synaptic plasticity to the coding of fear memories. Behavioural experiments suggest that the amygdala serves to store long‐term fear memories. Here we provide electrophysiological evidence showing that synaptic alterations in rats induced by fear conditioning are evident in vitro 10 days after fear conditioning. We show that synaptic transmission was facilitated and that high‐frequency stimulation dependent LTP (HFS–LTP) of the cortico‐lateral amygdala pathway remained attenuated 10 days following fear conditioning. Additionally, we found that the low‐frequency stimulation dependent LTP (LFS–LTP) measured 24 h after fear conditioning was absent 10 days post‐training. The persistent facilitation of synaptic transmission and occlusion of HFS–LTP suggests that, unlike hippocampal coding of contextual fear memory, the cortico‐lateral amygdala synapse is involved in the storage of long‐term fear memories. However, the absence of LFS–LTP 10 days following fear conditioning suggests that amygdala physiology 1 day following fear learning may reflect a dynamic state during memory stabilization that is inactive during the long‐term storage of fear memory. Results from these experiments have significant implications regarding the locus of storage for maladaptive fear memories and the synaptic alterations induced by these memories.

Decrease of GABA-immunoreactive neurons in the amygdala after electrical kindling in the rat

The present study was designed to investigate the effects of electrical kindling in vivo on GABA immunoreactivity (GABA-IR) of the lateral and basolateral amygdaloid nuclei 2-6 months post-stimulation. Male Sprague-Dawley rats were implanted with bipolar electrodes in the basolateral nucleus and stimulated once per day until 3-5 stage 5 seizures were observed. Coronal sections containing the amygdala were processed for GABA-IR using the contralateral side of the brain. Results indicate that, in comparison to controls, fully kindled animals showed a significant decrease in total number of GABA-IR amygdala neurons. Decreases in GABA-positive punctate structures surrounding unlabeled pyramidal cells were also observed, but not quantified. The present data suggest that epileptogenesis of the amygdala is associated with a significant reduction of GABA-IR in the lateral and basolateral areas throughout the contralateral amygdaloid nucleus.