A population of kisspeptin-GABA coexpressing neurons located in the rostral periventricular area of the third ventricle (RP3V) is believed to activate gonadotropin-releasing hormone (GnRH) neurons to generate the luteinizing hormone (LH) surge triggering ovulation. Selective optogenetic activation of RP3V kisspeptin (RP3VKISS) neurons in female mice for >30 s and ≥10 Hz in either a continuous or bursting mode was found to reliably generate a delayed and long-lasting activation of GnRH neuron firing in brain slices. Optogenetic activation of RP3VKISS neurons in vivo at 10 Hz generated substantial increments in LH secretion of similar amplitude to the endogenous LH surge. Studies using GABAA receptor antagonists and optogenetic activation of RP3V GABA (RP3VGABA) neurons in vitro revealed that low-frequency (2 Hz) stimulation generated immediate and transient GABAA receptor-mediated increases in GnRH neuron firing, whereas higher frequencies (10 Hz) recruited the long-lasting activation observed following RP3VKISS neuron stimulation. In vivo, 2 Hz activation of RP3VGABA neurons did not alter LH secretion, whereas 10 Hz stimulation evoked a sustained large increase in LH identical to RP3VKISS neuron activation. Optogenetic activation of RP3VKISS neurons in which kisspeptin had been deleted did not alter LH secretion. These studies demonstrate the presence of parallel transmission streams from RP3V neurons to GnRH neurons that are frequency dependent and temporally distinct. This comprises a rapid and transient GABAA receptor-mediated activation and a slower onset kisspeptin-mediated stimulation of long duration. At the time of the LH surge, GABA release appears to be functionally redundant with the neuropeptide kisspeptin being the dominant cotransmitter influencing GnRH neuron output.SIGNIFICANCE STATEMENT Miscommunication between the brain and ovaries is thought to represent a major cause of infertility in humans. Studies in rodents suggest that a population of neurons located in the rostral periventricular area of the third ventricle (RP3V) are critical for activating the gonadotropin-releasing hormone (GnRH) neurons that trigger ovulation. The present study provides evidence that an RP3V neuron population coexpressing kisspeptin and GABA provides a functionally important excitatory input to GnRH neurons at the time of ovulation. This neural input releases GABA and/or kisspeptin in the classical frequency dependent and temporally distinct nature of amino acid-neuropeptide cotransmission. Unusually, however, the neuropeptide stream is found to be functionally dominant in activating GnRH neurons at the time of ovulation.

Stress-related psychiatric disorders such as anxiety and depression involve dysfunction of the serotonin [5-hydroxytryptamine (5-HT)] system. Previous studies have found that the stress neurohormone corticotropin-releasing factor (CRF) inhibits 5-HT neurons in the dorsal raphe nucleus (DRN) in vivo. The goals of the present study were to characterize the CRF receptor subtypes (CRF-R1 and -R2) and cellular mechanisms underlying CRF-5-HT interactions. Visualized whole-cell patch-clamp recording techniques in brain slices were used to measure spontaneous or evoked GABA synaptic activity in DRN neurons of rats and CRF effects on these measures. CRF-R1 and -R2-selective agonists were bath applied alone or in combination with receptor-selective antagonists. CRF increased presynaptic GABA release selectively onto 5-HT neurons, an effect mediated by the CRF-R1 receptor. CRF increased postsynaptic GABA receptor sensitivity selectively in 5-HT neurons, an effect to which both receptor subtypes contributed. CRF also had direct effects on DRN neurons, eliciting an inward current in 5-HT neurons mediated by the CRF-R2 receptor and in non-5-HT neurons mediated by the CRF-R1 receptor. These results indicate that CRF has direct membrane effects on 5-HT DRN neurons as well as indirect effects on GABAergic synaptic transmission that are mediated by distinct receptor subtypes. The inhibition of 5-HT DRN neurons by CRF in vivo may therefore be primarily an indirect effect via stimulation of inhibitory GABA synaptic transmission. These results regarding the cellular mechanisms underlying the complex interaction between CRF, 5-HT, and GABA systems could contribute to the development of novel treatments for stress-related psychiatric disorders.

We studied how histamine and GABA release from axons originating from the hypothalamic tuberomammillary nucleus (TMN) and projecting to the prefrontal cortex (PFC) influence circuit processing. We optostimulated histamine/GABA from genetically defined TMN axons that express the histidine decarboxylase gene (TMNHDC axons). Whole-cell recordings from PFC neurons in layer 2/3 of prelimbic, anterior cingulate, and infralimbic regions were used to monitor excitability before and after optostimulated histamine/GABA release in male and female mice. We found that histamine-GABA release influences the PFC through actions on distinct neuronal types: the histamine stimulates fast-spiking interneurons; and the released GABA enhances tonic (extrasynaptic) inhibition on pyramidal cells (PyrNs). For fast-spiking nonaccommodating interneurons, histamine released from TMNHDC axons induced additive gain changes, which were blocked by histamine H1 and H2 receptor antagonists. The excitability of other fast-spiking interneurons in the PFC was not altered. In contrast, the GABA released from TMNHDC axons predominantly produced divisive gain changes in PyrNs, increasing their resting input conductance, and decreasing the slope of the input-output relationship. This inhibitory effect on PyrNs was not blocked by histamine receptor antagonists but was blocked by GABAA receptor antagonists. Across the adult life span (from 3 to 18 months of age), the GABA released from TMNHDC axons in the PFC inhibited PyrN excitability significantly more in older mice. For individuals who maintain cognitive performance into later life, the increases in TMNHDC GABA modulation of PyrNs during aging could enhance information processing and be an adaptive mechanism to buttress cognition.SIGNIFICANCE STATEMENT The hypothalamus controls arousal state by releasing chemical neurotransmitters throughout the brain to modulate neuronal excitability. Evidence is emerging that the release of multiple types of neurotransmitters may have opposing actions on neuronal populations in key cortical regions. This study demonstrates for the first time that the neurotransmitters histamine and GABA are released in the prefrontal cortex from axons originating from the tuberomammillary nucleus of the hypothalamus. This work demonstrates how hypothalamic modulation of neuronal excitability is maintained throughout adult life, highlighting an unexpected aspect of the aging process that may help maintain cognitive abilities.

Nigrostriatal dopamine (DA) is critical to action selection and learning. Axonal DA release is locally influenced by striatal neurotransmitters. Striatal neurons are principally GABAergic projection neurons and interneurons, and a small minority of other neurons are cholinergic interneurons (ChIs). ChIs strongly gate striatal DA release via nicotinic receptors (nAChRs) identified on DA axons. Striatal GABA is thought to modulate DA, but GABA receptors have not been documented conclusively on DA axons. However, ChIs express GABA receptors and are therefore candidates for potential mediators of GABA regulation of DA. We addressed whether striatal GABA and its receptors can modulate DA release directly, independently from ChI regulation, by detecting DA in striatal slices from male mice using fast-scan cyclic voltammetry in the absence of nAChR activation. DA release evoked by single electrical pulses in the presence of the nAChR antagonist dihydro-β-erythroidine was reduced by GABA or agonists of GABAA or GABAB receptors, with effects prevented by selective GABA receptor antagonists. GABA agonists slightly modified the frequency sensitivity of DA release during short stimulus trains. GABA agonists also suppressed DA release evoked by optogenetic stimulation of DA axons. Furthermore, antagonists of GABAA and GABAB receptors together, or GABAB receptors alone, significantly enhanced DA release evoked by either optogenetic or electrical stimuli. These results indicate that striatal GABA can inhibit DA release through GABAA and GABAB receptors and that these actions are not mediated by cholinergic circuits. Furthermore, these data reveal that there is a tonic inhibition of DA release by striatal GABA operating through predominantly GABAB receptors.SIGNIFICANCE STATEMENT The principal inhibitory transmitter in the mammalian striatum, GABA, is thought to modulate striatal dopamine (DA) release, but definitive evidence for GABA receptors on DA axons is lacking. Striatal cholinergic interneurons regulate DA release via axonal nicotinic receptors (nAChRs) and also express GABA receptors, but they have not been eliminated as potentially critical mediators of DA regulation by GABA. Here, we found that GABAA and GABAB receptors inhibit DA release without requiring cholinergic interneurons. Furthermore, ambient levels of GABA inhibited DA release predominantly through GABAB receptors. These findings provide further support for direct inhibition of DA release by GABA receptors and reveal that striatal GABA operates a tonic inhibition on DA output that could critically influence striatal output.

We previously found that low-frequency stimulation of direct temperoammonic (TA) inputs to hippocampal area CA1 depotentiates previously established long-term potentiation in the Schaffer collateral (SC) pathway through complex signaling involving dopamine, endocannabinoids, neuregulin-1, GABA, and adenosine, with adenosine being the most distal modulator identified to date. In the present studies, we examined mechanisms contributing to the effects of adenosine in hippocampal slices from male albino rats. We found that extracellular conversion of ATP to adenosine via an ectonucleotidase contributes significantly to TA-mediated SC depotentiation and the depotentiation resulting from block of adenosine transport. Adenosine-mediated SC depotentiation does not involve activation of c-Jun N-terminal protein kinase, serine phosphatases, or nitric oxide synthase, unlike homosynaptic SC depotentiation. Rather, adenosine-induced depotentiation is inhibited by specific antagonists of p38 MAPK, but not by a structural analog that does not inhibit p38. Additionally, using antagonists with relative selectivity for p38 subtypes, it appears that TA-induced SC depotentiation most likely involves p38 MAPK β. These findings have implications for understanding the role of adenosine and other extrahippocampal and intrahippocampal modulators in regulating SC synaptic function and the contributions of these modulators to the cognitive dysfunction associated with neuropsychiatric illnesses.SIGNIFICANCE STATEMENT Low-frequency stimulation of temperoammonic (TA) inputs to stratum lacunosum moleculare of hippocampal area CA1 heterosynaptically depotentiates long-term potentiation of Schaffer collateral (SC) synapses. TA-induced SC depotentiation involves complex signaling including dopamine, endocannabinoids, GABA, and adenosine, with adenosine serving as the most downstream messenger in the cascade identified to date. The present results indicate that TA-induced depotentiation requires intact inputs from entorhinal cortex and that adenosine ultimately drives depotentiation via activation of p38 MAPK. These studies have implications for understanding the cognitive dysfunction of psychiatric illnesses and certain abused drugs.

GABAergic neurons in the ventral tegmental area (VTA) play a primary role in local inhibition of mesocorticolimbic dopamine (DA) neurons but are not physiologically or anatomically well characterized. We used in vivo extracellular and intracellular recordings in the rat VTA to identify a homogeneous population of neurons that were distinguished from DA neurons by their rapid-firing, nonbursting activity (19.1 +/- 1.4 Hz), short-duration action potentials (310 +/- 10 microseconds), EPSP-dependent spontaneous spikes, and lack of spike accommodation to depolarizing current pulses. These non-DA neurons were activated both antidromically and orthodromically by stimulation of the internal capsule (IC; conduction velocity, 2.4 +/- 0.2 m/sec; refractory period, 0.6 +/- 0.1 msec) and were inhibited by stimulation of the nucleus accumbens septi (NAcc). Their firing rate was moderately reduced, and their IC-driven activity was suppressed by microelectrophoretic application or systemic administration of NMDA receptor antagonists. VTA non-DA neurons were recorded intracellularly and showed relatively depolarized resting membrane potentials (-61.9 +/- 1.8 mV) and small action potentials (68.3 +/- 2.1 mV). They were injected with neurobiotin and shown by light microscopic immunocytochemistry to be multipolar cells and by electron microscopy to contain GABA but not the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH). Neurobiotin-filled dendrites containing GABA received asymmetric excitatory-type synapses from unlabeled terminals and symmetric synapses from terminals that also contained GABA. These findings indicate that VTA non-DA neurons are GABAergic, project to the cortex, and are controlled, in part, by a physiologically relevant NMDA receptor-mediated input from cortical structures and by GABAergic inhibition.

The substantia nigra pars reticulata (SNr) plays a key role in basal ganglia function. Projections from multiple basal ganglia nuclei converge at the SNr to regulate nigrothalamic output. The SNr is also characterized by abundant aminergic input, including dopaminergic dendrites and axons containing 5-hydroxytryptamine (5-HT) or histamine (HA). The functions of HA in the SNr include motor control via HA H3 receptors (H3Rs), although the mechanism remains far from elucidated. In Parkinson's disease, there is an increase in H3Rs and the density of HA-immunoreactive axons in the SN. We explored the role of H3Rs in the regulation of 5-HT release in SNr using fast-scan cyclic voltammetry at carbon-fiber microelectrodes in rat midbrain slices. Immunohistochemistry identified a similar distribution for histaminergic and serotonergic processes in the SNr: immunoreactive varicosities were observed in the vicinity of dopaminergic dendrites. Electrically evoked 5-HT release was dependent on extracellular Ca2+ and prevented by NaV+-channel blockade. Extracellular 5-HT concentration was enhanced by inhibition of uptake transporters for 5-HT but not dopamine. Selective H3R agonists (R)-(-)-alpha-methyl-histamine or immepip inhibited evoked 5-HT release by up to 60%. This inhibition was prevented by the H3R antagonist thioperamide but not by the 5-HT1B receptor antagonist isamoltane. H3R inhibition of 5-HT release prevailed in the presence of GABA or glutamate receptor antagonists (ionotropic and metabotropic), suggesting minimal involvement of GABA or glutamate synapses. The potent regulation of 5-HT by H3Rs reported here not only elucidates HA function in the SNr but also raises the possibility of novel targets for basal ganglia therapies.

The serotonin (5-HT) system and the amygdala are key regulators of emotional behavior. Several lines of evidence suggest that 5-HT transmission in the amygdala is implicated in the susceptibility and drug treatment of mood disorders. Therefore, elucidating the physiological mechanisms through which midbrain 5-HT neurons modulate amygdala circuits could be pivotal in understanding emotional regulation in health and disease. To shed light on these mechanisms, we performed patch-clamp recordings from basal amygdala (BA) neurons in brain slices from mice with channelrhodopsin genetically targeted to 5-HT neurons. Optical stimulation of 5-HT terminals at low frequencies (≤1 Hz) evoked a short-latency excitation of BA interneurons (INs) that was depressed at higher frequencies. Pharmacological analysis revealed that this effect was mediated by glutamate and not 5-HT because it was abolished by ionotropic glutamate receptor antagonists. Optical stimulation of 5-HT terminals at higher frequencies (10-20 Hz) evoked both slow excitation and slow inhibition of INs. These effects were mediated by 5-HT because they were blocked by antagonists of 5-HT2A and 5-HT1A receptors, respectively. These fast glutamate- and slow 5-HT-mediated responses often coexisted in the same neuron. Interestingly, fast-spiking and non-fast-spiking INs displayed differential modulation by glutamate and 5-HT. Furthermore, optical stimulation of 5-HT terminals did not evoke glutamate release onto BA principal neurons, but inhibited these cells directly via activation of 5-HT1A receptors and indirectly via enhanced GABA release. Collectively, these findings suggest that 5-HT neurons exert a frequency-dependent, cell-type-specific control over BA circuitry via 5-HT and glutamate co-release to inhibit the BA output.SIGNIFICANCE STATEMENT The modulation of the amygdala by serotonin (5-HT) is important for emotional regulation and is implicated in the pathogenesis and treatment of affective disorders. Therefore, it is essential to determine the physiological mechanisms through which 5-HT neurons in the dorsal raphe nuclei modulate amygdala circuits. Here, we combined optogenetic, electrophysiological, and pharmacological approaches to study the effects of activation of 5-HT axons in the basal nucleus of the amygdala (BA). We found that 5-HT neurons co-release 5-HT and glutamate onto BA neurons in a cell-type-specific and frequency-dependent manner. Therefore, we suggest that theories on the contribution of 5-HT neurons to amygdala function should be revised to incorporate the concept of 5-HT/glutamate cotransmission.

Three neuronal pentraxins are expressed in brain, the membrane-bound "neuronal pentraxin receptor" (NPR) and the secreted proteins NP1 and NARP (i.e., NP2). Neuronal pentraxins bind to AMPARs at excitatory synapses and play important, well-documented roles in the activity-dependent regulation of neural circuits via this binding activity. However, it is unknown whether neuronal pentraxins perform roles in synapses beyond modulating postsynaptic AMPAR-dependent plasticity, and whether they may even act in inhibitory synapses. Here, we show that NPR expressed in non-neuronal cells potently induces formation of both excitatory and inhibitory postsynaptic specializations in cocultured hippocampal neurons. Knockdown of NPR in hippocampal neurons, conversely, dramatically decreased assembly and function of both excitatory and inhibitory postsynaptic specializations. Overexpression of NPR rescued the NPR knockdown phenotype but did not in itself change synapse numbers or properties. However, the NPR knockdown decreased the levels of NARP, whereas NPR overexpression produced a dramatic increase in the levels of NP1 and NARP, suggesting that NPR recruits and stabilizes NP1 and NARP on the presynaptic plasma membrane. Mechanistically, NPR acted in excitatory synapse assembly by binding to the N-terminal domain of AMPARs; antagonists of AMPA and GABA receptors selectively inhibited NPR-induced heterologous excitatory and inhibitory synapse assembly, respectively, but did not affect neurexin-1β-induced synapse assembly as a control. Our data suggest that neuronal pentraxins act as signaling complexes that function as general trans-synaptic organizers of both excitatory and inhibitory synapses by a mechanism that depends, at least in part, on the activity of the neurotransmitter receptors at these synapses.

Noradrenaline (NA) from the locus coeruleus and GABA from intracortical nonpyramidal cells exert strong influences on cortical activity. To assess possible interaction between the two, the effects of noradrenergic agonists on spontaneous GABAergic IPSCs as well as on the activity of identified GABAergic cell types were investigated by in vitro whole-cell recordings from the frontal cortex of 18- to 22-d-old rats. NA (3-50 microM) and an alpha-adrenergic agonist, 6-fluoronorepinephrine (FNE; 30-50 microM), induced an increase of IPSC frequency in pyramidal cells, but a beta-adrenergic agonist did not. This increase was reduced by tetrodotoxin, bicuculline, and alpha-adrenergic antagonists, suggesting that GABAergic cells are excited via alpha-adrenoceptors. Fast-spiking or late-spiking cells were depolarized by application of NA or FNE, but none demonstrated spike firings. The former morphologically included common multipolar cells with extended axonal arborizations as well as chandelier cells, and the latter neurogliaform cells. Most somatostatin-immunoreactive regular or burst-spiking cells, including Martinotti cells and wide arbor cells, were depolarized and accompanied by spike firing. In a few cases this was preceded by hyperpolarization. Cholecystokinin-immunoreactive regular or burst-spiking nonpyramidal cells, including large basket cells, were affected heterogeneously: depolarization, hyperpolarization followed by depolarization, or hyperpolarization resulted. The findings suggest that, similar to the effects of acetylcholine, the excitability of cortical GABAergic cell types is differentially regulated by NA and that NA actions are similar to cholinergic ones in some GABAergic cell types but not in others.

Previously, studies using human neuroimaging and excitotoxic lesions in non-human primate have demonstrated an important role of ventrolateral prefrontal cortex (vlPFC) in higher order cognitive functions such as cognitive flexibility and the planning of behavioral sequences. In the present experiments, we tested effects on performance of temporary inactivation (using GABA receptor agonists) and dopamine (DA) D2 and 5-HT2A-receptor (R) blockade of vlPFC via local intracerebral infusions in the marmoset. We trained common marmosets to perform spatial self-ordered sequencing tasks in which one cohort of animals performed two and three response sequences on a continuously varying spatial array of response options on a touch-sensitive screen. Inactivation of vlPFC produced a marked disruption of accuracy of sequencing which also exhibited significant error perseveration. There were somewhat contrasting effects of D2 and 5-HT2A-R blockade, with the former producing error perseveration on incorrect trials, though not significantly impairing accuracy overall, and the latter significantly impairing accuracy but not error perseveration. A second cohort of marmosets were directly compared on performance of fixed versus variable spatial arrays. Inactivation of vlPFC again impaired self-ordered sequencing, but only with varying, and not fixed spatial arrays, the latter leading to the consistent use of fewer, preferred sequences. These findings add to evidence that vlPFC is implicated in goal-directed behavior that requires higher-order response heuristics that can be applied flexibly over different (variable), as compared with fixed stimulus exemplars. They also show that dopaminergic and serotonergic chemomodulation has distinctive effects on such performance.SIGNIFICANCE STATEMENT This investigation employing local intracerebral infusions to inactivate the lateral prefrontal cortex (PFC) of the New World marmoset reveals the important role of this region in self-ordered response sequencing in variable but not fixed spatial arrays. These novel findings emphasize the higher order functions of this region, contributing to cognitive flexibility and planning of goal directed behavior. The investigation also reports for the first time somewhat contrasting neuromodulatory deficits produced by infusions of dopamine (DA) D2 and 5-HT2A receptor (R) antagonists into the same region, of possible significance for understanding cognitive deficits produced by anti-psychotic drugs.

The morphological effects of several neuroleptics as well as other novel and prototypic sigma ligands were examined by addition to cultures of C6 glioma cells. Sigma ligands caused loss of processes, assumption of spherical shape, and cessation of cell division. The time course and magnitude of this effect were dependent on the concentration of sigma ligand. Continued exposure to sigma compounds ultimately resulted in cell death. However, the morphological effect was reversible when sigma ligand was removed shortly after rounding. The potency of compounds to produce these effects generally correlated with binding affinity at sigma receptors of C6 glioma cell membranes labeled with [3H](+)-pentazocine. At a concentration of 100 microM, haloperidol, reduced haloperidol, fluphenazine, perphenazine, trifluoperazine, BD737, LR172, BD1008, and SH344 produced significant effects in 3-6 hr of exposure. Other compounds, such as trifluperidol, thioridazine, and (-)-butaclamol, produced significant effects by 24 hr of exposure. Despite the requirement of micromolar concentrations of ligand (some compounds were effective at 30 microM), the effect showed a remarkable specificity for compounds exhibiting sigma receptor binding affinity. Neuroleptics lacking potent sigma affinity [e.g., (-)-sulpiride, (+)-butaclamol, and clozapine] and other compounds that lack significant sigma affinity but that are agonists or antagonists at dopamine, serotonin, adrenergic, glutamate, phencyclidine, GABA, opiate, or muscarinic cholinergic receptors were without effect on cellular morphology at concentrations up to 300 microM over a period of 72 hr. Likewise, blockers and activators of Na+, K+, and Ca2+ channels and a monoamine oxidase inhibitor devoid of sigma affinity were without effect. Interestingly, 1,3-di-o-tolylguanidine (DTG), (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine [(+)-3-PPP], (+)-pentazocine, (+)-cyclazocine, and other sigma-active benzomorphans and morphinans appeared inactive in up to 72 hr of culture. However, these compounds interacted synergistically with a subeffective dose of BD737 (30 microM) to produce effects usually in 6 hr or less. Also, the pH of the culture medium had a profound effect on the activity of sigma compounds. Increasing the pH from the normal range of 7.2-7.4 to pH 8.3-8.5 shifted the dose curves (30, 100, 300 microM) for all sigma compounds to the left. Under these conditions, DTG, (+)-3-PPP, and benzomorphans produced effects in 24 hr or less.(ABSTRACT TRUNCATED AT 400 WORDS)