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The prevalence of obesity and related consequences, including insulin resistance and Alzheimer's-like neuropathology, has increased dramatically. Contributing to this prevalence is the shift in lifestyle preference away from wholesome foods and exercise to the Western-style diet and sedentarism. Despite advances in drug development, a healthy diet and regular exercise remain the most effective approaches to mitigating the unwanted sequelae of diet-induced obesity on brain health. In this study, we used the high-fat high-sugar (HFHS) mouse model of neurodegeneration to examine the effects of exercise training (HFHS+Ex), genistein treatment (HFHS+Gen), and combination treatment (HFHS+Ex+Gen) on proteins relating to neurodegeneration in the brain of male mice. After a period of 12 weeks, as expected, HFHS feeding increased body weight, adipose tissue weight, and systemic plasma inflammation (TNF-α) compared to lean mice fed a standard diet. HFHS feeding also increased protein expression of brain markers of insulin resistance (pGSK-3β, p-IR), apoptosis (caspase 3), early neurofibrillary tangles (CP13), and amyloid-beta precursor (CT20). Compared to HFHS mice, Ex decreased body weight, plasma TNF-α, and expression of pGSK-3β, caspase 3, CP13, amyloid-β precursor (22c11), and ADAM10. Treatment with Gen was equally protective on these markers and decreased the expression of p-IR. Combination treatment with Ex and Gen afforded the greatest overall benefits, and this group exhibited the greatest reduction in body and adipose tissue weight and all brain markers, except for 22c11 and ADAM10, which were decreased compared to mice fed an HFHS diet. In addition, levels of 4G8, which detects protein levels of amyloid-β, were decreased with combination treatment. Our results indicate that exercise training, genistein supplementation, or combination treatment provide varying degrees of neuroprotection from HFHS feeding-induced Alzheimer's pathology. Future perspectives could include evaluating moderate exercise regimens in combination with dietary supplementation with genistein in humans to determine whether the same benefits translate clinically.

Fear conditioning evokes a physiologic release of glucocorticoids that assists learning. As a cochaperone in the glucocorticoid receptor complex, FKBP51 modulates stress-induced glucocorticoid signaling and may influence conditioned fear responses. This study combines molecular and behavioral approaches to examine whether locally reducing FKBP51 expression in the ventral hippocampus is sufficient to affect fear-related behaviors. We hypothesized that reducing FKBP51 expression in the VH would increase glucocorticoid signaling to alter auditory fear conditioning. Adult male rats were injected with an adeno-associated virus (AAV) vector expressing short hairpin - RNAs (shRNA) targeting FKBP5 into the ventral hippocampus to reduce FKBP5 levels or a control AAV. Infusion of FKBP5-shRNA into the ventral hippocampus decreased auditory fear acquisition and recall. Although animals injected with FKBP5-shRNA showed less freezing during extinction recall, the difference was due to a reduced fear recall rather than improved extinction. Reducing ventral hippocampus FKBP51 did not affect exploratory behavior in either the open field test or the elevated zero maze test but did increase passive behavior in the forced swim test, suggesting that the reduction in auditory fear recall was not due to more active responses to acute stress. Furthermore, lower ventral hippocampus FKBP51 levels did not alter corticosterone release in response to restraint stress, suggesting that the reduced fear recall was not due to lower corticosterone release. Our findings suggest FKBP51 in the ventral hippocampus plays a selective role in modulating fear-learning processes and passive behavioral responses to acute stress rather than hypothalamic-pituitary-adrenal axis reactivity or exploratory responses.

Biomedical graduate students receive intensive training in their scientific area of interest yet need additional skills for successful scientific careers. Our aim was to promote team building, improve collaborations and enhance communication skills. An off-site yearly retreat was organized for the graduate students in our NIH-funded Research Initiative for Scientific Enhancement (RISE) graduate training program. Retreat themes were addressed through short presentations, case studies, live podcasts, webinars, focus groups, role-play, and breakout sessions with various team building exercises to practice communication skills and identify abilities, knowledge, values, and behaviors. Trainees gave short presentations and served as discussion leaders on topics related to the central theme. Expert guest speakers participated in discussion sessions with the trainees. Trainees evaluated the retreats at the end. A total of 48 trainees, 12 RISE Program faculty and staff, and 26 external speakers from industry, academia, media/journalism, the arts, psychology, and holistic medical fields participated over 9 years. The overall average benefit of the in-person retreats was rated 4.80 on a Likert scale of 1-5 by trainees. Trainees particularly enjoyed the informal interactions with program faculty, staff, and fellow trainees. They appreciated the opportunity to learn soft skills, such as interpersonal communication, conflict resolution, and leadership. Two additional retreats conducted virtually because of the COVID-19 pandemic were perceived as less beneficial. We conclude that off-site interactive retreats are a valuable tool for enhancing soft skills and a sense of team identity in a biomedical sciences graduate program, while covering important issues related to scientific careers. NEW & NOTEWORTHY Off-site interactive science-related retreats are a valuable tool for enhancing soft skills and sense of team identity in a biomedical sciences graduate program, while covering important issues related to pursuing a career in science. There are many perceived benefits, so we encourage other training programs to include a similar type of regular activity in students' training with the goal of improving trainee well-being and supporting their academic and research productivity.

Post-traumatic stress disorder (PTSD) is a neuropsychiatric disorder that not only entails alterations in fear behavior and anxiety but also includes neuroendocrine dysfunctions involving the hypothalamic pituitary adrenal (HPA) axis and the renin-angiotensin system. Recent preclinical studies demonstrate that activation of the angiotensin type 1 receptor (AT1R) in the paraventricular region of the hypothalamus (PVR) promotes anxiety-like behaviors and enables microglia proliferation. An increase in microglia and anxiety-like behavior also occurs in the PTSD animal model single-prolonged stress (SPS). In the present study, we tested whether AT1Rs contribute to the effects of SPS on behavior and microglia in brain structures important for HPA axis regulation and fear behavior. To test this, male and female animals were exposed to SPS and then given the oral AT1R antagonist candesartan beginning one week later. Candesartan did not alter auditory fear conditioning or extinction in SPS-exposed male or female animals. However, we found that the male animals exposed to SPS showed increased anxiety-like behavior, which was reversed by candesartan. In contrast, neither SPS nor candesartan altered anxiety-like behavior in the female animals. At the molecular level, SPS increased the cellular expression of AT1Rs in the PVR of male animals and candesartan reversed this effect, whereas AT1Rs in the PVR of females were unaltered by either SPS or candesartan. Iba1-expressing microglia increased in the PVR after SPS exposure and was reversed by candesartan in both sexes suggesting that SPS stimulates AT1Rs to increase microglia in the PVR. Collectively, these results suggest that the contribution of AT1Rs to the molecular and behavioral effects of SPS is sex-dependent., Competing Interests: Conflict of Interest This research was conducted with no biomedical financial interest or potential conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Clinical evidence has linked increased peripheral pro-inflammatory cytokines with post-traumatic stress disorder (PTSD) symptoms. However, whether inflammation contributes to or is a consequence of PTSD is still unclear. Previous research shows that stress can activate purinergic P2X7 receptors (P2X7Rs) on microglia to induce inflammation and behavioral changes. In this investigation, we examined whether P2X7Rs contribute to the development of PTSD-like behaviors induced by single prolonged stress (SPS) exposure in rats. Consistent with the literature, exposing adult male and female rats to SPS produced a PTSD-like phenotype of impaired fear extinction and extinction of cue-induced center avoidance one week after exposure. Next, we examined if inflammation precedes the behavioral manifestations. Three days after SPS exposure, increased inflammatory cytokines were found in the blood and hippocampal microglia showed increased expression of the P2X7R, IL-1β, and TNF-α, suggesting increased peripheral and central inflammation before the onset of impaired fear extinction. In addition, SPS-exposed animals with impaired fear extinction recall also had more Iba1-positive microglia expressing the P2X7R in the ventral hippocampus. To determine whether P2X7Rs contribute to the PTSD-related behaviors induced by SPS exposure, we gave ICV infusions of the P2X7R antagonist, A-438079, for one week starting the day of SPS exposure. Blocking P2X7Rs prevented the SPS-induced impaired fear extinction and extinction of cue-induced center avoidance in male and female rats, suggesting that SPS activates P2X7Rs which increase inflammation to produce a PTSD-like phenotype., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Single prolonged stress (SPS) is a preclinical rodent model for studying post-traumatic stress disorder (PTSD)-like behaviors. Previously we found that increased expression of the microglial marker Iba-1 in the ventral hippocampus after SPS exposure was associated with impaired fear extinction, suggesting that microglial activity contributed to the SPS-induced behavioral changes. To test this, we examined whether reducing microglia with the colony-stimulating factor 1 receptor blocker, PLX3397, in the diet would prevent the SPS-induced extinction impairment. Male rats exposed to SPS showed enhanced fear acquisition and impaired fear extinction memory. Adding PLX3397 to the diet prevented these behavioral changes. In contrast, PLX3397 did not prevent SPS from impairing fear extinction memory in the female rats. Despite the sex-dependent behavioral effects, we found a reduced number and area fraction of Iba-1+ microglia in both male and female rats suggesting that PLX3397 had similar effects on microglia in both sexes. Altogether, these results suggest that microglia contribute to the behavioral changes induced by SPS in male but not female rats., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Torres-Rodriguez, Ortiz-Nazario, Rivera-Escobales, Velazquez, Colón and Porter.)

Increased long-chain C20:0 ceramides have been found in the serum of patients with depression. Moreover, ceramides are linked with increased microglia reactivity and inflammatory cytokine production, which are associated with depression. Since ceramides can readily cross the blood brain barrier, peripheral C20:0 ceramides could enter the brain, activate microglia, and induce depressive-like behavior. In this study, we determined whether localized infusion of C20:0 ceramides into the ventral hippocampus (VH) of rats is sufficient to activate microglia and induce depressive-like behaviors. Adult male and female rats received infusions of C20:0 ceramides or vehicle solution every other day for 2 weeks. After the third infusion, C20:0-infused animals showed reduced sucrose preference suggesting anhedonia-like behavior. In contrast, infusions of C20:0 ceramides did not affect immobility in the forced swim test or sucrose grooming suggesting that the behavioral effects of ceramides are task dependent. Furthermore, C20:0-infusions did not increase Iba-1 + microglia or inflammatory markers in the VH suggesting that localized increases in C20:0 ceramides in the VH are sufficient to induce anhedonia-like behavior without microglia activation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sambolín-Escobales, Feliciano-Quiñones, Tirado-Castro, Suárez, Pacheco-Cruz, Irizarry-Méndez, Fonseca-Ferrer, Hernández-López, Colón-Romero and Porter.)

Clinical and preclinical studies suggest that increases in long-chain ceramides in blood may contribute to the development of depressive-like behavior. However, which factors contribute to these increases and whether the increases are sufficient to induce depressive-like behaviors is unclear. To begin to address this issue, we examined the effects of high fat diet (HFD) and short-term unpredictable (STU) stress on long-chain ceramides in the serum of male and female rats. We found that brief exposure to HFD or unpredictable stress was sufficient to induce selective increases in the serum concentrations of long-chain ceramides, associated with depression in people. Furthermore, combined exposure to HFD and unpredictable stress caused a synergistic increase in C16:0, C16:1, and C18:0 ceramides in both sexes and C18:1 and C24:1 in males. However, the increased peripheral long-chain ceramides were not associated with increases in depressive-like behaviors suggesting that increases in serum long-chain ceramides may not be associated with the development of depressive-like behaviors in rodents., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sambolín-Escobales, Tirado-Castro, Suarez, Pacheco-Cruz, Fonseca-Ferrer, Deme, Haughey, Chompre and Porter.)

Although the infralimbic cortex (IL) is not thought to play a role in fear acquisition, recent experiments found evidence that synaptic plasticity is occurring at ventral hippocampal (vHPC) synapses in IL during auditory fear acquisition as measured by changes in the N -methyl-D-aspartate (NMDA) receptor-mediated currents in male rats. These electrophysiological data suggest that fear conditioning changes the expression of NMDA receptors on vHPC-to-IL synapses. To further evaluate the plasticity of NMDA receptors at this specific synapse, we injected AAV particles expressing channelrhodopsin-EYFP into the vHPC of male and female rats to label vHPC projections with EYFP. To test for NMDA receptor changes in vHPC-to-IL synapses after fear learning, we used fluorescence-activated cell sorting (FACS) to quantify synaptosomes isolated from IL tissue punches that were positive for EYFP and the obligatory GluN1 subunit. More EYFP+/GluN1+ synaptosomes with greater average expression of GluN1 were isolated from male rats exposed to auditory fear conditioning (AFC) than those exposed to context and tones only or to contextual fear conditioning (CFC), suggesting that AFC increased NMDA receptor expression in males. In a second experiment, we found that pairing the tones and shocks was required to induce the molecular changes and that fear extinction did not reverse the changes. In contrast, females showed similar levels of EYFP+/GluN1+ synaptosomes in all behavioral groups. These findings suggest that AFC induces synaptic plasticity of NMDA receptors in the vHPC-to-IL projection in males, while female rats rely on different synaptic mechanisms., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Castillo-Ocampo, Colón, Hernández, Lopez, Gerena and Porter.)

The Research Centers in Minority Institutions (RCMI) Program was congressionally mandated in 1985 to build research capacity at institutions that currently and historically recruit, train, and award doctorate degrees in the health professions and health-related sciences, primarily to individuals from underrepresented and minority populations. RCMI grantees share similar infrastructure needs and institutional goals. Of particular importance is the professional development of multidisciplinary teams of academic and community scholars (the "workforce") and the harnessing of the heterogeneity of thought (the "thinkforce") to reduce health disparities. The purpose of this report is to summarize the presentations and discussion at the RCMI Investigator Development Core (IDC) Workshop, held in conjunction with the RCMI Program National Conference in Bethesda, Maryland, in December 2019. The RCMI IDC Directors provided information about their professional development activities and Pilot Projects Programs and discussed barriers identified by new and early-stage investigators that limit effective career development, as well as potential solutions to overcome such obstacles. This report also proposes potential alignments of professional development activities, targeted goals and common metrics to track productivity and success.

In rodents, the anterior cingulate (ACC), prelimbic (PL), and infralimbic cortex (IL) comprise the medial prefrontal cortex (mPFC). Through extensive connections with cortical and subcortical structures, the mPFC plays a key modulatory role in the neuronal circuits underlying associative fear and reward learning. In this article, we have compiled the evidence that associative learning induces plasticity in both the intrinsic and synaptic excitability of mPFC neurons to modulate conditioned fear and cocaine seeking behavior. The literature highlights the accumulating evidence that plasticity in the intrinsic excitability of mPFC neurons represents a major cellular mechanism that interacts with synaptic changes to alter the impact of the mPFC on fear and reward circuits., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: HIV-1-associated neurocognitive disorders (HAND) progression is related to continued inflammation despite undetectable viral loads and may be caused by early viral proteins expressed by latently infected cells. Astrocytes represent an HIV reservoir in the brain where the early viral neurotoxin negative factor (Nef) is produced. We previously demonstrated that astrocytic expression of Nef in the hippocampus of rats causes inflammation, macrophage infiltration, and memory impairment. Since these processes are affected by TGFβ signaling pathways, and TGFβ-1 is found at higher levels in the central nervous system of HIV-1+ individuals and is released by astrocytes, we hypothesized a role for TGFβ-1 in our model of Nef neurotoxicity., Methods: To test this hypothesis, we compared cytokine gene expression by cultured astrocytes expressing Nef or green fluorescent protein. To determine the role of Nef and a TGFβRI inhibitor on memory and learning, we infused astrocytes expressing Nef into the hippocampus of rats and then treated them daily with an oral dose of SD208 (10 mg/kg) or placebo for 7 days. During this time, locomotor activity was recorded in an open field and spatial learning tested in the novel location recognition paradigm. Postmortem tissue analyses of inflammatory and signaling molecules were conducted using immunohistochemistry and immunofluorescence., Results: TGFβ-1 was induced in cultures expressing Nef at 24 h followed by CCL2 induction which was prevented by blocking TGFβRI with SD208 (competitive inhibitor). Interestingly, Nef seems to change the TGFβRI localization as suggested by the distribution of the immunoreactivity. Nef caused a deficit in spatial learning that was recovered upon co-administration of SD208. Brain tissue from Nef-treated rats given SD208 showed reduced CCL2, phospho-SMAD2, cluster of differentiation 163 (CD163), and GFAP immunoreactivity compared to the placebo group., Conclusions: Consistent with our previous findings, rats treated with Nef showed deficits in spatial learning and memory in the novel location recognition task. In contrast, rats treated with Nef + SD208 showed better spatial learning suggesting that Nef disrupts memory formation in a TGFβ-1-dependent manner. The TGFβRI inhibitor further reduced the induction of inflammation by Nef which was concomitant with decreased TGFβ signaling. Our findings suggest that TGFβ-1 signaling is an intriguing target to reduce neuroHIV.

The medial prefrontal cortex (mPFC) processes contextual information from the hippocampus to generate appropriate fear responses. In rodents, one path for sending contextual information to the mPFC is via the direct projections from the ventral hippocampus (vHC) to the infralimbic cortex (IL). Plasticity in the synaptic communication from the vHC to the IL could contribute to the behavioral changes produced by the acquisition and extinction of conditioned fear. To examine this possibility, we used optogenetic stimulation of vHC synapses in brain slices from trained rats. We found that fear acquisition reduced NMDA receptor (NMDAR) currents at vHC synapses onto IL pyramidal neurons. The depression of NMDAR currents reversed more efficiently after extinction in the conditioning context than extinction in a novel context. Moreover, a cohort of animals that exhibited poor extinction retrieval failed to reverse the plasticity induced by fear conditioning. In addition, ex vivo application of brain-derived neurotrophic factor (BDNF), which is known to simulate extinction in IL, reversed this conditioning-induced plasticity mimicking extinction. Therefore, we have identified a novel mechanism that modulates conditioned fear via changes in NMDAR current at vHC synapses onto IL pyramidal neurons. Disruption of this mechanism could contribute to the abnormal contextual modulation of fear seen in posttraumatic stress disorder (PTSD).

Mood disorders affect nearly a quarter of the world's population. Therefore, understanding the molecular mechanisms underlying these conditions is of great importance. FK-506 binding protein 5 ( FKBP5 ) encodes the FKBP51 protein, a heat shock protein 90 kDa (Hsp90) co-chaperone, and is a risk factor for several affective disorders. FKBP51, in coordination with Hsp90, regulates glucocorticoid receptor (GR) activity via a short negative feedback loop. This signalling pathway rapidly restores homeostasis in the hypothalamic-pituitary-adrenal (HPA) axis following stress. Expression of FKBP5 increases with age through reduced DNA methylation. High levels of FKBP51 are linked to GR resistance and reduced stress coping behaviour. Moreover, common allelic variants in the FKBP5 gene are associated with increased risk of developing affective disorders like anxiety, depression and post-traumatic stress disorder (PTSD). This review highlights the current understanding of the Hsp90 co-chaperone, FKBP5, in disease from both human and animal studies. In addition, FKBP5 genetic implications in the clinic involving life stress exposure, gender differences and treatment outcomes are discussed.This article is part of the theme issue 'Heat shock proteins as modulators and therapeutic targets of chronic disease: an integrated perspective'., (© 2017 The Author(s).)

The factors influencing resiliency to the development of post-traumatic stress disorder (PTSD) remain to be elucidated. Clinical studies associate PTSD with polymorphisms of the FK506 binding protein 5 (FKBP5). However, it is unclear whether changes in FKBP5 expression alone could produce resiliency or susceptibility to PTSD-like symptoms. In this study, we used rats as an animal model to examine whether FKBP5 in the infralimbic (IL) or prelimbic (PL) medial prefrontal cortex regulates fear conditioning or extinction. First, we examined FKBP5 expression in IL and PL during fear conditioning or extinction. In contrast to the stable expression of FKBP5 seen in PL, FKBP5 expression in IL increased after fear conditioning and remained elevated even after extinction suggesting that IL FKBP5 levels may modulate fear conditioning or extinction. Consistent with this possibility, reducing basal FKBP5 expression via local infusion of FKBP5-shRNA into IL reduced fear conditioning. Furthermore, reducing IL FKBP5, after consolidation of the fear memory, enhanced extinction memory indicating that IL FKBP5 opposed formation of the extinction memory. Our findings demonstrate that lowering FKBP5 expression in IL is sufficient to both reduce fear acquisition and enhance extinction, and suggest that lower expression of FKBP5 in the ventral medial prefrontal cortex could contribute to resiliency to PTSD., (© 2017 Criado-Marrero et al.; Published by Cold Spring Harbor Laboratory Press.)

Patients with posttraumatic stress disorder (PTSD) show hypo-active ventromedial prefrontal cortices (vmPFC) that correlate with their impaired ability to discriminate between safe and dangerous contexts and cues. Previously, we found that auditory fear conditioning depresses the excitability of neurons populating the homologous structure in rodents, the infralimbic cortex (IL). However, it is undetermined if IL depression was mediated by the cued or contextual information. The objective of this study was to examine whether contextual information was sufficient to depress IL neuronal excitability. After exposing rats to context-alone, pseudoconditioning, or contextual fear conditioning, we used whole-cell current-clamp recordings to examine the excitability of IL neurons in prefrontal brain slices. We found that contextual fear conditioning reduced IL neuronal firing in response to depolarizing current steps. In addition, neurons from contextual fear conditioned animals showed increased slow afterhyperpolarization potentials (sAHPs). Moreover, the observed changes in IL excitability correlated with contextual fear expression, suggesting that IL depression may contribute to the encoding of contextual fear., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Patients with post-traumatic stress disorder (PTSD) tend to show signs of a relatively increased inflammatory state suggesting that activation of the immune system may contribute to the development of PTSD. In the present study, we tested whether activation of the innate immune system can disrupt acquisition or recall of auditory fear extinction using an animal model of PTSD. Male adolescent rats received auditory fear conditioning in context A. The next day, an intraperitoneal injection of lipopolysaccharide (LPS; 100 μg/kg) prior to auditory fear extinction in context B impaired acquisition and recall of extinction. LPS (100 μg/kg) given after extinction training did not impair extinction recall suggesting that LPS did not affect consolidation of extinction. In contrast to cued fear extinction, contextual fear extinction was not affected by prior injection of LPS (100 μg/kg). Although LPS also reduced locomotion, we could dissociate the effects of LPS on extinction and locomotion by using a lower dose of LPS (50 μg/kg) which impaired locomotion without affecting extinction. In addition, 15 h after an injection of 250 μg/kg LPS in adult rats, extinction learning and recall were impaired without affecting locomotion. A sub-chronic treatment with candesartan, an angiotensin II type 1 receptor blocker, prevented the LPS-induced impairment of extinction in adult rats. Our results demonstrate that activation of the innate immune system can disrupt auditory fear extinction in adolescent and adult animals. These findings also provide direction for clinical studies of novel treatments that modulate the innate immune system for stress-related disorders like PTSD., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Adolescent rats are prone to impaired fear extinction, suggesting that mechanistic differences in extinction could exist in adolescent and adult rats. Since the infralimbic cortex (IL) is critical for fear extinction, we used PCR array technology to identify gene expression changes in IL induced by fear extinction in adolescent rats. Interestingly, the ephrin type B receptor 2 (EphB2), a tyrosine kinase receptor associated with synaptic development, was downregulated in IL after fear extinction. Consistent with the PCR array results, EphB2 levels of mRNA and protein were reduced in IL after fear extinction compared with fear conditioning, suggesting that EphB2 signaling in IL regulates fear extinction memory in adolescents. Finally, reducing EphB2 synthesis in IL with shRNA accelerated fear extinction learning in adolescent rats, but not in adult rats. These findings identify EphB2 in IL as a key regulator of fear extinction during adolescence, perhaps due to the increase in synaptic remodeling occurring during this developmental phase., (Copyright © 2015 the authors 0270-6474/15/3512394-10$15.00/0.)

The use and validity of the Graduate Record Examination General Test (GRE) to predict the success of graduate school applicants is heavily debated, especially for its possible impact on the selection of underrepresented minorities into science, technology, engineering, and math fields. To better identify candidates who would succeed in our program with less reliance on the GRE and grade point average (GPA), we developed and tested a composite score (CS) that incorporates additional measurable predictors of success to evaluate incoming applicants. Uniform numerical values were assigned to GPA, GRE, research experience, advanced course work or degrees, presentations, and publications. We compared the CS of our students with their achievement of program goals and graduate school outcomes. The average CS was significantly higher in those students completing the graduate program versus dropouts (p < 0.002) and correlated with success in competing for fellowships and a shorter time to thesis defense. In contrast, these outcomes were not predicted by GPA, science GPA, or GRE. Recent implementation of an impromptu writing assessment during the interview suggests the CS can be improved further. We conclude that the CS provides a broader quantitative measure that better predicts success of students in our program and allows improved evaluation and selection of the most promising candidates., (© 2015 W. I. Pacheco et al. CBE—Life Sciences Education © 2015 The American Society for Cell Biology. This article is distributed by The American Society for Cell Biology under license from the author(s). It is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Previously, we demonstrated that group II metabotropic glutamate receptors (mGluRs) reduce glutamate release from thalamocortical synapses during early postnatal development (P7-11). To further examine the role of group II mGluRs in the modulation of somatosensory circuitry, we determined whether group II mGluRs continue to modulate thalamocortical synapses until adulthood and whether these receptors also modulate intra-cortical synapses in the barrel cortex. To address these issues, we examined the effect of the group II mGluR agonists on thalamocortical excitatory postsynaptic currents (EPSCs) and intra-barrel EPSCs in slices from animals of different ages (P7-53). We found that the depression of thalamocortical EPSCs by group II mGluRs rapidly declined after the second postnatal week. In contrast, adenosine continued to depress thalamocortical EPSCs via a presynaptic mechanism in young adult mice (P30-50). Activation of group II mGluRs also reduced intra-barrel EPSCs through a postsynaptic mechanism in young mice (P7-11). Similar to the thalamocortical synapses, the group II mGluR modulation of intra-barrel excitatory synapses declined with development. In young adult animals (P30-50), group II mGluR stimulation had little effect on intra-barrel EPSCs but did hyperpolarize the neurons. Together our results demonstrate that group II mGluRs modulate barrel cortex circuitry by presynaptic and postsynaptic mechanisms depending on the source of the synapse and that this modulation declines with development., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

In rodents, the infralimbic (IL) region of the medial prefrontal cortex plays a key role in the recall of fear extinction. Previously we showed that fear conditioning decreases the intrinsic excitability of IL neurons, and that fear extinction reverses the depressed excitability. In the current study, we examined the time course of the extinction-induced changes in adolescent rats. Immediately after extinction, IL neurons continued to show depressed excitability. However 4 hours after extinction, IL neurons showed an increase in evoked spikes that correlated with a reduced fast afterhyperpolarizing potential. This suggests that acquisition of fear extinction induces an increase in spike firing 4 hours later, during the consolidation of extinction. We also examined IL excitability in a group of rats that showed spontaneous recovery of fear 17 days after extinction (SR group). Similar to neurons after fear conditioning, IL neurons from the SR group showed depressed intrinsic excitability compared to neurons 4 hours after extinction, suggesting that extinction-induced enhancement in intrinsic excitability decreases with time reverting back to a depressed state. These results suggest that plasticity in IL contributes to the spontaneous recovery of fear and preventing this depression of IL excitability could prolong fear extinction.

HIV-1 infection can lead to neurocognitive impairment collectively known as HIV-associated neurocognitive disorders (HAND). Although combined antiretroviral treatment (cART) has significantly ameliorated HIV's morbidity and mortality, persistent neuroinflammation and neurocognitive dysfunction continue. This review focuses on the current clinical and molecular evidence of the viral and host factors that influence glutamate-mediated neurotoxicity and neuropathogenesis as an important underlying mechanism during the course of HAND development. In addition, discusses potential pharmacological strategies targeting the glutamatergic system that may help prevent and improve neurological outcomes in HIV-1-infected subjects.

Fear extinction correlates with increased infralimbic (IL) neuronal excitability. Since small conductance Ca(2+)-dependent K(+) (SK) channels modulate neuronal excitability and certain types of learning and memory, pharmacological modulation of SK channels could be used to regulate IL excitability and fear extinction. To test this, we first determined the effect of blocking SK channels with apamin on the intrinsic excitability of IL pyramidal neurons in brain slices. In whole-cell patch-clamp recordings, apamin increased the number of spikes evoked by a depolarizing current pulse, increased the firing frequency, and reduced the fast afterhyperpolarizing potential (fAHP) indicating that blockade of SK channels could be used to enhance the intrinsic excitability of IL neurons. Next, we assessed whether SK channels in IL regulate extinction of conditioned fear by infusing apamin into IL of fear conditioned rats prior to extinction training. Apamin infusion did not affect conditioned freezing at the beginning of the extinction session or within-session extinction. However, the following day, apamin-infused rats showed significantly less conditioned freezing. To further examine the importance of SK channels in IL in fear extinction, we assessed the effect of the SK channel activator DCEBIO on IL neuronal excitability and fear extinction. Activation of SK channels with DCEBIO decreased the number of evoked spikes, reduced the firing frequency, and enhanced the fAHP of IL neurons. Infusion of DCEBIO into IL prior to fear extinction impaired recall of fear extinction without affecting acquisition of extinction. Taken together, these findings suggest that SK channels are involved in regulating IL excitability and extinction-induced plasticity. Therefore, SK channels are a potential target for the development of new pharmacological treatments to facilitate extinction in patients suffering from anxiety disorders.

Studies suggest that plasticity in the infralimbic prefrontal cortex (IL) in rodents and its homolog in humans is necessary for inhibition of fear during the recall of fear extinction. The recall of extinction is impaired by locally blocking metabotropic glutamate receptor type 5 (mGluR5) activation in IL during extinction training. This finding suggests that mGluR5 stimulation may lead to IL plasticity needed for fear extinction. To test this hypothesis, we recorded AMPA and NMDA currents, AMPA receptor (AMPAR) rectification, and intrinsic excitability in IL pyramidal neurons in slices from trained rats using whole-cell patch-clamp recording. We observed that fear extinction increases the AMPA/NMDA ratio, consistent with insertion of AMPARs into IL synapses. In addition, extinction training increased inward rectification, suggesting that extinction induces the insertion of calcium-permeable (GluA2-lacking) AMPARs into IL synapses. Consistent with this, selectively blocking calcium-permeable AMPARs with Naspm reduced the AMPA EPSCs in IL neurons to a larger degree after extinction. Extinction-induced changes in AMPA/NMDA ratio, rectification, and intrinsic excitability were blocked with an mGluR5 antagonist. These findings suggest that mGluR5 activation leads to consolidation of fear extinction by regulating the intrinsic excitability of IL neurons and modifying the composition of AMPARs in IL synapses. Therefore, impaired mGluR5 activity in IL synapses could be one factor that causes inappropriate modulation of fear expression leading to anxiety disorders.

Despite the widespread use of antiretroviral therapy that effectively limits viral replication, memory impairment remains a dilemma for HIV infected people. In the CNS, HIV infection of astrocytes leads to the production of the HIV-1 Nef protein without viral replication. Post mortem studies have found Nef expression in hippocampal astrocytes of people with HIV associated dementia suggesting that astrocytic Nef may contribute to HIV associated cognitive impairment even when viral replication is suppressed. To test whether astrocytic expression of Nef is sufficient to induce cognitive deficits, we examined the effect of implanting primary rat astrocytes expressing Nef into the hippocampus on spatial and recognition memory. Rats implanted unilaterally with astrocytes expressing Nef showed impaired novel location and novel object recognition in comparison with controls implanted with astrocytes expressing green fluorescent protein (GFP). This impairment was correlated with an increase in chemokine ligand 2 (CCL2) expression and the infiltration of peripheral macrophages into the hippocampus at the site of injection. Furthermore, the Nef exposed rats exhibited a bilateral loss of CA3 neurons. These results suggest that Nef protein expressed by the implanted astrocytes activates the immune system leading to neuronal damage and spatial and recognition memory deficits. Therefore, the continued expression of Nef by astrocytes in the absence of viral replication has the potential to contribute to HIV associated cognitive impairment., (Copyright © 2012 Elsevier Inc. All rights reserved.)

There is considerable interest in identifying pharmacological compounds that could be used to facilitate fear extinction. Recently, we showed that the modulation of M-type K(+) channels regulates the intrinsic excitability of infralimbic (IL) neurons and fear expression. As muscarinic acetylcholine receptors inhibit M-type K(+) channels, cholinergic inputs to IL may have an important role in controlling IL excitability and, thereby, fear expression and extinction. To test this model, we combined whole-cell patch-clamp electrophysiology and auditory fear conditioning. In prefrontal brain slices, muscarine enhanced the intrinsic excitability of IL neurons by reducing the M-current and the slow afterhyperpolarization, resulting in an increased number of spikes with shorter inter-spike intervals. Next, we examined the role of endogenous activation of muscarinic receptors in fear extinction. Systemic injected scopolamine (Scop) (muscarinic receptor antagonist) before or immediately after extinction training impaired recall of extinction 24-h later, suggesting that muscarinic receptors are critically involved in consolidation of extinction memory. Similarly, infusion of Scop into IL before extinction training also impaired recall of extinction 24-h later. Finally, we demonstrated that systemic injections of the muscarinic agonist, cevimeline (Cev), given before or immediately after extinction training facilitated recall of extinction the following day. Taken together, these findings suggest that cholinergic inputs to IL have a critical role in modulating consolidation of fear extinction and that muscarinic agonists such as Cev might be useful for facilitating extinction memory in patients suffering from anxiety disorders.

Consolidation of fear extinction involves enhancement of N-methyl D aspartate (NMDA) receptor-dependent bursting in the infralimbic region (IL) of the medial prefrontal cortex (mPFC). Previous studies have shown that systemic blockade of metabotropic glutamate receptor type 5 (mGluR5) reduces bursting in the mPFC and mGluR5 agonists enhance NMDA receptor currents in vitro, suggesting that mGluR5 activation in IL may contribute to fear extinction. In the current study, rats injected with the mGluR5 antagonist 2-methyl-6-(phenylethyl)-pyridine (MPEP) systemically, or intra-IL, prior to extinction exhibited normal within-session extinction, but were impaired in their ability to recall extinction the following day. To directly determine whether mGluR5 stimulation enhances the burst firing of IL neurons, we used patch-clamp electrophysiology in prefrontal slices. The mGluR5 agonist, (RS)-2-chloro-5-hydroxyphenylglycine (CHPG), increased intrinsic bursting in IL neurons. Increased bursting was correlated with a reduction in the slow after hyperpolarizing potential and was prevented by coapplication of MPEP. CHPG did not increase NMDA currents, suggesting that an NMDA receptor-independent enhancement of IL bursting via stimulation of mGluR5 receptors contributes to fear extinction. Therefore, the mGluR5 receptor could be a suitable target for pharmacological adjuncts to extinction-based therapies for anxiety disorders.

Growing evidence indicates that the activity of infralimbic prefrontal cortex (IL) is critical for inhibiting inappropriate fear responses following extinction learning. Recently, we showed that fear conditioning and extinction alter the intrinsic excitability and bursting of IL pyramidal neurons in brain slices. IL neurons from Sprague Dawley rats expressing high fear had lower intrinsic excitability and bursting than those from rats expressing low fear, suggesting that regulating the intrinsic excitability and bursting of IL neurons would modulate fear expression. To test this, we combined patch-clamp electrophysiology, auditory fear conditioning, and IL infusions of M-type K(+) channel modulators. Patch-clamp recordings from IL neurons showed that the M-type K(+) channel blocker, XE-991, increased the number of spikes evoked by a depolarizing pulse and reduced the first interspike interval indicating enhanced bursting. To test whether pharmacological enhancement of IL excitability and bursting reduces fear expression and facilitates extinction, fear-conditioned rats were infused with XE-991 into IL before extinction training. XE-infused rats showed reduced freezing and facilitated extinction compared to vehicle-infused rats. The following day, recall of extinction memory was enhanced. Reducing IL excitability and bursting with the M-type K(+) channel agonist, flupirtine, had the opposite effect. Flupirtine reduced IL spike count and bursting in brain slices. Fear-conditioned rats infused with flupirtine into IL before extinction showed significantly higher levels of freezing, indicating that stimulation of M-channels enhanced fear expression. Our findings suggest that the intrinsic excitability and bursting of IL neurons regulate fear expression even before extinction.

Extinction of conditioned fear is an active learning process involving inhibition of fear expression. It has been proposed that fear extinction potentiates neurons in the infralimbic (IL) prefrontal cortex, but the cellular mechanisms underlying this potentiation remain unknown. It is also not known whether this potentiation occurs locally in IL neurons as opposed to IL afferents. To determine whether extinction enhances the intrinsic excitability of IL pyramidal neurons in layers II/III and V, we performed whole-cell patch-clamp recordings in slices from naive, conditioned, or conditioned-extinguished rats. We observed that conditioning depressed IL excitability compared with slices from naive animals, as evidenced by a decreased number of spikes evoked by injected current and an increase in the slow afterhyperpolarizing potential (sAHP). Extinction reversed these conditioning-induced effects. Furthermore, IL neurons from extinguished rats showed increased burst spiking compared with naive rats, which was correlated with extinction recall. These changes were specific to IL prefrontal cortex and were not observed in prelimbic prefrontal cortex. Together, these findings suggest that IL intrinsic excitability is reduced to allow for expression of conditioning memory and enhanced for expression of extinction memory through the modulation of Ca(2+)-gated K(+) channels underlying the sAHP. Inappropriate modulation of these intrinsic mechanisms may underlie anxiety disorders, characterized by exaggerated fear and deficient extinction.

Emotional arousal strengthens memory. This is most apparent in aversive conditioning, in which the stress-related neurotransmitter norepinephrine (NE) enhances associations between sensory stimuli and fear-inducing events. In contrast to conditioning, extinction decreases fear responses, and is thought to form a new memory. It is not known, however, whether NE is necessary for extinction learning. Previous work has shown that the infralimbic prefrontal cortex (IL) is a site of extinction consolidation. Here, we show that blocking noradrenergic beta-receptors in IL before extinction training impaired retrieval of extinction the following day, consistent with a weakened extinction memory. We further found that the sequelae of beta-receptor activation, including protein kinase A (PKA), gene transcription and translation in IL, are necessary for extinction. To determine whether activation of this cascade modulates IL excitability, we measured the response of IL pyramidal neurons to injected current. NE increased the excitability of IL neurons in a beta-receptor- and PKA-dependent manner. We suggest that NE released in IL during fear extinction activates a PKA-mediated molecular cascade that strengthens extinction memory. Thus, emotional arousal evoked by conditioned fear paradoxically promotes the subsequent extinction of that fear, thereby ensuring behavioral flexibility.

Thalamocortical synapses provide a strong glutamatergic excitation to cortical neurons that is critical for processing sensory information. Unit recordings in vivo indicate that metabotropic glutamate receptors (mGluRs) reduce the effect of thalamocortical input on cortical circuits. However, it is not known whether this reduction is due to a reduction in glutamate release from thalamocortical terminals or from a decrease in cortical neuron excitability. To directly determine whether mGluRs act as autoreceptors on thalamocortical terminals, we examined the effect of mGluR agonists on thalamocortical synapses in slices. Thalamocortical excitatory postsynaptic currents (EPSCs) were recorded in layer IV cortical neurons in developing mouse brain slices. The activation of group II mGluRs with (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG IV) reduced thalamocortical EPSCs in both excitatory and inhibitory neurons, while the stimulation of group I or group III mGluRs had no effect on thalamocortical EPSCs. Consistent with a reduction in glutamate release, DCG IV increased the paired pulse ratio and the coefficient of variation of the EPSCs. The reduction induced by DCG IV was reversed by the group II mGluR antagonist, LY341495, and mimicked by another selective group II agonist, (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylic acid (APDC). The mGluR2 subtype appears to mediate the reduction of thalamocortical EPSCs, since the selective mGluR3 agonist, N-acetylaspartylglutamate (NAAG), had no effect on the EPSCs. Consistent with this, we showed that mGluR2 is expressed in the barrels. Furthermore, blocking group II mGluRs with LY341495 reduced the synaptic depression induced by a short stimulus train, indicating that synaptically released glutamate activates these receptors. These results indicate that group II mGluRs modulate thalamocortical processing by inhibiting glutamate release from thalamocortical synapses. This inhibition provides a feedback mechanism for preventing excessive excitation of cortical neurons that could play a role in the plasticity and refinement of thalamocortical connections during this early developmental period.

Caffeine is consumed worldwide to enhance wakefulness, but the cellular mechanisms are poorly understood. Caffeine blocks adenosine receptors suggesting that adenosine decreases cortical arousal. Given the widespread innervation of the cerebral cortex by thalamic fibers, adenosine receptors on thalamocortical terminals could provide an efficient method of limiting thalamic activation of the cortex. Using a mouse thalamocortical slice preparation and whole-cell patch clamp recordings, we examined whether thalamocortical terminals are modulated by adenosine receptors. Bath application of adenosine decreased excitatory postsynaptic currents elicited by stimulation of the ventrobasal thalamus. Thalamocortical synapses onto inhibitory and excitatory neurons were equally affected by adenosine. Adenosine also increased the paired pulse ratio and the coefficient of variation of the excitatory postsynaptic currents, suggesting that adenosine decreased glutamate release. The inhibition produced by adenosine was reversed by a selective antagonist of adenosine A1 receptors (8-cyclopentyltheophylline) and mimicked by a selective A1 receptor agonist (N6-cyclopentyladenosine). Our results indicate that thalamocortical excitation is regulated by presynaptic adenosine A1 receptors and provide a mechanism by which increased adenosine levels can directly reduce cortical excitability.

Cortical inhibition plays an important role in the processing of sensory information, and the enlargement of receptive fields by the in vivo application of GABAB receptor antagonists indicates that GABAB receptors mediate some of this cortical inhibition. Although there is evidence of postsynaptic GABAB receptors on cortical neurons, there is no evidence of GABAB receptors on thalamocortical terminals. Therefore to determine if presynaptic GABAB receptors modulate the thalamic excitation of layer IV inhibitory neurons and excitatory neurons in layers II-III and IV of the somatosensory "barrel" cortex of mice, we used a thalamocortical slice preparation and patch-clamp electrophysiology. Stimulation of the ventrobasal thalamus elicited excitatory postsynaptic currents (EPSCs) in cortical neurons. Bath application of baclofen, a selective GABAB receptor agonist, reversibly decreased AMPA receptor-mediated and N-methyl-D-aspartate (NMDA) receptor-mediated EPSCs in inhibitory and excitatory neurons. The GABAB receptor antagonist, CGP 35348, reversed the inhibition produced by baclofen. Blocking the postsynaptic GABAB receptor-mediated effects with a Cs+ -based recording solution did not affect the inhibition, suggesting a presynaptic effect of baclofen. Baclofen reversibly increased the paired-pulse ratio and the coefficient of variation, consistent with the presynaptic inhibition of glutamate release. Our results indicate that the presynaptic activation of GABAB receptors modulates thalamocortical excitation of inhibitory and excitatory neurons and provide another mechanism by which cortical inhibition can modulate the processing of sensory information.

Sensory information, relayed through the thalamus, arrives in the neocortex as excitatory input, but rapidly induces strong disynaptic inhibition that constrains the cortical flow of excitation both spatially and temporally. This feedforward inhibition is generated by intracortical interneurons whose precise identity and properties were not known. To characterize interneurons generating feedforward inhibition, neurons in layers IV and V of mouse somatosensory ("barrel") cortex in vitro were tested in the cell-attached configuration for thalamocortically induced firing and in the whole-cell mode for synaptic responses. Identification as inhibitory or excitatory neurons was based on intrinsic firing patterns and on morphology revealed by intracellular staining. Thalamocortical stimulation evoked action potentials in approximately 60% of inhibitory interneurons but in <5% of excitatory neurons. The inhibitory interneurons that fired received fivefold larger thalamocortical inputs compared with nonfiring inhibitory or excitatory neurons. Thalamocortically evoked spikes in inhibitory interneurons followed at short latency the onset of excitatory monosynaptic responses in the same cells and slightly preceded the onset of inhibitory responses in nearby neurons, indicating their involvement in disynaptic inhibition. Both nonadapting (fast-spiking) and adapting (regular-spiking) inhibitory interneurons fired on thalamocortical stimulation, as did interneurons expressing parvalbumin, calbindin, or neither calcium-binding protein. Morphological analysis revealed that some interneurons might generate feedforward inhibition within their own layer IV barrel, whereas others may convey inhibition to upper layers, within their own or in adjacent columns. We conclude that feedforward inhibition is generated by diverse classes of interneurons, possibly serving different roles in the processing of incoming sensory information.

In the adult cerebral cortex, the neurotransmitter GABA is strongly inhibitory, as it profoundly decreases neuronal excitability and suppresses the network propensity for synchronous activity. When fast, GABA(A) receptor (GABA(A)R)-mediated neurotransmission is blocked in the mature cortex, neuronal firing is synchronized via recurrent excitatory (glutamatergic) synaptic connections, generating population discharges manifested extracellularly as spontaneous paroxysmal field potentials (sPFPs). This epileptogenic effect of GABA(A)R antagonists has rarely been observed in the neonatal cortex, and indeed, GABA in the neonate has been proposed to have an excitatory, rather than inhibitory, action. In contrast, we show here that when fast GABAergic neurotransmission was blocked in slices of neonatal mouse and rat hippocampus and neocortex, sPFPs occurred in nearly half the slices from postnatal day 4 (P4) to P7 neocortex and in most slices from P2 to P7 hippocampus. In Mg(2+)-free solution, GABA(A)R antagonists elicited sPFPs in nearly all slices of P2 and older neocortex and P0 and older hippocampus. Mg(2+)-free solution alone induced spontaneous events in the majority of P2 and older slices from both regions; addition of GABA(A)R antagonists caused a dramatic increase in the mean amplitude, but not frequency, of these events in the hippocampus and in their mean frequency, but not amplitude, in the neocortex. In the hippocampus, GABA(A)R agonists suppressed amplitudes, but not frequency, of sPFPs, whereas glutamate antagonists suppressed frequency but not amplitudes. We conclude that neonatal rodent cerebral cortex possesses glutamatergic circuits capable of generating synchronous network activity and that, as in the adult, tonic GABA(A)R-mediated inhibition prevents this activity from becoming paroxysmal.

A classification of fusiform neocortical interneurons (n = 60) was performed with an unsupervised cluster analysis based on the comparison of multiple electrophysiological and molecular parameters studied by patch-clamp and single-cell multiplex reverse transcription-PCR in rat neocortical acute slices. The multiplex reverse transcription-PCR protocol was designed to detect simultaneously the expression of GAD65, GAD67, calbindin, parvalbumin, calretinin, neuropeptide Y, vasoactive intestinal peptide (VIP), somatostatin (SS), cholecystokinin, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, kainate, N-methyl-d-aspartate, and metabotropic glutamate receptor subtypes. Three groups of fusiform interneurons with distinctive features were disclosed by the cluster analysis. The first type of fusiform neuron (n = 12), termed regular spiking nonpyramidal (RSNP)-SS cluster, was characterized by a firing pattern of RSNP cells and by a high occurrence of SS. The second type of fusiform neuron (n = 32), termed RSNP-VIP cluster, predominantly expressed VIP and also showed firing properties of RSNP neurons with accommodation profiles different from those of RSNP-SS cells. Finally, the last type of fusiform neuron (n = 16) contained a majority of irregular spiking-VIPergic neurons. In addition, the analysis of glutamate receptors revealed cell-type-specific expression profiles. This study shows that combinations of multiple independent criteria define distinct neocortical populations of interneurons potentially involved in specific functions.

The cellular mechanisms by which neuronal nicotinic cholinergic receptors influence many aspects of physiology and pathology in the neocortex remain primarily unknown. Whole-cell recordings and single-cell reverse transcription (RT)-PCR were combined to analyze the effect of nicotinic receptor agonists on different types of neurons in acute slices of rat neocortex. Nicotinic receptor agonists had no effect on pyramidal neurons and on most types of interneurons, including parvalbumin-expressing fast spiking interneurons and somatostatin-expressing interneurons, but selectively excited a subpopulation of interneurons coexpressing the neuropeptides vasoactive intestinal peptide (VIP) and cholecystokinin. This excitation persisted in the presence of glutamate, GABA, and muscarinic receptor antagonists and in the presence of tetrodotoxin and low extracellular calcium, suggesting that the depolarization was mediated through the direct activation of postsynaptic nicotinic receptors. The responses were blocked by the nicotinic receptor antagonists dihydro-beta-erythroidine and mecamylamine and persisted in the presence of the alpha7 selective nicotinic receptor antagonist methyllycaconitine, suggesting that the involved nicotinic receptors lacked the alpha7 subunit. Single-cell RT-PCR analysis indicated that the majority of the interneurons that responded to nicotinic stimulation coexpressed the alpha4, alpha5, and beta2 nicotinic receptor subunits. Therefore, these results provide a role for non-alpha7 nicotinic receptors in the selective excitation of a subpopulation of neocortical interneurons. Because the neocortical interneurons expressing VIP have been proposed previously to regulate regional cortical blood flow and metabolism, these results also provide a cellular basis for the neuronal regulation of cortical blood flow mediated by acetylcholine.

In the rat neocortex, a subset of GABAergic interneurons express the neuropeptide vasoactive intestinal peptide (VIP). Previously, we demonstrated that a population of VIPergic interneurons could be accurately identified by their irregular spiking (IS) pattern and their bipolar morphology. IS interneurons were studied in neocortical slices from 16-22-day-old rats using whole-cell recordings, intracellular labelling and single-cell RT-PCR. In response to a depolarizing pulse, IS interneurons typically discharged a burst of action potentials followed by spikes emitted at an irregular frequency. Several seconds of depolarization, micromolar concentrations of 4-aminopyridine, and nanomolar concentrations of either dendrotoxin I or K converted this irregular pattern to a sustained discharge, suggesting the involvement of an ID-like K+ current. The main glutamate receptor subunits detected in IS cells were GluR1 flop and GluR2 flop, GluR5 and GluR6, and NR2B and NR2D for the alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA), kainate and N-methyl-D-aspartic acid (NMDA) subtypes, respectively. Paired whole-cell patch-clamp recordings indicated that pyramidal neurons provide intracortical glutamatergic inputs onto IS interneurons. Most connections had high probabilities of response and exhibited frequency-dependent paired pulse depression. Comparison of the amplitude distribution of paired responses suggested that most of these connections consisted of multiple functional release sites. Finally, two discrete subpopulations of IS cells could be identified based on the duration of the initial burst of action potentials and the differential expression of calretinin and choline acetyltransferase.

In the brain, astrocytes are associated intimately with neurons and surround synapses. Due to their close proximity to synaptic clefts, astrocytes are in a prime location for receiving synaptic information from released neurotransmitters. Cultured astrocytes express a wide range of neurotransmitter receptors, but do astrocytes in vivo also express neurotransmitter receptors and, if so, are the receptors activated by synaptically released neurotransmitters? In recent years, considerable efforts has gone into addressing these issues. The experimental results of this effort have been compiled and are presented in this review. Although there are many different receptors which have not been identified on astrocytes in situ, it is clear that astrocytes in situ express a number of different receptors. There is evidence of glutamatergic, GABAergic, adrenergic, purinergic, serotonergic, muscarinic, and peptidergic receptors on protoplasmic, fibrous, or specialized (Bergmann glia, pituicytes, Müller glia) astrocytes in situ and in vivo. These receptors are functionally coupled to changes in membrane potential or to intracellular signaling pathways such as activation of phospholipase C or adenylate cyclase. The expression of neurotransmitter receptors by astrocytes in situ exhibits regional and intraregional heterogeneity and changes during development and in response to injury. There is also evidence that receptors on astrocytes in situ can be activated by neurotransmitter(s) released from synaptic terminals. Given the evidence of extra-synaptic signaling and the expression of neurotransmitter receptors by astrocytes in situ, direct communication between neurons and astrocytes via neurotransmitters could be a widespread form of communication in the brain which may affect many different aspects of brain function, such as glutamate uptake and the modulation of extracellular space.

A long-standing question in neurobiology is whether astrocytes respond to the neuronal release of neurotransmitters in vivo. To address this question, acutely isolated hippocampal slices were loaded with the calcium-sensitive dye Calcium Green-1 and the responses of the astrocytes to electrical stimulation of the Schaffer collaterals were monitored by confocal microscopy. To confirm that the responsive cells were astrocytes, the slices were immunostained for the astrocytic marker glial fibrillary acidic protein. Stimulation of the Schaffer collaterals (50 Hz, 2 sec) resulted in increases in the concentration of intracellular calcium ([Ca2+]i) in the astrocytes located in the stratum radiatum of CA1. The astrocytic responses were blocked by the sodium channel blocker tetrodotoxin, the voltage-dependent calcium channel blocker omega-conotoxin-MVIIC, and the selective metabotropic glutamate receptor antagonist alpha-methyl-4-carboxyphenylglycine (MCPG). These results suggest that the astrocytic responses were induced by stimulation of metabotropic glutamate receptors on the astrocytes by neuronally released glutamate. The astrocytic responses to neuronal stimulation were enhanced in the presence of the K+ channel antagonist 4-aminopyridine (4-AP). Inhibition of the astrocytic responses in the presence of 4-AP required the presence of both MCPG and the ionotropic glutamate receptor antagonist kynurenic acid. These results suggest that higher levels of neuronal activity result in stimulation of both metabotropic and ionotropic glutamate receptors on the astrocytes. Overall, the results indicate that hippocampal astrocytes in situ are able to respond to the neuronal release of the neurotransmitter glutamate with increases in [Ca2+]i.

Cultured astroglia express both adenosine and ATP purinergic receptors that are coupled to increases in intracellular calcium concentration ([Ca2+]i). Currently, there is little evidence that such purinergic receptors exist on astrocytes in vivo. To address this issue, calcium-sensitive fluorescent dyes were used in conjunction with confocal microscopy and immunocytochemistry to examine the responsiveness of astrocytes in acutely isolated hippocampal slices to purinergic neuroligands. Both ATP and adenosine induced dynamic increases in astrocytic [Ca2+]i that were blocked by the adenosine receptor antagonist 8-(p-sulfophenyl)theophylline. The responses to adenosine were not blocked by tetrodotoxin, 8-cyclopentyltheophylline, 8-(3-chlorostyryl)caffeine, dipyridamole, or removal of extracellular calcium. The P2Y-selective agonist 2-methylthioadenosine triphosphate was unable to induce increases in astrocytic [Ca2+]i, whereas the P2 agonist adenosine 5'-O-(2-thiodiphosphate) induced astrocytic responses in a low percentage of astrocytes. These results indicate that the majority of hippocampal astrocytes in situ contain P1 purinergic receptors coupled to increases in [Ca2+]i, whereas a small minority appear to contain P2 purinergic receptors. Furthermore, individual hippocampal astrocytes responded to adenosine, glutamate, and depolarization with increases in [Ca2+]i. The existence of both purinergic and glutamatergic receptors on individual astrocytes in situ suggests that astrocytes in vivo are able to integrate information derived from glutamate and adenosine receptor stimulation.

It is becoming increasingly clear that astrocytes play very dynamic and interactive roles that are important for the normal functioning of the central nervous system. In culture, astrocytes express many receptors coupled to increases in intracellular calcium ([Ca2+]i). In vivo, it is likely that these receptors are important for the modulation of astrocytic functions such as the uptake of neurotransmitters and ions. Currently, however, very little is known about the expression or stimulation of such astrocytic receptors in vivo. To address this issue, confocal microscopy and calcium-sensitive fluorescent dyes were used to examine the dynamic changes in astrocytic [Ca2+]i within acutely isolated hippocampal slices. Astrocytes were subsequently identified by immunocytochemistry for glial fibrillary acidic protein. In this paper, we present data indicating that hippocampal astrocytes in situ respond to glutamate, kainate, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), 1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD), N-methyl-D-aspartate (NMDA), and depolarization with increases in [Ca2+]i. The increases in [Ca2+]i occurred in both the astrocytic cell bodies and the processes. Temporally the changes in [Ca2+]i were very dynamic, and various patterns ranging from sustained elevations to oscillations of [Ca2+]i were observed. Individual astrocytes responded to neuroligands selective for both ionotropic and metabotropic glutamate receptors with increases in [Ca2+]i. These findings indicate that astrocytes in vivo contain glutamatergic receptors coupled to increases in [Ca2+]i and are able to respond to neuronally released neurotransmitters.

Astroglia are remarkedly diverse cells with respect to their pharmacological responsiveness and the processes regulated by neuroligand receptors. The results of single cell analyses indicate that astroglia continue to diversify in vitro. For example, the two daughter cells of a single division can exhibit qualitatively distinct responses to neuroligand application. These findings indicate that in contrast to most cells that "lock" their neuroligand receptor phenotype early in development, astroglia exhibit unusual plasticity. It is possible that this plasticity is necessary for immature astrocytes to respond to a changing neuronal environment during development. Interestingly, the increase in astroglial calcium following addition of neuroligands tends to originate from a localized area of the cell and then spreads as a wave that moves through the cell in a nondecremental manner. In situations in which astroglia are connected by gap junctions, the wave of calcium in one cell readily moves into the second without any obvious decrease in magnitude. Furthermore, astroglial calcium responses occur in an "all-or-none" manner reminiscent of neuronal action potentials. That is, the magnitude of an astroglial cell's calcium response appears relatively independent of the concentration of the ligand used or the density of receptors present on the cell. However, the probability of an astroglial cell responding to a given neuroligand is related to the density of receptors expressed and the concentration of the neuroligand applied. These characteristics suggest that, like neurons, signals received by an astrocyte are integrated and that when a threshold is reached the cell responds with a localized response that then propagates through that cell and into adjacent cells in a nondecremental manner. In neurons this is accomplished via depolarizations leading to action potentials and release of neurotransmitters. In astrocytes, distant signaling appears to occur via an "all-or-none" release of calcium, which then propagates through the cell and neighboring cells as a calcium wave. Our recent findings that neuroligands can modulate the opening and closing of astrocytic gap junctions suggest that specific pathways of astrocytic communication may exist. Overall, it is becoming evident that neuronal-glial signaling may be far more complex than previously imagined.

With the trend toward nonphysician health professionals has come the pressure to grant these specialists hospital clinical privileges and, in some cases, medical staff membership. The varying degrees of education, training, and licensure that exist bring about a number of questions concerning the hospital's potential liability, the choice of disciplines to receive clinical privileges, and the establishment of review criteria.