Functional Dissection of Glutamatergic and GABAergic Neurons in the Bed Nucleus of the Stria Terminalis

The bed nucleus of the stria terminalis (BNST)—a key part of the extended amygdala—has been implicated in the regulation of diverse behavioral states, ranging from anxiety and reward processing to feeding behavior. Among the host of distinct types of neurons within the BNST, recent investigations employing cell type- and projection-specific circuit dissection techniques (such as optogenetics, chemogenetics, deep-brain calcium imaging, and the genetic and viral methods for targeting specific types of cells) have highlighted the key roles of glutamatergic and GABAergic neurons and their axonal projections. As anticipated from their primary roles in excitatory and inhibitory neurotransmission, these studies established that the glutamatergic and GABAergic subpopulations of the BNST oppositely regulate diverse behavioral states. At the same time, these studies have also revealed unexpected functional specificity and heterogeneity within each subpopulation. In this Minireview, we introduce the body of studies that investigated the function of glutamatergic and GABAergic BNST neurons and their circuits. We also discuss unresolved questions and future directions for a more complete understanding of the cellular diversity and functional heterogeneity within the BNST.     

Sodium Salicylate Suppresses GABAergic Inhibitory Activity in Neurons of Rodent Dorsal Raphe Nucleus

Sodium salicylate (NaSal), a tinnitus inducing agent, can activate serotonergic (5-HTergic) neurons in the dorsal raphe nucleus (DRN) and can increase serotonin (5-HT) level in the inferior colliculus and the auditory cortex in rodents. To explore the underlying neural mechanisms, we first examined effects of NaSal on neuronal intrinsic properties and the inhibitory synaptic transmissions in DRN slices of rats by using whole-cell patch-clamp technique. We found that NaSal hyperpolarized the resting membrane potential, decreased the input resistance, and suppressed spontaneous and current-evoked firing in GABAergic neurons, but not in 5-HTergic neurons. In addition, NaSal reduced GABAergic spontaneous and miniature inhibitory postsynaptic currents in 5-HTergic neurons. We next examined whether the observed depression of GABAergic activity would cause an increase in the excitability of 5-HTergic neurons using optogenetic technique in DRN slices of the transgenic mouse with channelrhodopsin-2 expressed in GABAergic neurons. When the GABAergic inhibition was enhanced by optical stimulation to GABAergic neurons in mouse DRN, NaSal significantly depolarized the resting membrane potential, increased the input resistance and increased current-evoked firing of 5-HTergic neurons. However, NaSal would fail to increase the excitability of 5-HTergic neurons when the GABAergic synaptic transmission was blocked by picrotoxin, a GABA receptor antagonist. Our results indicate that NaSal suppresses the GABAergic activities to raise the excitability of local 5-HTergic neural circuits in the DRN, which may contribute to the elevated 5-HT level by NaSal in the brain.     

Activation of Metabotropic Glutamate Receptors Regulates Ribosomes of Cochlear Nucleus Neurons

The brain stem auditory system of the chick is an advantageous model for examining changes that occur as a result of deafness. Elimination of acoustic input through cochlear ablation results in the eventual death of approximately 30% of neurons in the chick cochlear nucleus, nucleus magnocellularis (NM). One early change following deafness is an alteration in NM ribosomes, evidenced both by a decrease in protein synthesis and reduction in antigenicity for Y10B, a monoclonal antibody that recognizes a ribosomal epitope. Previous studies have shown that mGluR activation is necessary to maintain Y10B antigenicity and NM viability. What is still unclear, however, is whether or not mGluR activation is sufficient to prevent deafness-induced changes in these neurons, or if other activity-dependent factors are also necessary. The current study investigated the ability of mGluR activation to regulate cochlear nucleus ribosomes in the absence of auditory nerve input. In vitro methods were employed to periodically pressure eject glutamate or mGluR agonists over neurons on one side of a slice preparation leaving the opposite side of the same slice untreated. Immunohistochemistry was then performed using Y10B in order to assess ribosomal changes. Application of glutamate and both group I and II selective mGluR agonists effectively rescued ribosomal antigenicity on the treated side of the slice in comparison to ribosomes on the untreated side. These findings suggest that administration of mGluR agonists is sufficient to reduce the early interruption of normal ribosomal integrity that is typically seen following loss of auditory nerve activity.     

Galanin Alters GABAergic Neurotransmission in the Dorsal Raphe Nucleus

The neuropeptide galanin and its three receptor subtypes (Gal R1-3) are highly expressed in the dorsal raphe nucleus (DRN), a region of the brain that contains a large population of serotonergic neurons. Galanin is co-expressed with serotonin in approximately 40% of the DRN neurons, and galanin and GALR2 expression are elevated by antidepressants like the SSRI fluoxetine, suggesting an interaction between serotonin and galanin. The present study examines the effect of galanin (Gal 1–29), a pan ligand for GalR (1–3) and the GalR2/GalR3-selective ligand, Gal 2–11, on the electrophysiological properties of DRN serotonergic neurons in a slice preparation. We recorded from cells in the DRN with electrophysiological characteristics consistent with those of serotonergic neurons that exhibit high input resistance, large after-hyperpolarizations and long spike duration as defined by Aghajanian and Vandermaelen. Both Gal 1–29 and Gal 2–11 decreased the amplitudes pharmacologically-isolated GABAergic inhibitory postsynaptic potentials (IPSPs) in these putative serotonergic neurons. Furthermore, based on paired pulse facilitation studies, we show that Gal 1–29 likely decreases GABA release through a presynaptic mechanism, whereas Gal 2–11 may act postsynaptically. These findings may enhance understanding of the cellular mechanisms underlying the effects of antidepressant treatments on galanin and galanin receptors in DRN. Special issue article in honor of Dr. Frode Fonnum.     

Hyperbaric Oxygen Attenuates Withdrawal Symptoms by Regulating Monoaminergic Neurotransmitters and NO Signaling Pathway at Nucleus Accumbens in Morphine-Dependent Rats

In this study, we examined whether hyperbaric oxygen (HBO₂) plays a detoxification role in withdrawal symptoms in a morphine-dependent rat model. The model was established through injections of morphine at increasing doses for 7 days. Withdrawal symptoms were induced by naloxone injection on the 8th day. The detoxification effect of HBO₂ was evaluated using the withdrawal symptom scores, biochemical indices and neurotransmitters. Compared with the model group, HBO₂ therapy significantly attenuated the withdrawal symptom scores, body weight loss and the level of norepinephrine level, whereas it increased the dopamine level and tyrosine hydroxylase expression in the nucleus accumbens. Moreover, HBO₂ therapy substantially alleviated the NO, NOS, cAMP, and cGMP levels. Our findings indicate that HBO₂ can effectively alleviate withdrawal symptoms induced by morphine dependence, and these effects may be attributed to the modulation of monoaminergic neurotransmitters and the suppression of the NO–cGMP signaling pathway.     

Positive Reinforcement Mediated by Midbrain Dopamine Neurons Requires D1 and D2 Receptor Activation in the Nucleus Accumbens

The neural basis of positive reinforcement is often studied in the laboratory using intracranial self-stimulation (ICSS), a simple behavioral model in which subjects perform an action in order to obtain exogenous stimulation of a specific brain area. Recently we showed that activation of ventral tegmental area (VTA) dopamine neurons supports ICSS behavior, consistent with proposed roles of this neural population in reinforcement learning. However, VTA dopamine neurons make connections with diverse brain regions, and the specific efferent target(s) that mediate the ability of dopamine neuron activation to support ICSS have not been definitively demonstrated. Here, we examine in transgenic rats whether dopamine neuron-specific ICSS relies on the connection between the VTA and the nucleus accumbens (NAc), a brain region also implicated in positive reinforcement. We find that optogenetic activation of dopaminergic terminals innervating the NAc is sufficient to drive ICSS, and that ICSS driven by optical activation of dopamine neuron somata in the VTA is significantly attenuated by intra-NAc injections of D1 or D2 receptor antagonists. These data demonstrate that the NAc is a critical efferent target sustaining dopamine neuron-specific ICSS, identify receptor subtypes through which dopamine acts to promote this behavior, and ultimately help to refine our understanding of the neural circuitry mediating positive reinforcement.     

Nicotine Effects on Dopamine Clearance in Rat Nucleus Accumbens

Abstract: In vivo voltammetry was used to measure the clearance of exogenously applied dopamine (DA) in the nucleus accumbens following acute systemic nicotine administration in urethane-anesthetized rats. The IVEC-5 system was used for continuous in vivo electrochemical measurements. A finite amount of DA was pressure-ejected (25–100 nl, 200 µ M barrel concentration) at 5-min intervals from micropipettes (tip diameter, 10–15 µm) positioned 250 ± 50 µm from the recording electrode. The peak DA concentration after each DA ejection was significantly decreased in rats following nicotine, but not in rats given saline. In addition, when mecamylamine was administered 20 min before nicotine it clearly antagonized nicotine effects. These results suggest that nicotine may actually facilitate DA transporter systems within the nucleus accumbens.     

Hormonal Regulation of Opioid Peptide Neurons in the Anteroventral Periventricular Nucleus

Steroid hormones provide a means of coordinating the activity of widespread neural systems that mediate endocrine, autonomic, and somatomotor aspects of reproductive processes that are essential for the propagation of mammalian species. Because these processes are quite different in each sex, the neural pathways that control them are also sexually differentiated. The anteroventral periventricular nucleus (AVPV) of the preoptic region occupies a nodal point in sexually dimorphic forebrain circuits and appears to play a critical role in regulating gonadotropin secretion. The AVPV contains sexually dimorphic populations of opioid peptide containing neurons that display different patterns of development and are differentially regulated in adult animals by gonadal steroids. Moreover, estrogen (ER) and progesterone (PR) receptors are expressed in AVPV neurons in a transmitter-specific way, and the expression of these nuclear transacting factors is differentially regulated by sex steroids. Thus, neurons in the AVPV show distinct patterns of hormonal regulation of gene expression, and distinct hormone receptor profiles.     

GABAergic Neurons in the Embryonic Olfactory Pit/Vomeronasal Organ: Maintenance of Functional GABAergic Synapses in Olfactory Explants

In previous work, we showed a robust γ-aminobutyric acid (GABAergic) synaptic input onto embryonic luteinizing hormone-releasing hormone (LHRH) neurons maintained in olfactory explants. In this study, we identify GABAergic neurons in olfactory pit (OP) of embryonic micein vivoand study, using patch-pipet whole-cell current and voltage clamp techniques, synaptic interactions of these neurons in explant cultures.In vivo,glutamate decarboxylase (GAD, the enzyme which synthesizes GABA) mRNA was first detected in nasal regions on Embryonic Day (E) 11.5. From E12.5 to E13.5, robust GAD expression was localized to cells primarily in the ventral aspect of the OP. GAD mRNA was not detected over dorsally located cells in olfactory sensory or respiratory epithelium. In addition, GAD mRNA was not observed in cells along olfactory axons. GAD mRNA was dramatically reduced in the OP/vomeronasal organ by E16.5. Using antibodies against both GABA and GAD, immunopositive axonal-like tracts were detected in the nasal septum on E12.5. GABAergic staining decreased by E13.5. To examine synaptic interactions of these GABAergic cells, embryonic olfactory explants were generated and maintained in serum-free media. As explants spread, neuron-like cells migrated into the periphery, sometimes forming ganglion-like clusters. Cells were recorded, marked intracellularly with Lucifer Yellow and post-fixation, immunocytochemically examined. Forty-six cells, typically multipolar, were GABAergic, had resting potentials around −50 mV, and exhibited spontaneous action potentials which were generated by spontaneous depolarizing GABAergic (GABA_A) synaptic activity. OP neurons depolarized in response to GABA by increasing Cl⁻conductance. The biophysical properties of OP-derived GABAergic neurons were distinct from those reported for olfactory receptor neurons but similar to embryonic LHRH neurons. However, unlike LHRH neurons, GABAergic neurons did not migrate large distances in olfactory explants or appear to leave the olfactory pitin vivo.     

Fractal Stochastic Modeling of Spiking Activity in Suprachiasmatic Nucleus Neurons

Individual neurons in the suprachiasmatic nucleus (SCN), the master biological clock in mammals, autonomously produce highly complex patterns of spikes. We have shown that most (~90%) SCN neurons exhibit truly stochastic interspike interval (ISI) patterns. The aim of this study was to understand the stochastic nature of the firing patterns in SCN neurons by analyzing the ISI sequences of 150 SCN neurons in hypothalamic slices. Fractal analysis, using the periodogram, Fano factor, and Allan factor, revealed the presence of a 1/f-type power-law (fractal) behavior in the ISI sequences. This fractal nature was persistent after the application of the GABA_A receptor antagonist bicuculline, suggesting that the fractal stochastic activity is an intrinsic property of individual SCN neurons. Based on these physiological findings, we developed a computational model for the stochastic SCN neurons to find that their stochastic spiking activity was best described by a gamma point process whose mean firing rate was modulated by a fractal binomial noise. Taken together, we suggest that SCN neurons generate temporal spiking patterns using the fractal stochastic point process.Action Editor: Carson C. Chow     

Reliable Activation of Immature Neurons in the Adult Hippocampus

Neurons born in the adult dentate gyrus develop, mature, and connect over a long interval that can last from six to eight weeks. It has been proposed that, during this period, developing neurons play a relevant role in hippocampal signal processing owing to their distinctive electrical properties. However, it has remained unknown whether immature neurons can be recruited into a network before synaptic and functional maturity have been achieved. To address this question, we used retroviral expression of green fluorescent protein to identify developing granule cells of the adult mouse hippocampus and investigate the balance of afferent excitation, intrinsic excitability, and firing behavior by patch clamp recordings in acute slices. We found that glutamatergic inputs onto young neurons are significantly weaker than those of mature cells, yet stimulation of cortical excitatory axons elicits a similar spiking probability in neurons at either developmental stage. Young neurons are highly efficient in transducing ionic currents into membrane depolarization due to their high input resistance, which decreases substantially in mature neurons as the inward rectifier potassium (Kir) conductance increases. Pharmacological blockade of Kir channels in mature neurons mimics the high excitability characteristic of young neurons. Conversely, Kir overexpression induces mature-like firing properties in young neurons. Therefore, the differences in excitatory drive of young and mature neurons are compensated by changes in membrane excitability that render an equalized firing activity. These observations demonstrate that the adult hippocampus continuously generates a population of highly excitable young neurons capable of information processing.     

Acidosis-Induced Dysfunction of Cortical GABAergic Neurons through Astrocyte-Related Excitotoxicity

Background

Acidosis impairs cognitions and behaviors presumably by acidification-induced changes in neuronal metabolism. Cortical GABAergic neurons are vulnerable to pathological factors and their injury leads to brain dysfunction. How acidosis induces GABAergic neuron injury remains elusive. As the glia cells and neurons interact each other, we intend to examine the role of the astrocytes in acidosis-induced GABAergic neuron injury.

Results

Experiments were done at GABAergic cells and astrocytes in mouse cortical slices. To identify astrocytic involvement in acidosis-induced impairment, we induced the acidification in single GABAergic neuron by infusing proton intracellularly or in both neurons and astrocytes by using proton extracellularly. Compared the effects of intracellular acidification and extracellular acidification on GABAergic neurons, we found that their active intrinsic properties and synaptic outputs appeared more severely impaired in extracellular acidosis than intracellular acidosis. Meanwhile, extracellular acidosis deteriorated glutamate transporter currents on the astrocytes and upregulated excitatory synaptic transmission on the GABAergic neurons. Moreover, the antagonists of glutamate NMDA-/AMPA-receptors partially reverse extracellular acidosis-induced injury in the GABAergic neurons.

Conclusion

Our studies suggest that acidosis leads to the dysfunction of cortical GABAergic neurons by astrocyte-mediated excitotoxicity, in addition to their metabolic changes as indicated previously.     

A Two-Day Continuous Nicotine Infusion Is Sufficient to Demonstrate Nicotine Withdrawal in Rats as Measured Using Intracranial Self-Stimulation

Avoidance of the negative affective (emotional) symptoms of nicotine withdrawal (e.g., anhedonia, anxiety) contributes to tobacco addiction. Establishing the minimal nicotine exposure conditions required to demonstrate negative affective withdrawal signs in animals, as well as understanding moderators of these conditions, could inform tobacco addiction-related research, treatment, and policy. The goal of this study was to determine the minimal duration of continuous nicotine infusion required to demonstrate nicotine withdrawal in rats as measured by elevations in intracranial self-stimulation (ICSS) thresholds (anhedonia-like behavior). Administration of the nicotinic acetylcholine receptor antagonist mecamylamine (3.0 mg/kg, s.c.) on alternate test days throughout the course of a 2-week continuous nicotine infusion (3.2 mg/kg/day via osmotic minipump) elicited elevations in ICSS thresholds beginning on the second day of infusion. Magnitude of antagonist-precipitated withdrawal did not change with further nicotine exposure and mecamylamine injections, and was similar to that observed in a positive control group receiving mecamylamine following a 14-day nicotine infusion. Expression of a significant withdrawal effect was delayed in nicotine-infused rats receiving mecamylamine on all test days rather than on alternate test days. In a separate study, rats exhibited a transient increase in ICSS thresholds following cessation of a 2-day continuous nicotine infusion (3.2 mg/kg/day). Magnitude of this spontaneous withdrawal effect was similar to that observed in rats receiving a 9-day nicotine infusion. Our findings demonstrate that rats exhibit antagonist-precipitated and spontaneous nicotine withdrawal following a 2-day continuous nicotine infusion, at least under the experimental conditions studied here. Magnitude of these effects were similar to those observed in traditional models involving more prolonged nicotine exposure. Further development of these models, including evaluation of more clinically relevant nicotine dosing regimens and other measures of nicotine withdrawal (e.g., anxiety-like behavior, somatic signs), may be useful for understanding the development of the nicotine withdrawal syndrome.     

Electrical Remodeling of Preoptic GABAergic Neurons Involves the Kv1.5 Subunit

The electrogenic machinery of an excitable cell can adapt in response to changes in input, genetic deficit or in pathological conditions, however the underlying molecular mechanisms are not understood. In cases of genetic deletion it is commonly observed that a channel subunit from the same family replaces the missing one. We have previously reported that Kv4.2−/− preoptic GABAergic neurons display identical firing characteristics to those of wild-type neurons despite having reduced A-type currents, and that, surprisingly, they present a robust upregulation of a delayed rectifier current, the nature of which is unknown. Here, using pharmacology, qPCR and Western blots we report that, although the wild-type neurons express several Kv subunits, the upregulated current is conducted by the Kv1.5 subunit exclusively. Thus, this study reveals the molecular nature of a novel mechanism of electrical remodeling in central neurons.