Luke R. Johnson

LESIONS IN THE BED NUCLEUS OF THE STRIA TERMINALIS DISRUPT CORTICOSTERONE AND FREEZING RESPONSES ELICITED BY A CONTEXTUAL BUT NOT BY A SPECIFIC CUE-CONDITIONED FEAR STIMULUS

The bed nucleus of the stria terminalis (BNST) is believed to be a critical relay between the central nucleus of the amygdala (CE) and the paraventricular nucleus of the hypothalamus in the control of hypothalamic-pituitary-adrenal (HPA) responses elicited by conditioned fear stimuli. If correct, lesions of CE or BNST should block expression of HPA responses elicited by either a specific conditioned fear cue or a conditioned context. To test this, rats were subjected to cued (tone) or contextual classical fear conditioning. Two days later, electrolytic or sham lesions were placed in CE or BNST. After 5 days, the rats were tested for both behavioral (freezing) and neuroendocrine (corticosterone) responses to tone or contextual cues. CE lesions attenuated conditioned freezing and corticosterone responses to both tone and context. In contrast, BNST lesions attenuated these responses to contextual but not tone stimuli. These results suggest CE is indeed an essential output of the amygdala for the expression of conditioned fear responses, including HPA responses, regardless of the nature of the conditioned stimulus. However, because lesions of BNST only affected behavioral and endocrine responses to contextual stimuli, the results do not support the notion that BNST is critical for HPA responses elicited by conditioned fear stimuli in general. Instead, the BNST may be essential specifically for contextual conditioned fear responses, including both behavioral and HPA responses, by virtue of its connections with the hippocampus, a structure essential to contextual conditioning. The results are also not consistent with the hypothesis that BNST is only involved in unconditioned aspects of fear and anxiety.

Localization of glucocorticoid receptors at postsynaptic membranes in the lateral amygdala.

Glucocorticoids, released in high concentrations from the adrenal cortex during stressful experiences, bind to glucocorticoid receptors in nuclear and peri-nuclear sites in neuronal somata. Their classically known mode of action is to induce gene promoter receptors to alter gene transcription. Nuclear glucocorticoid receptors are particularly dense in brain regions crucial for memory, including memory of stressful experiences, such as the hippocampus and amygdala. While it has been proposed that glucocorticoids may also act via membrane bound receptors, the existence of the latter remains controversial. Using electron microscopy, we found glucocorticoid receptors localized to non-genomic sites in rat lateral amygdala, glia processes, presynaptic terminals, neuronal dendrites, and dendritic spines including spine organelles and postsynaptic membrane densities. The lateral nucleus of the amygdala is a region specifically implicated in the formation of memories for stressful experiences. These newly observed glucocorticoid receptor immunoreactive sites were in addition to glucocorticoid receptor immunoreactive signals observed using electron and confocal microscopy in lateral amygdala principal neuron and GABA neuron soma and nuclei, cellular domains traditionally associated with glucocorticoid immunoreactivity. In lateral amygdala, glucocorticoid receptors are thus also localized to non-nuclear-membrane translocation sites, particularly dendritic spines, where they show an affinity for postsynaptic membrane densities, and may have a specialized role in modulating synaptic transmission plasticity related to fear and emotional memory.

Input from the amygdala to the rat nucleus accumbens: Its relationship with tyrosine hydroxylase immunoreactivity and identified neurons

Both tyrosine hydroxylase-positive fibres from the mesolimbic dopamine system and amygdala projection fibres from the basolateral nucleus are known to terminate heavily in the nucleus accumbens. Caudal amygdala fibres travelling dorsally via the stria terminalis project densely to the nucleus accumbens shell, especially in the dopamine rich septal hook. The amygdala has been associated with the recognition of emotionally relevant stimuli while the mesolimbic dopamine system is implicated with reward mechanisms. There is behavioural and electrophysiological evidence that the amygdala input to the nucleus accumbens is modulated by the mesolimbic dopamine input, but it is not known how these pathways interact anatomically within the nucleus accumbens. Using a variety of neuroanatomical techniques including anterograde and retrograde tracing, immunocytochemistry and intracellular filling, we have demonstrated convergence of these inputs on to medium-sized spiny neurons. The terminals of the basolateral amygdala projection make asymmetrical synapses predominantly on the heads of spines which also receive on their necks or adjacent dendrites, symmetrical synaptic input from the mesolimbic dopamine system. Some of these neurons have also been identified as projection neurons, possibly to the ventral pallidum. We have shown a synaptic level how dopamine is positioned to modulate excitatory limbic input in the nucleus accumbens.

Stress at the Synapse: Signal Transduction Mechanisms of Adrenal Steroids at Neuronal Membranes

Adrenal steroids regulate neuronal excitability by altering ion channel conductance or gene transcription. All of us have vivid memories of past stressful events. Although decades of research have identified some of the behavioral principles of the effects of stress on memory, our understanding of the underlying neurobiology is incomplete. Stress itself is a complex phenomenon that can be described on multiple levels, one of which is the activation of the hypothalamic-pituitary-adrenal axis and cortisol release into the blood. The timing of stress on memory formation and memory recall are important, and the effects of cortisol on memory can be rapid, occurring within minutes. Increased cortisol just before and during memory formation enhances memory of the event. In contrast, cortisol elevation during memory recall reduces memory. Cortisol acts on glucocorticoid receptors (GRs) and mineralocorticoid receptors (MRs), which have long been known to function as transcription factors. A growing body of subcellular and electrophysiological evidence indicates that cortisol may also promote rapid effects on neuron-to-neuron communication through membrane-located MRs and GRs. The site of neuron-to-neuron communication is the synapse; thus, stress may directly and rapidly regulate the synapse itself. Low concentrations of cortisol at the synapse can increase neurotransmitter release, whereas higher concentrations may dampen excitability by reducing current flow through ion channels. These data may help to explain how stress can be so influential on memory and cognitive performance. This Review, with three figures and 111 citations, highlights the signaling processes by which cortisol may influence neuronal excitability and thereby memory and cognitive performance. As the key neuron-to-neuron interface, the synapse is involved in learning and memory, including traumatic memories during times of stress. However, the signal transduction mechanisms by which stress mediates its lasting effects on synapse transmission and on memory are not fully understood. A key component of the stress response is the increased secretion of adrenal steroids. Adrenal steroids (e.g., cortisol) bind to genomic mineralocorticoid and glucocorticoid receptors (gMRs and gGRs) in the cytosol. In addition, they may act through membrane receptors (mMRs and mGRs), and signal transduction through these receptors may allow for rapid modulation of synaptic transmission as well as modulation of membrane ion currents. mMRs increase synaptic and neuronal excitability; mechanisms include the facilitation of glutamate release through extracellular signal–regulated kinase signal transduction. In contrast, mGRs decrease synaptic and neuronal excitability by reducing calcium currents through N-methyl-d-aspartate receptors and voltage-gated calcium channels by way of protein kinase A– and G protein–dependent mechanisms. This body of functional data complements anatomical evidence localizing GRs to the postsynaptic membrane. Finally, accumulating data also suggest the possibility that mMRs and mGRs may show an inverted U–shaped dose response, whereby glutamatergic synaptic transmission is increased by low doses of corticosterone acting at mMRs and decreased by higher doses acting at mGRs. Thus, synaptic transmission is regulated by mMRs and mGRs, and part of the stress signaling response is a direct and bidirectional modulation of the synapse itself by adrenal steroids.

Oral Testosterone Self-Administration in Male Hamsters

The addiction potential of anabolic steroids remains largely unexplored. Here, we demonstrate voluntary oral testosterone intake in hamsters. Using a 2-bottle choice test, males preferred an aqueous solution of 200 µg/ml testosterone over vehicle. However, the taste of testosterone is not highly preferred. Addition of testosterone at 400 µg/ml increased fluid consumption from the nonpreferred bottle in a 2-bottle choice test, but cholesterol at the same concentration reduced drinking, suggesting that testosterone reward is not common to all sterols. With food-induced drinking, testosterone maintained fluid intake when food was withdrawn. These data demonstrate that oral self-administration of testosterone is reinforcing in hamsters, suggesting the potential for dependence in human users.

A light and electron microscopic study of NADPH-diaphorase-, calretinin- and parvalbumin-containing neurons in the rat nucleus accumbens.

The rat nucleus accumbens contains medium-sized, spiny projection neurons and intrinsic, local circuit neurons, or interneurons. Sub-classes of interneurons, revealed by calretinin (CR) or parvalbumin (PV) immunoreactivity or reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry, were compared in the nucleus accumbens core, shell and rostral pole. CR, PV and NADPH-diaphorase-containing neurons are shown to form three non-co-localising populations in these three areas. No significant differences in neuronal population densities were found between the subterritories. NADPH-diaphorase-containing neurons could be further separated morphologically into three sub-groups, but CR- and PV-immunoreactive neurons form homogeneous populations. Ultrastructurally, NADPH-diaphorase-, CR- and PV-containing neurons in the nucleus accumbens all possess nuclear indentations. These are deeper and fewer in neurons immunoreactive for PV than in CR- and NADPH-diaphorase-containing neurons. CR-immunoreactive boutons form asymmetrical and symmetrical synaptic specialisations on spines, dendrites and somata, while PV-immunoreactive boutons make only symmetrical synaptic specialisations. Both CR- and PV-immunoreactive boutons form symmetrical synaptic specialisations with medium-sized spiny neurons and contact other CR- and PV-immunoreactive somata, respectively. A novel non-carcinogenic substrate for the peroxidase reaction (Vector Slate Grey, SG) was found to be characteristically electron-dense and may be distinguishable from the diaminobenzidine reaction product. We conclude that the three markers used in this study are localised in distinct populations of nucleus accumbens interneurons. Our studies of their synaptic connections contribute to an increased understanding of the intrinsic circuitry of this area.

Distribution of NMDA and AMPA receptor subunits at thalamo-amygdaloid dendritic spines.

Synapses onto dendritic spines in the lateral amygdala formed by afferents from the auditory thalamus represent a site of plasticity in Pavlovian fear conditioning. Previous work has demonstrated that thalamic afferents synapse onto LA spines expressing glutamate receptor (GluR) subunits, but the GluR subunit distribution at the synapse and within the cytoplasm has not been characterized. Therefore, we performed a quantitative analysis for alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunits GluR2 and GluR3 and N-methyl-D-aspartate (NMDA) receptor subunits NR1 and NR2B by combining anterograde labeling of thalamo-amygdaloid afferents with postembedding immunoelectron microscopy for the GluRs in adult rats. A high percentage of thalamo-amygdaloid spines was immunoreactive for GluR2 (80%), GluR3 (83%), and NR1 (83%), while a smaller proportion of spines expressed NR2B (59%). To compare across the various subunits, the cytoplasmic to synaptic ratios of GluRs were measured within thalamo-amygdaloid spines. Analyses revealed that the cytoplasmic pool of GluR2 receptors was twice as large compared to the GluR3, NR1, and NR2B subunits. Our data also show that in the adult brain, the NR2B subunit is expressed in the majority of in thalamo-amygdaloid spines and that within these spines, the various GluRs are differentially distributed between synaptic and non-synaptic sites. The prevalence of the NR2B subunit in thalamo-amygdaloid spines provides morphological evidence supporting its role in the fear conditioning circuit while the differential distribution of the GluR subtypes may reflect distinct roles for their involvement in this circuitry and synaptic plasticity.

Associative Pavlovian conditioning leads to an increase in spinophilin-immunoreactive dendritic spines in the lateral amygdala

Changes in dendritic spine number and shape are believed to reflect structural plasticity consequent to learning. Previous studies have strongly suggested that the dorsal subnucleus of the lateral amygdala is an important site of physiological plasticity in Pavlovian fear conditioning. In the present study, we examined the effect of auditory fear conditioning on dendritic spine numbers in the dorsal subnucleus of the lateral amygdala using an immunolabelling procedure to visualize the spine‐associated protein spinophilin. Associatively conditioned rats that received paired tone and shock presentations had 35% more total spinophilin‐immunoreactive spines than animals that had unpaired stimulation, consistent with the idea that changes in the number of dendritic spines occur during learning and account in part for memory.

The importance of reporting housing and husbandry in rat research.

In 1963, the National Institutes of Health (NIH) first issued guidelines for animal housing and husbandry. The most recent 2010 revision emphasizes animal care “in ways judged to be scientifically, technically, and humanely appropriate” (National Institutes of Health, 2010, p. XIII). The goal of these guidelines is to ensure humanitarian treatment of animals and to optimize the quality of research. Although these animal care guidelines cover a substantial amount of information regarding animal housing and husbandry, researchers generally do not report all these variables (see Table ​Table1).1). The importance of housing and husbandry conditions with respect to standardization across different research laboratories has been debated previously (Crabbe et al., 1999; Van Der Staay and Steckler, 2002; Wahlsten et al., 2003; Wolfer et al., 2004; Van Der Staay, 2006; Richter et al., 2010, 2011). This paper focuses on several animal husbandry and housing issues that are particularly relevant to stress responses in rats, including transportation, handling, cage changing, housing conditions, light levels and the light–dark cycle. We argue that these key animal housing and husbandry variables should be reported in greater detail in an effort to raise awareness about extraneous experimental variables, especially those that have the potential to interact with the stress response.

Posttraumatic stress disorder and traumatic stress: from bench to bedside, from war to disaster

War is a tragic event and its mental health consequences can be profound. Recent studies indicate substantial rates of posttraumatic stress disorder and other behavioral alterations because of war exposure. Understanding the psychological, behavioral, and neurobiological mechanism of mental health and behavioral changes related to war exposure is critical to helping those in need of care. Substantial work to encourage bench to bedside to community knowledge and communication is a core component of addressing this world health need.