Cara L. Wellman

Brief Uncontrollable Stress Causes Dendritic Retraction in Infralimbic Cortex and Resistance to Fear Extinction in Mice

Extinction of conditioned fear responses is an active learning process resulting from the repeated presentation of a conditioned stimulus in the absence of the unconditioned aversive stimulus. Recent research implicates the medial prefrontal cortex (mPFC) in the mediation of fear extinction in rodents and the pathophysiology of posttraumatic stress disorder. However, there is currently little understanding of precisely how stress can impact fear extinction and the neural circuitry subserving this behavior. The present study examined the effects of brief exposure to an uncontrollable stressor on (1) fear conditioning and fear extinction, and (2) dendritic morphology of pyramidal neurons in the infralimbic (IL) and prelimbic (PL) regions of the mPFC in mice. Exposure to three episodes of stress ending 24 h before fear conditioning significantly attenuated the rate of cued fear extinction relative to nonstressed controls, but did not affect fear conditioning or cue or context recall. Analysis of Golgi-stained neurons showed that one or three exposures to daily swim stress caused significant retraction of terminal branches of apical, but not basilar, dendrites of IL neurons. In contrast, PL neuronal morphology was unaltered by stress. These data demonstrate that IL, but not PL, neurons are highly sensitive to even brief exposure to stress, and that this same form of stress impairs fear extinction. Present findings suggest that trauma may compromise the functional integrity of the mPFC with implications for the pathophysiology of certain neuropsychiatric disorders.

Impaired Stress-Coping and Fear Extinction and Abnormal Corticolimbic Morphology in Serotonin Transporter Knock-Out Mice

A lesser-expressing form of the human 5-HT transporter (5-HTT) gene has been associated with increased fear and anxiety and vulnerability to the effects of stress. These phenotypic abnormalities are linked to functional and anatomical disturbances in a neural pathway connecting the prefrontal cortex (PFC) and amygdala. Likewise, rodent and nonhuman primate studies indicate a major role for PFC and amygdala in the mediation of fear- and stress-related behaviors. We used a 5-HTT knock-out (KO) mouse to examine the effects of genetically driven loss of 5-HTT function for the following: (1) depression-related behavior in response to repeated stress, and pavlovian fear conditioning, extinction, and extinction recall; and (2) dendritic morphology and spine density of Golgi-stained pyramidal neurons in the infralimbic cortex (IL) and the basolateral amygdala (BLA). 5-HTT KO mice exhibited increased depressive-like immobility after repeated exposure to forced swim stress, compared with wild-type (WT) controls. Whereas fear conditioning and fear extinction was normal, 5-HTT KO mice exhibited a significant deficit in extinction recall. The apical dendritic branches of IL pyramidal neurons in 5-HTT KO mice were significantly increased in length relative to WT mice. Pyramidal neurons in BLA had normal dendritic morphology but significantly greater spine density in 5-HT KO mice compared with WT mice. Together, the present findings demonstrate a specific phenotypic profile of fear- and stress-related deficits in 5-HTT KO mice, accompanied by morphological abnormalities in two key neural loci. These data provide insight into the behavioral sequelae of loss of 5-HTT gene function and identify potential neural substrates underlying these phenotypes.

Characteristics of BDNF-induced weight loss

There is evidence that central infusion of brain-derived neurotrophic factor (BDNF) induces weight loss in rats. We have begun to investigate the physiological basis for BDNF-induced weight loss by assessing its relationship to (a) appetite, (b) serum indices of metabolic and renal toxicity, and (c) brain monoamine activity in areas associated with feeding or motor function. BDNF (0-6 microgram/day) was infused into the lateral ventricle (LV) of male Long-Evans rats for 14 days. Body weight and food intake were monitored throughout infusion and recovery periods. BDNF induced severe, dose-dependent appetite suppression and weight loss. Although appetite began to recover after the 10th infusion day, body weight had not returned to control values at the end of the recovery period. The weight loss observed in BDNF-infused rats was related to appetite suppression, since uninfused rats that were pair-fed to high dose BDNF-treated rats showed comparable weight loss. Despite severe weight loss, serum BUN, creatinine, thyroxine, glucose, and total protein were not affected by BDNF infusion. Striatal DO-PAC/DA was similarly unaffected by BDNF. In contrast, BDNF-infused rats showed a dose-dependent increase in hypothalamic 5-HIAA/5-HT that was not observed in pair-fed rats, suggesting that the observed increase in hypothalamic 5-HIAA/5-HT was a direct effect of BDNF infusion rather than a secondary effect of food restriction. These data suggest that BDNF may induce appetite suppression and weight loss through a central mechanism.

Strain Differences in Stress Responsivity Are Associated with Divergent Amygdala Gene Expression and Glutamate-Mediated Neuronal Excitability

Stress is a major risk factor for numerous neuropsychiatric diseases. However, susceptibility to stress and the qualitative nature of stress effects on behavior differ markedly among individuals. This is partly because of the moderating influence of genetic factors. Inbred mouse strains provide a relatively stable and restricted range of genetic and environmental variability that is valuable for disentangling gene–stress interactions. Here, we screened a panel of inbred strains for anxiety- and depression-related phenotypes at baseline (trait) and after exposure to repeated restraint. Two strains, DBA/2J and C57BL/6J, differed in trait and restraint-induced anxiety-related behavior (dark/light exploration, elevated plus maze). Gene expression analysis of amygdala, medial prefrontal cortex, and hippocampus revealed divergent expression in DBA/2J and C57BL/6J both at baseline and after repeated restraint. Restraint produced strain-dependent expression alterations in various genes including glutamate receptors (e.g., Grin1, Grik1). To elucidate neuronal correlates of these strain differences, we performed ex vivo analysis of glutamate excitatory neurotransmission in amygdala principal neurons. Repeated restraint augmented amygdala excitatory postsynaptic signaling and altered metaplasticity (temporal summation of NMDA receptor currents) in DBA/2J but not C57BL/6J. Furthermore, we found that the C57BL/6J-like changes in anxiety-related behavior after restraint were absent in null mutants lacking the modulatory NMDA receptor subunit Grin2a, but not the AMPA receptor subunit Gria1. Grin2a null mutants exhibited significant (∼30%) loss of dendritic spines on amygdala principal neurons under nonrestraint conditions. Collectively, our data support a model in which genetic variation in glutamatergic neuroplasticity in corticolimbic circuitry underlies phenotypic variation in responsivity to stress.

Chronic Stress Effects on Dendritic Morphology in Medial Prefrontal Cortex: Sex Differences and Estrogen Dependence

A growing body of work has documented sex differences in many behavioral, neurochemical, and morphological responses to stress. Chronic stress alters morphology of dendrites in medial prefrontal cortex in male rats. However, potential sex differences in stress-induced morphological changes in medial prefrontal cortex have not been examined. Thus, in Experiment 1 we assessed dendritic morphology in medial prefrontal cortex in male and female rats after chronic stress. Male and female rats underwent either 3 hours of restraint daily for 1 week or were left unhandled except for weighing. On the final day of restraint, all rats were euthanized and brains were stained using a Golgi-Cox procedure. Pyramidal neurons in layer II-III of medial prefrontal cortex were drawn in three dimensions, and morphology of apical and basilar arbors was quantified. In males, stress decreased apical dendritic branch number and length, whereas in females, stress increased apical dendritic length. In Experiment 2, we assessed whether estradiol mediates this stress-induced dendritic hypertrophy in females by assessing the effects of restraint stress on female rats that had received either ovariectomy with or without 17-beta-estradiol replacement or sham ovariectomy. Brains were processed and neurons reconstructed as described in Experiment 1. Both sham-operated and ovariectomized rats with estradiol implants showed stress-induced increases in apical dendritic material, whereas ovariectomy without estradiol replacement prevented the stress-induced increase. Thus, the stress-induced increase in apical dendritic material in females is estradiol-dependent.

NMDA Receptor Blockade Alters Stress-Induced Dendritic Remodeling in Medial Prefrontal Cortex

The development and relapse of many psychopathologies can be linked to both stress and prefrontal cortex dysfunction. Glucocorticoid stress hormones target medial prefrontal cortex (mPFC) and either chronic stress or chronic administration of glucocorticoids produces dendritic remodeling in prefrontal pyramidal neurons. Exposure to stress also causes an increase in the release of the excitatory amino acid glutamate, which binds to N-methyl-D-aspartate (NMDA) receptors, which are plentiful in mPFC. NMDA receptor activation is crucial for producing hippocampal dendritic remodeling due to stress and for dendritic reorganization in frontal cortex after cholinergic deafferentation. Thus, NMDA receptors could mediate stress-induced dendritic retraction in mPFC. To test this hypothesis, dendritic morphology of pyramidal cells in mPFC was assessed after blocking NMDA receptors with the competitive NMDA antagonist ±3-(2-carboxypiperazin-4yl)propyl-1-phosphonic acid (CPP) during restraint stress. Administration of CPP prevented stress-induced dendritic atrophy. Instead, CPP-injected stressed rats showed hypertrophy of apical dendrites compared with controls. These results suggest that NMDA activation is crucial for stress-induced dendritic atrophy in mPFC. Furthermore, NMDA receptor blockade uncovers a new pattern of stress-induced dendritic changes, suggesting that other neurohormonal changes in concert with NMDA receptor activation underlie the net dendritic retraction seen after chronic stress.

Daily injections alter spine density in rat medial prefrontal cortex

Apical dendrites of pyramidal neurons in medial prefrontal cortex are reorganized after chronic corticosterone treatment. In this study, we assessed the effects of chronic corticosterone administration on dendritic spines. Adult male rats received sc injections of either corticosterone or vehicle daily for 3 weeks or were left untreated. Layer II-III neurons in medial prefrontal cortex were stained using a Golgi-Cox procedure and spines were counted on the portions of apical dendrites where maximal corticosterone-induced dendritic changes occur. In vehicle- and corticosterone-treated rats, spine density proximal to the soma was increased relative to untreated rats, while spine density in the distal portion of the apical arbor was unchanged, suggesting that daily handling alone altered spine density. This stress-induced change in spine density likely reflects important functional changes in excitatory synaptic inputs in prefrontal cortex.

Chronic stress-induced hippocampal dendritic retraction requires CA3 NMDA receptors

Chronic stress induces dendritic retraction in the hippocampal CA3 subregion, but the mechanisms responsible for this retraction and its impact on neural circuitry are not well understood. To determine the role of NMDA (N-methyl-d-aspartic acid) receptor (NMDAR)-mediated signaling in this process, we compared the effects of chronic immobilization stress (CIS) on hippocampal dendritic morphology, hypothalamic-pituitary-adrenal (HPA) axis activation, and anxiety-related and hippocampus-dependent behaviors, in transgenic male mice in which the NMDAR had been selectively deleted in CA3 pyramidal cells and in non-mutant littermates. We found that CIS exposure for 10 consecutive days in non-mutant mice effectively induces HPA axis activation and dendritic retraction of CA3 short-shaft pyramidal neurons, but not CA3 long-shaft pyramidal neurons, suggesting a differential cellular stress response in this region. Dendritic reorganization of short-shaft neurons occurred throughout the longitudinal axis of the hippocampus and, in particular, in the ventral pole of this structure. We also observed a robust retraction of dendrites in dorsal CA1 pyramidal neurons in the non-mutant C57BL/6 mouse strain. Strikingly, chronic stress-induced dendritic retraction was not evident in any of the neurons in either CA3 or CA1 in the mutant mice that had a functional lack of NMDARs restricted to CA3 pyramidal neurons. Interestingly, the prevention of dendritic retraction in the mutant mice had a minimal effect on HPA axis activation and behavioral alterations that were induced by chronic stress. These data support a role for NMDAR-dependent glutamatergic signaling in CA3 in the cell-type specific induction of dendritic retraction in two hippocampal subregions following chronic stress.

CHRONIC STRESS ALTERS NEURAL ACTIVITY IN MEDIAL PREFRONTAL CORTEX DURING RETRIEVAL OF EXTINCTION

Chronic restraint stress produces morphological changes in medial prefrontal cortex and disrupts a prefrontally mediated behavior, retrieval of extinction. To assess potential physiological correlates of these alterations, we compared neural activity in infralimbic and prelimbic cortex of unstressed versus stressed rats during fear conditioning and extinction. After implantation of microwire bundles into infralimbic or prelimbic cortex, rats were either unstressed or stressed via placement in a plastic restrainer (3 h/day for 1 week). Rats then underwent fear conditioning and extinction while activity of neurons in infralimbic or prelimbic cortex was recorded. Percent freezing and neural activity were assessed during all phases of training. Chronic stress enhanced freezing during acquisition of conditioned fear, and altered both prelimbic and infralimbic activity during this phase. Stress did not alter initial extinction or conditioned stimulus (CS)-related activity during this phase. However, stress impaired retrieval of extinction assessed 24 h later, and this was accompanied by alterations in neuronal activity in both prelimbic and infralimbic cortex. In prelimbic cortex, unstressed rats showed decreased activity in response to CS presentation, whereas stressed rats showed no change. In infralimbic cortex, neurons in unstressed rats exhibited increased firing in response to the CS, whereas stressed rats showed no increase in infralimbic firing during the tone. Finally, CS-related firing in infralimbic but not prelimbic cortex was correlated with extinction retrieval. Thus, the stress-induced alteration of neuronal activity in infralimbic cortex may be responsible for the stress-induced deficit in retrieval of extinction.

The effects of intrahippocampal BDNF and NGF on spatial learning in aged long evans rats

Spatial learning rate was compared in cognitively impaired aged rats infused with either brain-derived neurotrophic factor (BDNF) or nerve growth factor (NGF). BDNF or NGF was infused into the dorsal hippocampus/third ventricle while animals were being trained on the Morris water maze. Training continued until all rats met a spatial learning criterion. Seven weeks later, they were tested for retention of the task, and sacrificed for assessment of hippocampal high-affinity choline uptake (HACU) or hypothalamic biogenic amine levels. NGF, but not BDNF, improved spatial learning rate in aged rats and increased hippocampal choline uptake weeks after withdrawal of NGF. Although BDNF did not improve spatial learning, it did induce a partial, long-term normalization of the elevated hypothalamic 5-HT levels observed in our aged rats. These data suggest that (1) intrahippocampal/intraventricular infusion of NGF can improve the learning rate of aged, spatial learning-impaired rats, and that this improvement in acquisition could be associated with increased hippocampal cholinergic activity, and (2) that the BDNF-induced normalization of hypothalamic 5-HT levels in aged rats was not sufficient to improve learning rate in aged, spatial learning-impaired rats.