Alleviating Hypertension by Selectively Targeting Angiotensin Receptor-Expressing Vagal Sensory Neurons.

Cardiovascular homeostasis is maintained, in part, by neural signals arising from arterial baroreceptors that apprise the brain of blood volume and pressure. Here, we test whether neurons within the nodose ganglia that express angiotensin type-1a receptors (referred to as NGAT1aR) serve as baroreceptors that differentially influence blood pressure (BP) in male and female mice. Using Agtr1a-Cre mice and Cre-dependent AAVs to direct tdTomato to NGAT1aR, neuroanatomical studies revealed that NGAT1aR receive input from the aortic arch, project to the caudal nucleus of the solitary tract (NTS), and synthesize mechanosensitive ion channels, Piezo1/2 To evaluate the functionality of NGAT1aR, we directed the fluorescent calcium indicator (GCaMP6s) or the light-sensitive channelrhodopsin-2 (ChR2) to Agtr1a-containing neurons. Two-photon intravital imaging in Agtr1a-GCaMP6s mice revealed that NGAT1aR couple their firing to elevated BP, induced by phenylephrine (i.v.). Furthermore, optical excitation of NGAT1aR at their soma or axon terminals within the caudal NTS of Agtr1a-ChR2 mice elicited robust frequency-dependent decreases in BP and heart rate, indicating that NGAT1aR are sufficient to elicit appropriate compensatory responses to vascular mechanosensation. Optical excitation also elicited hypotensive and bradycardic responses in ChR2-expressing mice that were subjected to deoxycorticosterone acetate (DOCA)-salt hypertension; however, the duration of these effects was altered, suggestive of hypertension-induced impairment of the baroreflex. Similarly, increased GCaMP6s fluorescence observed after administration of phenylephrine was delayed in mice subjected to DOCA-salt or chronic delivery of angiotensin II. Collectively, these results reveal the structure and function of NGAT1aR and suggest that such neurons may be exploited to discern and relieve hypertension.

Circuit and Cell-Specific Contributions to Decision Making Involving Risk of Explicit Punishment in Male and Female Rats.

Decision making is a complex cognitive process that recruits a distributed network of brain regions, including the basolateral amygdala (BLA) and nucleus accumbens shell (NAcSh). Recent work suggests that communication between these structures, as well as activity of cells expressing dopamine (DA) D2 receptors (D2R) in the NAcSh, are necessary for some forms of decision making; however, the contributions of this circuit and cell population during decision making under risk of punishment are unknown. The current experiments addressed this question using circuit-specific and cell type-specific optogenetic approaches in rats during a decision making task involving risk of punishment. In experiment 1, Long-Evans rats received intra-BLA injections of halorhodopsin or mCherry (control) and in experiment 2, D2-Cre transgenic rats received intra-NAcSh injections of Cre-dependent halorhodopsin or mCherry. Optic fibers were implanted in the NAcSh in both experiments. Following training in the decision making task, BLA→NAcSh or D2R-expressing neurons were optogenetically inhibited during different phases of the decision process. Inhibition of the BLA→NAcSh during deliberation (the time between trial initiation and choice) increased preference for the large, risky reward (increased risk taking). Similarly, inhibition during delivery of the large, punished reward increased risk taking, but only in males. Inhibition of D2R-expressing neurons in the NAcSh during deliberation increased risk taking. In contrast, inhibition of these neurons during delivery of the small, safe reward decreased risk taking. These findings extend our knowledge of the neural dynamics of risk taking, revealing sex-dependent circuit recruitment and dissociable activity of selective cell populations during decision making.SIGNIFICANCE STATEMENT Until recently, the ability to dissect the neural substrates of decision making involving risk of punishment (risk taking) in a circuit-specific and cell-specific manner has been limited by the tools available for use in rats. Here, we leveraged the temporal precision of optogenetics, together with transgenic rats, to probe contributions of a specific circuit and cell population to different phases of risk-based decision making. Our findings reveal basolateral amygdala (BLA)→nucleus accumbens shell (NAcSh) is involved in evaluation of punished rewards in a sex-dependent manner. Further, NAcSh D2 receptor (D2R)-expressing neurons make unique contributions to risk taking that vary across the decision making process. These findings advance our understanding of the neural principles of decision making and provide insight into how risk taking may become compromised in neuropsychiatric diseases.

Circuit and cell-specific contributions to decision making involving risk of explicit punishment in male and female rats.

Decision making is a complex cognitive process that recruits a distributed network of brain regions, including the basolateral amygdala (BLA) and nucleus accumbens shell (NAcSh). Recent work suggests that communication between these structures, as well as activity of cells expressing dopamine D2 receptors (D2R) in the NAcSh, are necessary for some forms of decision making; however, the contributions of this circuit and cell population during decision making under risk of punishment are unknown. The current experiments addressed this question using circuit- and cell type-specific optogenetic approaches in rats during a decision-making task involving risk of punishment. In Experiment 1, Long-Evans rats received intra-BLA injections of halorhodopsin or mCherry (control) and in Experiment 2, D2-Cre transgenic rats received intra-NAcSh injections of Cre-dependent halorhodopsin or mCherry. Optic fibers were implanted in the NAcSh in both experiments. Following training in the decision-making task, BLA→NAcSh or D2R-expressing neurons were optogenetically inhibited during different phases of the decision process. Inhibition of the BLA→NAcSh during deliberation (the time between trial initiation and choice) increased choice of the large, risky reward (increased risk taking). Similarly, inhibition during delivery of the large, punished reward increased risk taking, but only in males. Inhibition of D2R-expressing neurons in the NAcSh during deliberation increased risk taking. In contrast, inhibition of these neurons during delivery of the small, safe reward decreased risk taking. These findings extend our knowledge of the neural dynamics of risk taking, revealing sex-dependent circuit recruitment and dissociable activity of selective cell populations during decision making.

A rapid in vitro assay for evaluating the effects of acetylcholinesterase inhibitors and reactivators in the rat basolateral amygdala.

We established a novel brain slice assay to test the ability of acetylcholinesterase (AChE) reactivators to prevent ACh-induced M1 muscarinic acetylcholine receptor (mAChR) dependent hyperexcitability observed after exposure to the organophosphate (OP)-based AChE inhibitor and sarin surrogate 4-nitrophenyl isopropyl methylphosphonate (NIMP). Whole-cell patch clamp recordings were used to evaluate the response of pyramidal neurons in the rat basolateral amygdala (BLA) to brief (1 min) bath application of ACh (100 µM), either in control conditions, or after exposure to NIMP ± an AChE reactivator. Bath application of ACh produced atropine- and pirenzepine-sensitive inward currents in voltage clamped BLA pyramidal neurons, and increased the frequency of spontaneous EPSCs, suggesting robust activation of M1 mAChRs. Responses to ACh were increased ~3-5 fold in slices that had been preincubated in NIMP, and these effects were reversed in a concentration dependent manner by exposure to a commercially available AChE reactivator. The current work outlines a simple assay that can be used to evaluate the efficacy of both known and novel AChE reactivators in an area of the limbic system that likely contributes to seizures after acute exposure to OP-based AChE inhibitors.

A Novel Organ-Specific Approach to Selectively Target Sensory Afferents Innervating the Aortic Arch.

The brain maintains cardiovascular homeostasis, in part, via the arterial baroreflex which senses changes in blood pressure (BP) at the level of the aortic arch. Sensory afferents innervating the aortic arch employ baroreceptors to convert stretch exerted on the arterial wall into action potentials carried by the vagus nerve to second order neurons residing within the nucleus of the solitary tract (NTS). Although the baroreflex was described more than 80 years ago, the specific molecular, structural, and functional phenotype of the baroreceptors remain uncharacterized. This is due to the lack of tools that provide the genetic and target organ specificity that is required to selectively characterize baroreceptor afferents. Here, we use a novel approach to selectively target baroreceptors. Male mice on a C57BL/6J background were anesthetized with isoflurane, intubated, and artificially ventilated. Following sternotomy, the aortic arch was exposed, and a retrograde adeno-associated virus was applied to the aortic arch to direct the expression of channelrhoropsin-2 (ChR2) and/or tdTomato (tdTom) to sensory afferents presumably functioning as baroreceptors. Consistent with the structural characteristics of arterial baroreceptors, robust tdTom expression was observed in nerve endings surrounding the aortic arch, within the fibers of the aortic depressor and vagus nerves, cell bodies of the nodose ganglia (NDG), and neural projections to the caudal NTS (cNTS). Additionally, the tdTom labeled cell bodies within the NDG also expressed mRNAs coding for the mechanically gated ion channels, PIEZO-1 and PIEZO-2. In vitro electrophysiology revealed that pulses of blue light evoked excitatory post-synaptic currents in a subset of neurons within the cNTS, suggesting a functional connection between the labeled aortic arch sensory afferents and second order neurons. Finally, the in vivo optogenetic stimulation of the cell bodies of the baroreceptor expressing afferents in the NDG produced robust depressor responses. Together, these results establish a novel approach for selectively targeting sensory neurons innervating the aortic arch. This approach may be used to investigate arterial baroreceptors structurally and functionally, and to assess their role in the etiology or reversal of cardiovascular disease.

Intrahypothalamic effects of oxytocin on PVN CRH neurons in response to acute stress.

Much of the centrally available oxytocin (OT) is synthesized in magnocellular neurons located in the paraventricular nucleus of the hypothalamus. This same area is home to parvocellular corticotropin-releasing hormone (CRH) synthesizing neurons that regulate activation of the hypothalamic-pituitary-adrenal (HPA) axis. A large body of data indicates that complex interactions between these systems inextricably link central OT signaling with the neuroendocrine response to stress. This review focuses on a small but diverse set of cellular and synaptic mechanisms that have been proposed to underlie intrahypothalamic OT/CRF interactions during the response to acute stress.

Targeting angiotensin type-2 receptors located on pressor neurons in the nucleus of the solitary tract to relieve hypertension in mice.

AIMS: These studies evaluate whether angiotensin type-2 receptors (AT2Rs) that are expressed on γ-aminobutyric acid (GABA) neurons in the nucleus of the solitary tract (NTS) represent a novel endogenous blood pressure-lowering mechanism. METHODS AND RESULTS: Experiments combined advanced genetic and neuroanatomical techniques, pharmacology, electrophysiology, and optogenetics in mice to define the structure and cardiovascular-related function of NTS neurons that contain AT2R. Using mice with Cre-recombinase directed to the AT2R gene, we discovered that optogenetic stimulation of AT2R-expressing neurons in the NTS increases GABA release and blood pressure. To evaluate the role of the receptor, per se, in cardiovascular regulation, we chronically delivered C21, a selective AT2R agonist, into the brains of normotensive mice and found that central AT2R activation reduces GABA-related gene expression and blunts the pressor responses induced by optogenetic excitation of NTS AT2R neurons. Next, using in situ hybridization, we found that the levels of Agtr2 mRNAs in GABAergic NTS neurons rise during experimentally induced hypertension, and we hypothesized that this increased expression may be exploited to ameliorate the disease. Consistent with this, final experiments revealed that central administration of C21 attenuates hypertension, an effect that is abolished in mice lacking AT2R in GABAergic NTS neurons. CONCLUSION: These studies unveil novel hindbrain circuits that maintain arterial blood pressure, and reveal a specific population of AT2R that can be engaged to alleviate hypertension. The implication is that these discrete receptors may serve as an access point for activating an endogenous depressor circuit.

Attenuated NMDAR signaling on fast-spiking interneurons in prefrontal cortex contributes to age-related decline of cognitive flexibility.

Ionotropic glutamate receptors of the NMDA and AMPA subtypes transduce excitatory signaling on neurons in the prefrontal cortex (PFC) in support of cognitive flexibility. Cognitive flexibility is reliably observed to decline at advanced ages, coinciding with changes in PFC glutamate receptor expression and neuronal physiology. However, the relationship between age-related impairment of cognitive flexibility and changes to excitatory signaling on distinct classes of PFC neurons is not known. In this study, one cohort of young adult (4 months) and aged (20 months) male F344 rats were characterized for cognitive flexibility on an operant set-shifting task. Expression of the essential NMDAR subunit, NR1, was correlated with individual differences in set-shifting abilities such that lower NR1 in the aged PFC was associated with worse set-shifting. In contrast, lower expression of two AMPAR subunits, GluR1 and GluR2, was not associated with set-shift abilities in aging. As NMDARs are expressed by both pyramidal cells and fast-spiking interneurons (FSI) in PFC, whole-cell patch clamp recordings were performed in a second cohort of age-matched rats to compare age-associated changes on these neuronal subtypes. Evoked excitatory postsynaptic currents were generated using a bipolar stimulator while AMPAR vs. NMDAR-mediated components were isolated using pharmacological tools. The results revealed a clear increase in AMPA/NMDA ratio in FSIs that was not present in pyramidal neurons. Together, these data indicate that loss of NMDARs on interneurons in PFC contributes to age-related impairment of cognitive flexibility.

Dendritic osmosensors modulate activity-induced calcium influx in oxytocinergic magnocellular neurons of the mouse PVN.

Hypothalamic oxytocinergic magnocellular neurons have a fascinating ability to release peptide from both their axon terminals and from their dendrites. Existing data indicates that the relationship between somatic activity and dendritic release is not constant, but the mechanisms through which this relationship can be modulated are not completely understood. Here, we use a combination of electrical and optical recording techniques to quantify activity-induced calcium influx in proximal vs. distal dendrites of oxytocinergic magnocellular neurons located in the paraventricular nucleus of the hypothalamus (OT-MCNs). Results reveal that the dendrites of OT-MCNs are weak conductors of somatic voltage changes; however, activity-induced dendritic calcium influx can be robustly regulated by both osmosensitive and non-osmosensitive ion channels located along the dendritic membrane. Overall, this study reveals that dendritic conductivity is a dynamic and endogenously regulated feature of OT-MCNs that is likely to have substantial functional impact on central oxytocin release.

Oxytocin is often referred to as a 'love hormone' because it can be released during activities such as hugging, snuggling, or sex. Reality, of course, can be a bit more complicated. In the brain, oxytocin can have powerful and diverse effects on mood, stress, anxiety, and social interactions. In the body it helps regulate fluid balance, promotes contractions during childbirth, and stimulates the letdown of milk during breastfeeding. Much of the oxytocin produced in both humans and rodents comes from oxytocin-synthetizing magnocellular neurons located in an area of the brain called the hypothalamus. These very specialized neurons have separate, but overlapping, mechanisms for releasing oxytocin into the brain and into the rest of the body. This means that while certain signals cause the neurons to release oxytocin into the body and the brain at the same time, others can cause them to release the hormone preferentially into the body or the brain. Sheng et al. wanted to better understand how these different release mechanisms work, and, in particular, to learn more about how release of oxytocin into the brain is regulated. This is important, because when oxytocin is given as a medicine, much of it fails to reach the brain. A lot of the oxytocin that acts in the brain is released from a specific part of the oxytocin-synthesizing magnocellular neurons called the dendrites. When these neurons are stimulated, calcium enters the dendrites, triggering the release of oxytocin directly into the brain. Sheng et al. used electrical and optical tools on brain tissue extracted from mice to measure how different signals change the amount of calcium that enters the dendrites of oxytocin-synthesizing magnocellular neurons in response to a consistent stimulus. The results showed that increasing the osmolarity, the amount of water-soluble particles that cannot spontaneously cross the cell membrane, in the liquid surrounding the neurons reduced the amount of calcium that flowed into the dendrites during stimulation. Meanwhile, decreasing osmolarity had the opposite effect. Sheng et al. also found that the influx of calcium induced by stimulating the neurons can be strongly regulated by activating receptors in the dendrites that detect a common molecule in the brain called GABA. This occurs even absent a change in osmolarity. These results shed light on some of the physiological processes that control the release of oxytocin into the brain. Understanding these processes is a necessary step towards developing new drugs intended to regulate levels of oxytocin in the brain. Such drugs could be useful in the treatment of several types of mental health disorders.

Alteration of the cholinergic system and motor deficits in cholinergic neuron-specific Dyt1 knockout mice.

Dystonia is a neurological movement disorder characterized by sustained or intermittent muscle contractions, repetitive movement, and sometimes abnormal postures. DYT1 dystonia is one of the most common genetic dystonias, and most patients carry heterozygous DYT1 ∆GAG mutations causing a loss of a glutamic acid of the protein torsinA. Patients can be treated with anticholinergics, such as trihexyphenidyl, suggesting an abnormal cholinergic state. Early work on the cell-autonomous effects of Dyt1 deletion with ChI-specific Dyt1 conditional knockout mice (Dyt1 Ch1KO) revealed abnormal electrophysiological responses of striatal ChIs to muscarine and quinpirole, motor deficits, and no changes in the number or size of the ChIs. However, the Chat-cre line that was used to derive Dyt1 Ch1KO mice contained a neomycin cassette and was reported to have ectopic cre-mediated recombination. In this study, we generated a Dyt1 Ch2KO mouse line by removing the neomycin cassette in Dyt1 Ch1KO mice. The Dyt1 Ch2KO mice showed abnormal paw clenching behavior, motor coordination and balance deficits, impaired motor learning, reduced striatal choline acetyltransferase protein level, and a reduced number of striatal ChIs. Furthermore, the mutant striatal ChIs had a normal muscarinic inhibitory function, impaired quinpirole-mediated inhibition, and altered current density. Our findings demonstrate a cell-autonomous effect of Dyt1 deletion on the striatal ChIs and a critical role for the striatal ChIs and corticostriatal pathway in the pathogenesis of DYT1 dystonia.

An Angiotensin-Responsive Connection from the Lamina Terminalis to the Paraventricular Nucleus of the Hypothalamus Evokes Vasopressin Secretion to Increase Blood Pressure in Mice.

Blood pressure is controlled by endocrine, autonomic, and behavioral responses that maintain blood volume and perfusion pressure at levels optimal for survival. Although it is clear that central angiotensin type 1a receptors (AT1aR; encoded by the Agtr1a gene) influence these processes, the neuronal circuits mediating these effects are incompletely understood. The present studies characterize the structure and function of AT1aR neurons in the lamina terminalis (containing the median preoptic nucleus and organum vasculosum of the lamina terminalis), thereby evaluating their roles in blood pressure control. Using male Agtr1a-Cre mice, neuroanatomical studies reveal that AT1aR neurons in the area are largely glutamatergic and send projections to the paraventricular nucleus of the hypothalamus (PVN) that appear to synapse onto vasopressin-synthesizing neurons. To evaluate the functionality of these lamina terminalis AT1aR neurons, we virally delivered light-sensitive opsins and then optogenetically excited or inhibited the neurons while evaluating cardiovascular parameters or fluid intake. Optogenetic excitation robustly elevated blood pressure, water intake, and sodium intake, while optogenetic inhibition produced the opposite effects. Intriguingly, optogenetic excitation of these AT1aR neurons of the lamina terminalis also resulted in Fos induction in vasopressin neurons within the PVN and supraoptic nucleus. Further, within the PVN, selective optogenetic stimulation of afferents that arise from these lamina terminalis AT1aR neurons induced glutamate release onto magnocellular neurons and was sufficient to increase blood pressure. These cardiovascular effects were attenuated by systemic pretreatment with a vasopressin-1a-receptor antagonist. Collectively, these data indicate that excitation of lamina terminalis AT1aR neurons induces neuroendocrine and behavioral responses that increase blood pressure.SIGNIFICANCE STATEMENT Hypertension is a widespread health problem and risk factor for cardiovascular disease. Although treatments exist, a substantial percentage of patients suffer from "drug-resistant" hypertension, a condition associated with increased activation of brain angiotensin receptors, enhanced sympathetic nervous system activity, and elevated vasopressin levels. The present study highlights a role for angiotensin Type 1a receptor expressing neurons located within the lamina terminalis in regulating endocrine and behavioral responses that are involved in maintaining cardiovascular homeostasis. More specifically, data presented here reveal functional excitatory connections between angiotensin-sensitive neurons in the lamina terminals and vasopressin neurons in the paraventricular nucleus of the hypothalamus, and further indicate that activation of this circuit raises blood pressure. These neurons may be a promising target for antihypertensive therapeutics.

Endogenous oxytocin inhibits hypothalamic corticotrophin-releasing hormone neurones following acute hypernatraemia.

Significant prior evidence indicates that centrally acting oxytocin robustly modulates stress responsiveness and anxiety-like behaviour, although the neural mechanisms behind these effects are not entirely understood. A plausible neural basis for oxytocin-mediated stress reduction is via inhibition of corticotrophin-releasing hormone (CRH) neurones in the paraventricular nucleus of the hypothalamus (PVN) that regulate activation of the hypothalamic-pituitary-adrenal axis. Previously, we have shown that, following s.c. injection of 2.0 mol L-1 NaCl, oxytocin synthesising neurones are activated in the rat PVN, an oxytocin receptor (Oxtr)-dependent inhibitory tone develops on a subset of parvocellular neurones and stress-mediated increases in plasma corticosterone levels are blunted. In the present study, we utilised transgenic male CRH-reporter mice to selectively target PVN CRH neurones for whole-cell recordings. These experiments reveal that acute salt loading produces tonic inhibition of PVN CRH neurones through a mechanism that is largely independent of synaptic activity. Further studies reveal that a subset of CRH neurones within the PVN synthesise mRNA for Oxtr(s). Salt induced Oxtr-dependent inhibitory tone was eliminated in individual PVN CRH neurones filled with GDP-ß-S. Additional electrophysiological studies suggest that reduced excitability of PVN CRH neurones in salt-loaded animals is associated with increased activation of inwardly rectifying potassium channels. Nevertheless, substantial effort to recapitulate the core effects of salt loading by activating Oxtr(s) with an exogenous agonist produced mixed results. Collectively, these results enhance our understanding of how oxytocin receptor-mediated signalling modulates the function of CRH neurones in the PVN.

Region-specific effects of HIV-1 Tat on intrinsic electrophysiological properties of pyramidal neurons in mouse prefrontal cortex and hippocampus.

Human immunodeficiency virus (HIV)-1 transactivator of transcription protein (Tat) is a viral protein that promotes transcription of the HIV genome and possesses cell-signaling properties. Long-term exposure of central nervous system (CNS) tissue to HIV-1 Tat is theorized to contribute to HIV-associated neurodegenerative disorder (HAND). In the current study, we sought to directly evaluate the effect of HIV-1 Tat expression on the intrinsic electrophysiological properties of pyramidal neurons located in layer 2/3 of the medial prefrontal cortex and in area CA1 of the hippocampus. Toward that end, we drove Tat expression with doxycycline (100 mg·kg-1·day-1 ip) in inducible Tat (iTat) transgenic mice for 7 days and then performed single-cell electrophysiological studies in acute tissue slices made through the prefrontal cortex and hippocampus. Control experiments were performed in doxycycline-treated G-tg mice, which retain the tetracycline-sensitive promoter but do not express Tat. Our results indicated that the predominant effects of HIV-1 Tat expression are excitatory in medial prefrontal cortical pyramidal neurons yet inhibitory in hippocampal pyramidal neurons. Notably, in these two populations, HIV-1 Tat expression produced differential effects on neuronal gain, membrane time constant, resting membrane potential, and rheobase. Similarly, we also observed distinct effects on action potential kinetics and afterhyperpolarization, as well as on the current-voltage relationship in subthreshold voltage ranges. Collectively, these data provide mechanistic evidence of complex and region-specific changes in neuronal physiology by which HIV-1 Tat protein may promote cognitive deficits associated with HAND.NEW & NOTEWORTHY We drove expression of human immunodeficiency virus (HIV)-1 transactivator of transcription protein (Tat) protein in inducible Tat (iTat) transgenic mice for 7 days and then examined the effects on the intrinsic electrophysiological properties of pyramidal neurons located in the medial prefrontal cortex (mPFC) and in the hippocampus. Our results reveal a variety of specific changes that promote increased intrinsic excitability of layer II/III mPFC pyramidal neurons and decreased intrinsic excitability of hippocampal CA1 pyramidal neurons, highlighting both cell type and region-specific effects.

Optogenetic dissection of basolateral amygdala contributions to intertemporal choice in young and aged rats.

Across species, aging is associated with an increased ability to choose delayed over immediate gratification. These experiments used young and aged rats to test the role of the basolateral amygdala (BLA) in intertemporal decision making. An optogenetic approach was used to inactivate the BLA in young and aged rats at discrete time points during choices between levers that yielded a small, immediate vs. a large, delayed food reward. BLA inactivation just prior to decisions attenuated impulsive choice in both young and aged rats. In contrast, inactivation during receipt of the small, immediate reward increased impulsive choice in young rats but had no effect in aged rats. BLA inactivation during the delay or intertrial interval had no effect at either age. These data demonstrate that the BLA plays multiple, temporally distinct roles during intertemporal choice, and show that the contribution of BLA to choice behavior changes across the lifespan.

Oxytocin Receptors Are Expressed by Glutamatergic Prefrontal Cortical Neurons That Selectively Modulate Social Recognition.

Social recognition, the ability to recognize individuals that were previously encountered, requires complex integration of sensory inputs with previous experience. Here, we use a variety of approaches to discern how oxytocin-sensitive neurons in the PFC exert descending control over a circuit mediating social recognition in mice. Using male mice with Cre-recombinase directed to the oxytocin receptor gene (Oxtr), we revealed that oxytocin receptors (OXTRs) are expressed on glutamatergic neurons in the PFC, optogenetic stimulation of which elicited activation of neurons residing in several mesolimbic brain structures. Optogenetic stimulation of axons in the BLA arising from OXTR-expressing neurons in the PFC eliminated the ability to distinguish novel from familiar conspecifics, but remarkably, distinguishing between novel and familiar objects was unaffected. These results suggest that an oxytocin-sensitive PFC to BLA circuit is required for social recognition. The implication is that impaired social memory may manifest from dysregulation of this circuit.SIGNIFICANCE STATEMENT Using mice, we demonstrate that optogenetic activation of the neurons in the PFC that express the oxytocin receptor gene (Oxtr) impairs the ability to distinguish between novel and familiar conspecifics, but the ability to distinguish between novel and familiar objects remains intact. Subjects with autism spectrum disorders (ASDs) have difficulty identifying a person based on remembering facial features; however, ASDs and typical subjects perform similarly when remembering objects. In subjects with ASD, viewing the same face increases neural activity in the PFC, which may be analogous to the optogenetic excitation of oxytocin receptor (OXTR) expressing neurons in the PFC that impairs social recognition in mice. The implication is that overactivation of OXTR-expressing neurons in the PFC may contribute to ASD symptomology.

Optogenetic Inhibition Reveals Distinct Roles for Basolateral Amygdala Activity at Discrete Time Points during Risky Decision Making.

Decision making is a multifaceted process, consisting of several distinct phases that likely require different cognitive operations. Previous work showed that the basolateral amygdala (BLA) is a critical substrate for decision making involving risk of punishment; however, it is unclear how the BLA is recruited at different stages of the decision process. To this end, the current study used optogenetics to inhibit the BLA during specific task phases in a model of risky decision making (risky decision-making task) in which rats choose between a small, "safe" reward and a large reward accompanied by varying probabilities of footshock punishment. Male Long-Evans rats received intra-BLA microinjections of viral vectors carrying either halorhodopsin (eNpHR3.0-mCherry) or mCherry alone (control) followed by optic fiber implants and were trained in the risky decision-making task. Laser delivery during the task occurred during intertrial interval, deliberation, or reward outcome phases, the latter of which was further divided into the three possible outcomes (small, safe; large, unpunished; large, punished). Inhibition of the BLA selectively during the deliberation phase decreased choice of the large, risky outcome (decreased risky choice). In contrast, BLA inhibition selectively during delivery of the large, punished outcome increased risky choice. Inhibition had no effect during the other phases, nor did laser delivery affect performance in control rats. Collectively, these data indicate that the BLA can either inhibit or promote choice of risky options, depending on the phase of the decision process in which it is active.SIGNIFICANCE STATEMENT To date, most behavioral neuroscience research on neural mechanisms of decision making has used techniques that preclude assessment of distinct phases of the decision process. Here we show that optogenetic inhibition of the BLA has opposite effects on choice behavior in a rat model of risky decision making, depending on the phase in which inhibition occurs. BLA inhibition during a period of deliberation between small, safe and large, risky outcomes decreased risky choice. In contrast, BLA inhibition during receipt of the large, punished outcome increased risky choice. These findings highlight the importance of temporally targeted approaches to understand neural substrates underlying complex cognitive processes. More importantly, they reveal novel information about dynamic BLA modulation of risky choice.

Chronic salt-loading reduces basal excitatory input to CRH neurons in the paraventricular nucleus and accelerates recovery from restraint stress in male mice.

Neurons synthesizing corticotrophin-releasing hormone (CRH) in the paraventricular nucleus of the hypothalamus (PVN) are activated during acute stress and act via the hypothalamic-pituitary-adrenal (HPA) axis to increase systemic levels of corticosterone (CORT). Recent data indicates that CRH neurons in the PVN are inhibited by acute salt-loading, and that this inhibition blunts the response to restraint stress as measured by increases in plasma CORT. The current study evaluates the effects of chronic rather than acute salt-loading on stress-induced activation of the HPA axis. Relative to euhydrated controls, chronic salt-loading over a 5-day period elevated plasma sodium and fluid intake without eliciting hypovolemia or substantial alterations in food intake or body weight. Chronic salt-loading also decreased expression of CRH mRNA in the anterior but not posterior portion of the PVN. Similarly, whole cell patch clamp recordings revealed that salt-loading effectively decreases spontaneous excitatory input to CRH neurons in the PVN without altering spontaneous inhibitory input. Generally consistent with these observations, chronic salt attenuated HPA axis activation as indicated by a significant reduction of plasma CORT during recovery from restraint stress.

NR2A-Containing NMDARs in the Prefrontal Cortex Are Required for Working Memory and Associated with Age-Related Cognitive Decline.

Working memory, the ability to temporarily maintain representational knowledge, is a foundational cognitive process that can become compromised in aging and neuropsychiatric disease. NMDA receptor (NMDAR) activation in prefrontal cortex (PFC) is necessary for the pyramidal neuron activity believed to enable working memory; however, the distinct biophysical properties and localization of NMDARs containing NR2A and NR2B subunits suggest unique roles for NMDAR subtypes in PFC neural activity and working memory. Experiments herein show that working memory depends on NR2A- but not NR2B-NMDARs in PFC of rats and that NR2A-NMDARs mediate the majority of evoked NMDAR currents on layer 2/3 PFC pyramidal neurons. Moreover, attenuated expression of the NR2A but not the NR2B subunit in PFC associates with naturally occurring working memory impairment in aged rats. Finally, NMDAR currents and working memory are enhanced in aged rats by promoting activation of the NR2A-enriched synaptic pool of PFC NMDARs. These results implicate NR2A-NMDARs in normal working memory and suggest novel treatment strategies for improving working memory in cognitive disorders. SIGNIFICANCE STATEMENT: Working memory, the ability to hold information "in mind," requires persistent activity of pyramidal neurons in prefrontal cortex (PFC) mediated by NMDA receptor (NMDAR) activation. NMDAR loss in PFC may account for working memory impairments in aging and psychiatric disease. Our studies demonstrate that NMDARs containing the NR2A subunit, but not the NR2B subunit, are required for working memory and that loss of NR2A predicts severity of age-related working memory impairment. The importance of NR2A to working memory is likely due its abundant contribution to pyramidal neuron activity and location at synaptic sites in PFC. This information is useful in designing new therapies to treat working memory impairments by enhancing the function of NR2A-containing NMDARs.

Age-related changes in tonic activation of presynaptic versus extrasynaptic γ-amniobutyric acid type B receptors in rat medial prefrontal cortex.

The present study examined the effect of age on both glutamatergic and γ-aminobutyric acid mediated (GABAergic) signaling in the rodent medial prefrontal cortex (mPFC), with an emphasis on revealing novel changes contributing to increased inhibition in age. Whole-cell patch clamp recordings were obtained from layer 2/3 mPFC pyramidal neurons in acute cortical slices prepared from either young (4 months) or aged (20-24 months) male F344 rats. Results indicated that GABAB receptors on GABAergic, but not on glutamatergic, inputs to layer 2/3 pyramidal cells are tonically activated by ambient GABA in young animals and further demonstrated that this form of tonic inhibition is significantly attenuated in aged mPFC. Moreover, concurrent with loss of tonic presynaptic GABAB autoreceptor activation, layer 2/3 pyramidal cells in aged mPFC are subjected to increased tonic activation of extrasynaptic GABAA and GABAB receptors. These data demonstrate a shift in the site of GABAB receptor-mediated inhibitory tone in the aged mPFC that clearly promotes increased inhibition of pyramidal cells in aged animals, and that may plausibly contribute to impaired executive function.

Alcohol consumption increases basal extracellular glutamate in the nucleus accumbens core of Sprague-Dawley rats without increasing spontaneous glutamate release.

Glutamate neurotransmission in the nucleus accumbens core (NAc) mediates ethanol consumption. Previous studies using non-contingent and voluntary alcohol administration in inbred rodents have reported increased basal extracellular glutamate levels in the NAc. Here, we assessed basal glutamate levels in the NAc following intermittent alcohol consumption in male Sprague-Dawley rats that had access to ethanol for 7 weeks on alternating days. We found increased basal NAc glutamate at 24 h withdrawal from ethanol and thus sought to identify the source of this glutamate. To do so, we employed a combination of microdialysis, slice electrophysiology and western blotting. Reverse dialysis of the voltage-gated sodium channel blocker tetrodotoxin did not affect glutamate levels in either group. Electrophysiological recordings in slices made after 24 h withdrawal revealed a decrease in spontaneous excitatory postsynaptic current (sEPSC) frequency relative to controls, with no change in sEPSC amplitude. No change in metabotropic glutamate receptor 2/3 (mGlu2/3) function was detected as bath application of the mGlu2/3 agonist LY379268 decreased spontaneous and miniature EPSC frequency in slices from both control and ethanol-consuming rats. The increase in basal glutamate was not associated with changes in the surface expression of GLT-1, however, a decrease in slope of the no-net-flux dialysis function was observed following ethanol consumption, indicating a potential decrease in glutamate reuptake. Taken together, these findings indicate that the increase in basal extracellular glutamate occurring after chronic ethanol consumption is not mediated by an increase in action potential-dependent glutamate release or a failure of mGlu2/3 autoreceptors to regulate such release.