Neurons In Brain Slices Research Articles

Amplitude Normalization of Dendritic EPSPs at the Soma of Binaural Coincidence Detector Neurons of the Medial Superior Olive.

The principal neurons of the medial superior olive (MSO) encode cues for horizontal sound localization through comparisons of the relative timing of EPSPs. To understand how the timing and amplitude of EPSPs are maintained during propagation in the dendrites, we made dendritic and somatic whole-cell recordings from MSO principal neurons in brain slices from Mongolian gerbils. In somatic recordings, EPSP amplitudes were largely uniform following minimal stimulation of excitatory synapses at visualized locations along the dendrites. Similar results were obtained when excitatory synaptic transmission was eliminated in a low calcium solution and then restored at specific dendritic sites by pairing input stimulation and focal application of a higher calcium solution. We performed dual dendritic and somatic whole-cell recordings to measure spontaneous EPSPs using a dual-channel template-matching algorithm to separate out those events initiated at or distal to the dendritic recording location. Local dendritic spontaneous EPSP amplitudes increased sharply in the dendrite with distance from the soma (length constant, 53.6 μm), but their attenuation during propagation resulted in a uniform amplitude of ∼0.2 mV at the soma. The amplitude gradient of dendritic EPSPs was also apparent in responses to injections of identical simulated excitatory synaptic currents in the dendrites. Compartmental models support the view that these results extensively reflect the influence of dendritic cable properties. With relatively few excitatory axons innervating MSO neurons, the normalization of dendritic EPSPs at the soma would increase the importance of input timing versus location during the processing of interaural time difference cues in vivoSIGNIFICANCE STATEMENT The neurons of the medial superior olive analyze cues for sound localization by detecting the coincidence of binaural excitatory synaptic inputs distributed along the dendrites. Previous studies have shown that dendritic voltages undergo severe attenuation as they propagate to the soma, potentially reducing the influence of distal inputs. However, using dendritic and somatic patch recordings, we found that dendritic EPSP amplitude increased with distance from the soma, compensating for dendritic attenuation and normalizing EPSP amplitude at the soma. Much of this normalization reflected the influence of dendritic morphology. As different combinations of presynaptic axons may be active during consecutive cycles of sound stimuli, somatic EPSP normalization renders spike initiation more sensitive to synapse timing than dendritic location.

Neuronal hyperexcitability in a mouse model of SCN8A epileptic encephalopathy

Patients with early infantile epileptic encephalopathy (EIEE) experience severe seizures and cognitive impairment and are at increased risk for sudden unexpected death in epilepsy (SUDEP). EIEE13 [Online Mendelian Inheritance in Man (OMIM) # 614558] is caused by de novo missense mutations in the voltage-gated sodium channel gene SCN8A Here, we investigated the neuronal phenotype of a mouse model expressing the gain-of-function SCN8A patient mutation, p.Asn1768Asp (Nav1.6-N1768D). Our results revealed regional and neuronal subtype specificity in the effects of the N1768D mutation. Acutely dissociated hippocampal neurons from Scn8aN1768D/+ mice showed increases in persistent sodium current (INa) density in CA1 pyramidal but not bipolar neurons. In CA3, INa,P was increased in both bipolar and pyramidal neurons. Measurement of action potential (AP) firing in Scn8aN1768D/+ pyramidal neurons in brain slices revealed early afterdepolarization (EAD)-like AP waveforms in CA1 but not in CA3 hippocampal or layer II/III neocortical neurons. The maximum spike frequency evoked by depolarizing current injections in Scn8aN1768D/+ CA1, but not CA3 or neocortical, pyramidal cells was significantly reduced compared with WT. Spontaneous firing was observed in subsets of neurons in CA1 and CA3, but not in the neocortex. The EAD-like waveforms of Scn8aN1768D/+ CA1 hippocampal neurons were blocked by tetrodotoxin, riluzole, and SN-6, implicating elevated persistent INa and reverse mode Na/Ca exchange in the mechanism of hyperexcitability. Our results demonstrate that Scn8a plays a vital role in neuronal excitability and provide insight into the mechanism and future treatment of epileptogenesis in EIEE13.

Control of Amygdala Circuits by 5-HT Neurons via 5-HT and Glutamate Cotransmission.

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.

Transcriptome Analysis Identifies Key Metabolic Changes in the Hooded Seal (Cystophora cristata) Brain in Response to Hypoxia and Reoxygenation

The brain of diving mammals tolerates low oxygen conditions better than the brain of most terrestrial mammals. Previously, it has been demonstrated that the neurons in brain slices of the hooded seal (Cystophora cristata) withstand hypoxia longer than those of mouse, and also tolerate reduced glucose supply and high lactate concentrations. This tolerance appears to be accompanied by a shift in the oxidative energy metabolism to the astrocytes in the seal while in terrestrial mammals the aerobic energy production mainly takes place in neurons. Here, we used RNA-Seq to compare the effect of hypoxia and reoxygenation in vitro on brain slices from the visual cortex of hooded seals. We saw no general reduction of gene expression, suggesting that the response to hypoxia and reoxygenation is an actively regulated process. The treatments caused the preferential upregulation of genes related to inflammation, as found before e.g. in stroke studies using mammalian models. Gene ontology and KEGG pathway analyses showed a downregulation of genes involved in ion transport and other neuronal processes, indicative for a neuronal shutdown in response to a shortage of O2 supply. These differences may be interpreted in terms of an energy saving strategy in the seal's brain. We specifically analyzed the regulation of genes involved in energy metabolism. Hypoxia and reoxygenation caused a similar response, with upregulation of genes involved in glucose metabolism and downregulation of the components of the pyruvate dehydrogenase complex. We also observed upregulation of the monocarboxylate transporter Mct4, suggesting increased lactate efflux. Together, these data indicate that the seal brain responds to the hypoxic challenge by a relative increase in the anaerobic energy metabolism.

Implantable microcoils for intracortical magnetic stimulation.

Neural prostheses that stimulate the neocortex have the potential to treat a wide range of neurological disorders. However, the efficacy of electrode-based implants remains limited, with persistent challenges that include an inability to create precise patterns of neural activity as well as difficulties in maintaining response consistency over time. These problems arise from fundamental limitations of electrodes as well as their susceptibility to implantation and have proven difficult to overcome. Magnetic stimulation can address many of these limitations, but coils small enough to be implanted into the cortex were not thought strong enough to activate neurons. We describe a new microcoil design and demonstrate its effectiveness for both activating cortical neurons and driving behavioral responses. The stimulation of cortical pyramidal neurons in brain slices in vitro was reliable and could be confined to spatially narrow regions (<60 μm). The spatially asymmetric fields arising from the coil helped to avoid the simultaneous activation of passing axons. In vivo implantation was safe and resulted in consistent and predictable behavioral responses. The high permeability of magnetic fields to biological substances may yield another important advantage because it suggests that encapsulation and other adverse effects of implantation will not diminish coil performance over time, as happens to electrodes. These findings suggest that a coil-based implant might be a useful alternative to existing electrode-based devices. The enhanced selectivity of microcoil-based magnetic stimulation will be especially useful for visual prostheses as well as for many brain-computer interface applications that require precise activation of the cortex.

Visualisation of Kiss1 Neurone Distribution Using a Kiss1‐CRE Transgenic Mouse

Kisspeptin neuropeptides are encoded by the Kiss1 gene and play a critical role in the regulation of the mammalian reproductive axis. Kiss1 neurones are found in two locations in the rodent hypothalamus: one in the arcuate nucleus (ARC) and another in the RP3V region, which includes the anteroventral periventricular nucleus (AVPV). Detailed mapping of the fibre distribution of Kiss1 neurones will help with our understanding of the action of these neurones in other regions of the brain. We have generated a transgenic mouse in which the Kiss1 coding region is disrupted by a CRE‐GFP transgene so that expression of the CRE recombinase protein is driven from the Kiss1 promoter. As expected, mutant mice of both sexes are sterile with hypogonadotrophic hypogonadism and do not show the normal rise in luteinising hormone after gonadectomy. Mutant female mice do not develop mature Graafian follicles or form corpora lutea consistent with ovulatory failure. Mutant male mice have low blood testosterone levels and impaired spermatogenesis beyond the meiosis stage. Breeding Kiss‐CRE heterozygous mice with CRE‐activated tdTomato reporter mice allows fluorescence visualisation of Kiss1 neurones in brain slices. Approximately 80‐90% of tdTomato positive neurones in the ARC were co‐labelled with kisspeptin and expression of tdTomato in the AVPV region was sexually dimorphic, with higher expression in females than males. A small number of tdTomato‐labelled neurones was also found in other locations, including the lateral septum, the anterodorsal preoptic nucleus, the amygdala, the dorsomedial and ventromedial hypothalamic nuclei, the periaquaductal grey, and the mammillary nucleus. Three dimensional visualisation of Kiss1 neurones and fibres by CLARITY processing of whole brains showed an increase in ARC expression during puberty and higher numbers of Kiss1 neurones in the caudal region of the ARC compared to the rostral region. ARC Kiss1 neurones sent fibre projections to several hypothalamic regions, including rostrally to the periventricular and pre‐optic areas and to the lateral hypothalamus.

Estrogens modulate ventrolateral ventromedial hypothalamic glucose-inhibited neurons

ObjectiveBrain regulation of glucose homeostasis is sexually dimorphic; however, the impact sex hormones have on specific neuronal populations within the ventromedial hypothalamic nucleus (VMN), a metabolically sensitive brain region, has yet to be fully characterized. Glucose-excited (GE) and -inhibited (GI) neurons are located throughout the VMN and may play a critical role in glucose and energy homeostasis. Within the ventrolateral portion of the VMN (VL-VMN), glucose sensing neurons and estrogen receptor (ER) distributions overlap. We therefore tested the hypothesis that VL-VMN glucose sensing neurons were sexually dimorphic and regulated by 17β-estradiol (17βE). MethodsElectrophysiological recordings of VL-VMN glucose sensing neurons in brain slices isolated from age- and weight-matched female and male mice were performed in the presence and absence of 17βE. ResultsWe found a new class of VL-VMN GI neurons whose response to low glucose was transient despite continued exposure to low glucose. Heretofore, we refer to these newly identified VL-VMN GI neurons as ‘adapting’ or AdGI neurons. We found a sexual dimorphic response to low glucose, with male nonadapting GI neurons, but not AdGI neurons, responding more robustly to low glucose than those from females. 17βE blunted the response of both nonadapting GI and AdGI neurons to low glucose in both males and females, which was mediated by activation of estrogen receptor β and inhibition of AMP-activated kinase. In contrast, 17βE had no impact on GE or non-glucose sensing neurons in either sex. ConclusionThese data suggest sex differences and estrogenic regulation of VMN hypothalamic glucose sensing may contribute to the sexual dimorphism in glucose homeostasis.

Firing Properties of Genetically Identified Dorsal Raphe Serotonergic Neurons in Brain Slices.

Tonic spiking of serotonergic neurons establishes serotonin levels in the brain. Since the first observations, slow regular spiking has been considered as a defining feature of serotonergic neurons. Recent studies, however, have revealed the heterogeneity of serotonergic neurons at multiple levels, comprising their electrophysiological properties, suggesting the existence of functionally distinct cellular subpopulations. In order to examine in an unbiased manner whether serotonergic neurons of the dorsal raphe nucleus (DRN) are heterogeneous, we used a non-invasive loose-seal cell-attached method to record α1 adrenergic receptor-stimulated spiking of a large sample of neurons in brain slices obtained from transgenic mice lines that express fluorescent marker proteins under the control of serotonergic system-specific Tph2 and Pet-1 promoters. We found wide homogeneous distribution of firing rates, well fitted by a single Gaussian function (r2 = 0.93) and independent of anatomical location (P = 0.45), suggesting that in terms of intrinsic firing properties, serotonergic neurons in the DRN represent a single cellular population. Characterization of the population in terms of spiking regularity was hindered by its dependence on the firing rate. For instance, the coefficient of variation of the interspike intervals (ISI), a common measure of spiking irregularity, is of limited usefulness since it correlates negatively with the firing rate (r = −0.33, P < 0.0001). Nevertheless, the majority of neurons exhibited regular, pacemaker-like activity, with coefficient of variance of the ISI lower than 0.5 in ~97% of cases. Unexpectedly, a small percentage of neurons (~1%) exhibited a particular spiking pattern, characterized by low frequency (~0.02–0.1 Hz) oscillations in the firing rate. Transitions between regular and oscillatory firing were observed, suggesting that the oscillatory firing is an alternative firing pattern of serotonergic neurons.

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices, a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular, when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

Whole-cell Patch-clamp Recordings in Brain Slices.

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of theelectrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

Excitability and Burst Generation of AVPV Kisspeptin Neurons Are Regulated by the Estrous Cycle Via Multiple Conductances Modulated by Estradiol Action.

The preovulatory secretory surge of gonadotropin-releasing hormone (GnRH) is crucial for fertility and is regulated by a switch of estradiol feedback action from negative to positive. GnRH neurons likely receive estradiol feedback signals via ERα-expressing afferents. Kisspeptin neurons in anteroventral periventricular nucleus (AVPV) are thought to be critical for estradiol-positive feedback induction of the GnRH surge. We examined the electrophysiological properties of GFP-identified AVPV kisspeptin neurons in brain slices from mice on the afternoon of diestrus (negative feedback) and proestrus (positive feedback, time of surge). Extracellular recordings revealed increased firing frequency and action potential bursts on proestrus versus diestrus. Whole-cell recordings were used to study the intrinsic mechanisms of bursting. Upon depolarization, AVPV kisspeptin neurons exhibited tonic firing or depolarization-induced bursts (DIB). Both tonic and DIB cells exhibited bursts induced by rebound from hyperpolarization. DIB occurred similarly on both cycle stages, but rebound bursts were observed more often on proestrus. DIB and rebound bursts were both sensitive to Ni2+, suggesting that T-type Ca2+ currents (ITs) are involved. IT current density was greater on proestrus versus diestrus. In addition to IT, persistent sodium current (INaP) facilitated rebound bursting. On diestrus, 4-aminopyridine-sensitive potassium currents contributed to reduced rebound bursts in both tonic and DIB cells. Manipulation of specific sex steroids suggests that estradiol induces the changes that enhance AVPV kisspeptin neuron excitability on proestrus. These observations indicate cycle-driven changes in circulating estradiol increased overall action potential generation and burst firing in AVPV kisspeptin neurons on proestrus versus diestrus by regulating multiple intrinsic currents.

Somatostatin triggers rhythmic electrical firing in hypothalamic GHRH neurons.

Hypothalamic growth hormone-releasing hormone (GHRH) neurons orchestrate body growth/maturation and have been implicated in feeding responses and ageing. However, the electrical patterns that dictate GHRH neuron functions have remained elusive. Since the inhibitory neuropeptide somatostatin (SST) is considered to be a primary oscillator of the GH axis, we examined its acute effects on GHRH neurons in brain slices from male and female GHRH-GFP mice. At the cellular level, SST irregularly suppressed GHRH neuron electrical activity, leading to slow oscillations at the population level. This resulted from an initial inhibitory action at the GHRH neuron level via K+ channel activation, followed by a delayed, sst1/sst2 receptor-dependent unbalancing of glutamatergic and GABAergic synaptic inputs. The oscillation patterns induced by SST were sexually dimorphic, and could be explained by differential actions of SST on both GABAergic and glutamatergic currents. Thus, a tripartite neuronal circuit involving a fast hyperpolarization and a dual regulation of synaptic inputs appeared sufficient in pacing the activity of the GHRH neuronal population. These “feed-forward loops” may represent basic building blocks involved in the regulation of GHRH release and its downstream sexual specific functions.

GABAB receptor plasticity in corticotrophin releasing hormone neurons in the paraventricular nucleus during chronic unpredictable stress

GABAB (γ‐aminobutytic acid type B) receptor is important in the regulation of neuronal excitability. Chronic stress leads to hyperactivity of corticotrophin releasing hormone (CRH) neurons. However, the role of GABAB receptors in the regulation of CRH neurons in paraventricular nucleus (PVN) of the hypothalamus in chronic stress is not known. Here, we determined pre‐ and postsynaptic GABAB receptor function in identified PVN‐CRH neurons expressing eGFP driven by crh premotor‐driven eGFP. Whole cell patch‐clamp recording were performed on PVN‐CRH neurons in brain slices from unstressed rats and rats with chronic unpredictable mild stress (CUMS). Bath application of the GABAB receptor agonist baclofen induced a significant lesser reduction of firing activity in PVN‐CRH neurons in CUMS than in unstressed rats. Furthermore, baclofen induced lesser outward currents in PVN‐CRH neurons in CUMS than in unstressed rats. Baclofen‐induced currents were eliminated by inclusion of GDP‐β‐s in internal recording solution or blocking G protein‐coupled inwardly‐rectifying K (GIRK) channels with tertiapin. GABAB1 receptor expression level in the PVN was significantly decreased in CUMS than in unstressed rats. Baclofen produced significant greater inhibition of the frequency of glutamatergic excitatory postsynaptic currents (EPSCs) and GABAergic inhibitor postsynaptic currents (IPSCs) without affecting the amplitude of EPSCs and IPSCs in PVN‐CRH neurons in CUMS and unstressed rats. These data suggest that down‐regulated postsynaptic GABAB receptor contributes to suppressing GABAB‐mediated inhibition of PVN‐CRH neurons in chronic stress.Support or Funding InformationNIMH grants MH096086.

Unitary synaptic connections among substantia nigra pars reticulata neurons

Neurons in substantia nigra pars reticulata (SNr) are synaptically coupled by local axon collaterals, providing a potential mechanism for local signal processing. Because SNr neurons fire spontaneously, these synapses are constantly active. To investigate their properties, we recorded spontaneous inhibitory postsynaptic currents (sIPSCs) from SNr neurons in brain slices, in which afferents from upstream nuclei are severed, and the cells fire rhythmically. The sIPSC trains contained a mixture of periodic and aperiodic events. Autocorrelation analysis of sIPSC trains showed that a majority of cells had one to four active unitary inputs. The properties of the unitary IPSCs (uIPSCs) were analyzed for cells with one unitary input, using a model of periodic presynaptic firing and stochastic synaptic transmission. The inferred presynaptic firing rates and coefficient of variation of interspike intervals (ISIs) corresponded well with direct measurements of spiking in SNr neurons. Methods were developed to estimate the success probability, amplitude distributions, and kinetics of the uIPSCs, while removing the contribution from aperiodic sIPSCs. The sIPSC amplitudes were not increased upon release from halorhodopsin silencing, suggesting that most synapses were not depressed at the spontaneous firing rate. Gramicidin perforated-patch recordings indicated that the average reversal potential of spontaneous inhibitory postsynaptic potentials was -64 mV. Because of the change in driving force across the ISI, the unitary inputs are predicted to have a larger postsynaptic impact when they arrive late in the ISI. Simulations of network activity suggest that this very sparse inhibitory coupling may act to desynchronize the activity of SNr neurons while having only a small effect on firing rate.

In vitro thermosensitivity of rat lateral parabrachial neurons

The lateral parabrachial (LPB) neurons play a pivotal role in the thermoregulatory afferent pathway by transmitting cutaneous thermosensory signals received from spinal neurons directly to the thermoregulatory command center, the preoptic area (POA). The present study was conducted to electrophysiologically characterize the local temperature responsiveness of rat LPB neurons in brain slices to evaluate their local thermosensitivity and permit comparison with thermosensitive neurons in POA and spinal cord slices under consistent experimental conditions. In current clamp, warm- and cold-sensitive neurons were recorded in LPBel, LPBc and LPBd, the three LPB subnuclei responsible for the transmission of cutaneous feedforward signals. Of the 92 spontaneously firing LPB neurons, 27% were warm sensitive, 10% were cold sensitive, and 63% were temperature insensitive, and the spontaneous firing rate of the warm-sensitive neurons was significantly greater than that of the temperature-insensitive neurons. These proportions and spontaneous activity are similar to results obtained in the POA and spinal cord. Furthermore, the thermosensitivity was also present in 38% of silent neurons evoked by injection of a small amount of depolarizing current. Warm-sensitive neurons in the LPB were similar in thermoresponsiveness to those in the POA and spinal cord. However, cold sensitivity in the LPB was distinct from that in the POA. The firing rate of most cold-sensitive neurons changed steeply at a relatively narrow band of temperature, and some of them were silent near thermoneutrality. The percentages of thermosensitive and insensitive neurons within the three LPB subnuclei were not significantly different, nor were the mean maximal thermal coefficients of the thermosensitive neurons. These results suggest that LPB have local thermosensory functions as POA and spinal cord, and might be an important extrahypothalamic “thermoregulator”.

Enzyme-luminescence method: Tool for real-time monitoring of natural neurotoxins in vitro and l-glutamate release from primary cortical neurons

Novel enzyme-luminescence method is used for the rapid and sensitive in vitro detection of natural neurotoxins (e.g., shellfish and mushroom toxins) using model brain cells. Paralytic shellfish poisons gonyautoxins (e.g., GTX2,3 and GTX1,4) were detected at 1nM level by their inhibition of glutamate release from C6 glioma cells upon drug stimulation (IC50: GTX2,3=30nM and GTX1,4=8nM). Activation of glutamate release from C6 cells by ibotenic acid (a mushroom toxin) was also evaluated (EC50=10nM). The method was tested for real-time detection of glutamate release from primary rat cortical neurons. Dose-dependent effects of KCl (0–200mM) and NMDA on glutamate release from primary cortical neurons were studied. The effects of different culture conditions on K+-depolarization-induced glutamate release were also investigated. The method may be applicable to screening of drugs and toxins, and finding glutamatergic neurons in brain slices without in situ staining.

Both Estrogen and Androgen Modify the Response to Activation of Neurokinin-3 and κ-Opioid Receptors in Arcuate Kisspeptin Neurons From Male Mice.

Gonadal steroids regulate the pattern of GnRH secretion. Arcuate kisspeptin (kisspeptin, neurokinin B, and dynorphin [KNDy]) neurons may convey steroid feedback to GnRH neurons. KNDy neurons increase action potential firing upon the activation of neurokinin B receptors (neurokinin-3 receptor [NK3R]) and decrease firing upon the activation of dynorphin receptors (κ-opioid receptor [KOR]). In KNDy neurons from intact vs castrated male mice, NK3R-mediated stimulation is attenuated and KOR-mediated inhibition enhanced, suggesting gonadal secretions are involved. Estradiol suppresses spontaneous GnRH neuron firing in male mice, but the mediators of the effects on firing in KNDy neurons are unknown. We hypothesized the same gonadal steroids affecting GnRH firing pattern would regulate KNDy neuron response to NK3R and KOR agonists. To test this possibility, extracellular recordings were made from KNDy neurons in brain slices from intact, untreated castrated or castrated adult male mice treated in vivo with steroid receptor agonists. As observed previously, the stimulation of KNDy neurons by the NK3R agonist senktide was attenuated in intact vs castrated mice and suppression by dynorphin was enhanced. In contrast to observations of steroid effects on the GnRH neuron firing pattern, both estradiol and DHT suppressed senktide-induced KNDy neuron firing and enhanced the inhibition caused by dynorphin. An estrogen receptor-α agonist but not an estrogen receptor-β agonist mimicked the effects of estradiol on NK3R activation. These observations suggest the steroid modulation of responses to activation of NK3R and KOR as mechanisms for negative feedback in KNDy neurons and support the contribution of these neurons to steroid-sensitive elements of a GnRH pulse generator.

T-type calcium channels cause bursts of spikes in motor but not sensory thalamic neurons during mimicry of natural patterns of synaptic input.

Although neurons within intact nervous systems can be classified as ‘sensory’ or ‘motor,’ it is not known whether there is any general distinction between sensory and motor neurons at the cellular or molecular levels. Here, we extend and test a theory according to which activation of certain subtypes of voltage-gated ion channel (VGC) generate patterns of spikes in neurons of motor systems, whereas VGC are proposed to counteract patterns in sensory neurons. We previously reported experimental evidence for the theory from visual thalamus, where we found that T-type calcium channels (TtCCs) did not cause bursts of spikes but instead served the function of ‘predictive homeostasis’ to maximize the causal and informational link between retinogeniculate excitation and spike output. Here, we have recorded neurons in brain slices from eight sensory and motor regions of rat thalamus while mimicking key features of natural excitatory and inhibitory post-synaptic potentials. As predicted by theory, TtCC did cause bursts of spikes in motor thalamus. TtCC-mediated responses in motor thalamus were activated at more hyperpolarized potentials and caused larger depolarizations with more spikes than in visual and auditory thalamus. Somatosensory thalamus is known to be more closely connected to motor regions relative to auditory and visual thalamus, and likewise the strength of its TtCC responses was intermediate between these regions and motor thalamus. We also observed lower input resistance, as well as limited evidence of stronger hyperpolarization-induced (‘H-type’) depolarization, in nuclei closer to motor output. These findings support our theory of a specific difference between sensory and motor neurons at the cellular level.

Suppressive Effects of Resveratrol Treatment on The Intrinsic Evoked Excitability of CA1 Pyramidal Neurons.

Resveratrol, a phytoalexin, has a wide range of desirable biological actions. Despite a growing body of evidence indicating that resveratrol induces changes in neu- ronal function, little effort, if any, has been made to investigate the cellular effect of res- veratrol treatment on intrinsic neuronal properties. This experimental study was performed to examine the acute effects of resveratrol (100 µM) on the intrinsic evoked responses of rat Cornu Ammonis (CA1) pyramidal neurons in brain slices, using whole cell patch clamp re- cording under current clamp conditions. Findings showed that resveratrol treatment caused dramatic changes in evoked responses of pyramidal neurons. Its treatment induced a significant (P<0.05) increase in the after hyperpolarization amplitude of the first evoked action potential. Resveratrol-treated cells displayed a significantly broader action potential (AP) when compared with either control or vehicle-treated groups. In addition, the mean instantaneous firing frequency between the first two action potentials was significantly lower in resveratrol-treated neurons. It also caused a significant reduction in the time to maximum decay of AP. The rheobase current and the utilization time were both significantly greater following resveratrol treatment. Neurons exhibited a significantly depolarized voltage threshold when exposed to resveratrol. Results provide direct electrophysiological evidence for the inhibitory effects of resveratrol on pyramidal neurons, at least in part, by reducing the evoked neural activity.

Morphological Characterization of the Action Potential Initiation Segment in GnRH Neuron Dendrites and Axons of Male Mice.

GnRH neurons are the final output neurons of the hypothalamic network controlling fertility in mammals. In the present study, we used ankyrin G immunohistochemistry and neurobiotin filling of live GnRH neurons in brain slices from GnRH-green fluorescent protein transgenic male mice to examine in detail the location of action potential initiation in GnRH neurons with somata residing at different locations in the basal forebrain. We found that the vast majority of GnRH neurons are bipolar in morphology, elaborating a thick (primary) and thinner (secondary) dendrite from opposite poles of the soma. In addition, an axon-like process arising predominantly from a proximal dendrite was observed in a subpopulation of GnRH neurons. Ankyrin G immunohistochemistry revealed the presence of a single action potential initiation zone ∼27 μm in length primarily in the secondary dendrite of GnRH neurons and located 30 to 140 μm distant from the cell soma, depending on the type of process and location of the cell body. In addition to dendrites, the GnRH neurons with cell bodies located close to hypothalamic circumventricular organs often elaborated ankyrin G-positive axon-like structures. Almost all GnRH neurons (>90%) had their action potential initiation site in a process that initially, or ultimately after a hairpin loop, was coursing in the direction of the median eminence. These studies indicate that action potentials are initiated in different dendritic and axonal compartments of the GnRH neuron in a manner that is dependent partly on the neuroanatomical location of the cell body.