Richard Piet

Physiological contribution of the astrocytic environment of neurons to intersynaptic crosstalk

Interactions between separate synaptic inputs converging on the same target appear to contribute to the fine-tuning of information processing in the central nervous system. Intersynaptic crosstalk is made possible by transmitter spillover from the synaptic cleft and its diffusion over a distance to neighboring synapses. This is the case for glutamate, which inhibits γ-aminobutyric acid (GABA)ergic transmission in several brain regions through the activation of presynaptic receptors. Such heterosynaptic modulation depends on factors that influence diffusion in the extracellular space (ECS). Because glial cells represent a physical barrier to diffusion and, in addition, are essential for glutamate uptake, we investigated the physiological contribution of the astrocytic environment of neurons to glutamate-mediated intersynaptic communication in the brain. Here we show that the reduced astrocytic coverage of magnocellular neurons occurring in the supraoptic nucleus of lactating rats facilitates diffusion in the ECS, as revealed by tortuosity and volume fraction measurements. Under these conditions, glutamate spillover, monitored through metabotropic glutamate receptor-mediated depression of GABAergic transmission, is greatly enhanced. Conversely, impeding diffusion with dextran largely prevents crosstalk between glutamatergic and GABAergic afferent inputs. Astrocytes, therefore, by hindering diffusion in the ECS, regulate intersynaptic communication between neighboring synapses and, probably, overall volume transmission in the brain.

Retrograde regulation of GABA transmission by the tonic release of oxytocin and endocannabinoids governs postsynaptic firing.

The probability of neurotransmitter release at the nerve terminal is an important determinant of synaptic efficacy. At some central synapses, the postsynaptic, or target, neuron determines neurotransmitter release probability (Pr) at the presynaptic terminal. The mechanisms responsible for this target-cell dependent control of Pr have not been elucidated. Using whole-cell patch-clamp recordings from magnocellular neurosecretory cells in the paraventricular and supraoptic nuclei of the hypothalamus, we demonstrate that inhibitory, GABA synapses specifically onto oxytocin (OT)-producing neurosecretory cells exhibit a low Pr that is relatively uniform at multiple synapses onto the same cell. This low Pr results from a two-step process that requires the tonic release of OT from the postsynaptic cell. The ambient extracellular levels of neuropeptide are sufficient to activate postsynaptic OT receptors and trigger the Ca2+-dependent production of endocannabinoids, which act in a retrograde manner at presynaptic cannabinoid CB1 receptors to decrease GABA release. The functional consequence of this tonic inhibition of GABA release is that all inhibitory inputs facilitate uniformly when activated at high rates of activity. This causes inhibition in the postsynaptic cell that is sufficiently powerful to disrupt firing. Blockade of CB1 receptors increases Pr at these synapses, resulting in a rapid depression of IPSCs at high rates of activity, thereby eliminating the ability of afferent inputs to inhibit postsynaptic firing. By playing a deterministic role in GABA release at the afferent nerve terminal, the postsynaptic OT neuron effectively filters synaptic signals and thereby modulates its own activity patterns.

Glial modulation of synaptic transmission: Insights from the supraoptic nucleus of the hypothalamus.

Astrocytes clear synaptically released glutamate from the extracellular space through high‐affinity transporters present on their plasma membrane. By controlling the extracellular level of the main excitatory transmitter in the central nervous system, astrocytes thus contribute prominently to the regulation of overall cellular excitability and synaptic information processing. We recently investigated the influence of the glial environment on glutamatergic and GABAergic neurotransmission in the supraoptic nucleus of the rat hypothalamus under physiological conditions such as lactation that significantly reduce astrocytic coverage of its neurons. By performing electrophysiological analyses on this unique model of dynamic neuronal‐glial interactions, we have been able to show that the fine astrocytic processes normally enwrapping synapses serve two important functions. First, they govern the level of activation of presynaptic metabotropic glutamate receptors on glutamatergic terminals, thereby regulating synaptic efficacy at excitatory synapses. Second, they act as a physical and functional barrier to diffusion in the extracellular space, limiting spillover of glutamate and other neuroactive substances and therefore contributing to the regulation of heterosynaptic transmission and intercellular communication.

Glutamatergic and Purinergic Receptor-Mediated Calcium Transients in Bergmann Glial Cells

Astrocytes respond to neuronal activity with [Ca2+]i increases after activation of specific receptors. Bergmann glial cells (BGs), astrocytes of the cerebellar molecular layer (ML), express various receptors that can mobilize internal Ca2+. BGs also express Ca2+ permeable AMPA receptors that may be important for maintaining the extensive coverage of Purkinje cell (PC) excitatory synapses by BG processes. Here, we examined Ca2+ signals in single BGs evoked by synaptic activity in cerebellar slices. Short bursts of high-frequency stimulation of the ML elicited Ca2+ transients composed of a small-amplitude fast rising phase, followed by a larger and slower rising phase. The first phase resulted from Ca2+ influx through AMPA receptors, whereas the second phase required release of Ca2+ from internal stores initiated by P2 purinergic receptor activation. We found that such Ca2+ responses could be evoked by direct activation of neurons releasing ATP onto BGs or after activation of metabotropic glutamate receptor 1 on these neurons. Moreover, examination of BG and PC responses to various synaptic stimulation protocols suggested that ML interneurons are likely the cellular source of ATP.

Spontaneous Kisspeptin Neuron Firing in the Adult Mouse Reveals Marked Sex and Brain Region Differences but No Support for a Direct Role in Negative Feedback

Kisspeptin-Gpr54 signaling is critical for the GnRH neuronal network controlling fertility. The present study reports on a kisspeptin (Kiss)-green fluorescent protein (GFP) mouse model enabling brain slice electrophysiological recordings to be made from Kiss neurons in the arcuate nucleus (ARN) and rostral periventricular region of the third ventricle (RP3V). Using dual immunofluorescence, approximately 90% of GFP cells in the RP3V of females, and ARN in both sexes, are shown to be authentic Kiss-synthesizing neurons in adult mice. Cell-attached recordings of ARN Kiss-GFP cells revealed a marked sex difference in their mean firing rates; 90% of Kiss-GFP cells in males exhibited slow irregular firing (0.17 ± 0.04 Hz) whereas neurons from diestrous (0.01 ± 0.01 Hz) and ovariectomized (0 Hz) mice were mostly or completely silent. In contrast, RP3V Kiss-GFP cells were all spontaneously active, exhibiting tonic, irregular, and bursting firing patterns. Mean firing rates were significantly (P < 0.05) higher in diestrus (2.1 ± 0.3 Hz) compared with ovariectomized (1.0 ± 0.2 Hz) mice without any changes in firing pattern. Recordings from RP3V Kiss-GFP neurons at the time of the proestrous GnRH surge revealed a significant decline in firing rate after the surge. Together, these observations demonstrate unexpected sex differences in the electrical activity of ARN Kiss neurons and markedly different patterns of firing by Kiss neurons in the ARN and RP3V. Although data supported a positive influence of gonadal steroids on RP3V Kiss neuron firing, no direct evidence was found to support the previously postulated role of ARN Kiss neurons in the estrogen-negative feedback mechanism.

Oxytocin and estrogen promote rapid formation of functional GABA synapses in the adult supraoptic nucleus

We here investigated inhibitory synapse turnover in the adult brain using the hypothalamic supraoptic nucleus where new synapses form during different physiological conditions, in particular on oxytocin neurons largely controlled by GABAergic inputs and locally released oxytocin. Patch clamp recordings and ultrastructural analysis of the nucleus in acute slices from late gestating rats showed that oxytocin and estrogen promoted rapid formation of inhibitory synapses. Thus, after 2-h exposure to a combination of oxytocin and 17-beta estradiol, the frequency of miniature inhibitory postsynaptic currents was significantly enhanced. Since their amplitude and presynaptic GABA release probability were unmodified, this indicated an increased number of synapses. Electron microscopy confirmed increased densities of symmetric, putative GABAergic synapses within 2-h exposure to the peptide or steroid, effects which were reversible and oxytocin receptor mediated. Our observations thus offer direct evidence that hypothalamic GABAergic microcircuitries can undergo rapid and functional remodeling under changing neuroendocrine conditions.

Electrical properties of kisspeptin neurons and their regulation of GnRH neurons.

Kisspeptin neurons are critical components of the neuronal network controlling the activity of the gonadotropin-releasing hormone (GnRH) neurons. A variety of genetically-manipulated mouse models have recently facilitated the study of the electrical activity of the two principal kisspeptin neuron populations located in the rostral periventricular area of the third ventricle (RP3V) and arcuate nucleus (ARN) in acute brain slices. We discuss here the mechanisms and pathways through which kisspeptin neurons regulate GnRH neuron activity. We then examine the different kisspeptin-green fluorescent protein mouse models being used for kisspeptin electrophysiology and the data obtained to date for RP3V and ARN kisspeptin neurons. In light of these new observations on the spontaneous firing rates, intrinsic membrane properties, and neurotransmitter regulation of kisspeptin neurons, we speculate on the physiological roles of the different kisspeptin populations.

Estrous Cycle Plasticity in the Hyperpolarization-Activated Current Ih Is Mediated by Circulating 17β-Estradiol in Preoptic Area Kisspeptin Neurons

Circulating gonadal steroid hormones are thought to modulate a wide range of brain functions. However, the effects of steroid fluctuations through the ovarian cycle on the intrinsic properties of neurons are not well understood. We examined here whether gonadal steroids modulated the excitability of kisspeptin neurons located in the rostral periventricular region of the third ventricle (RP3V) of female mice. These cells are strongly implicated in sensing the high levels of circulating estradiol on proestrus to activate gonadotropin-releasing hormone (GnRH) neurons that, in turn, trigger ovulation. Electrophysiological studies were undertaken in brain slices from ovariectomized (OVX), diestrous, and proestrous kisspeptin-GFP mice. RP3V kisspeptin neurons exhibited marked changes in the hyperpolarization-evoked depolarizing sag and rebound firing across these groups. The hyperpolarization-activated current Ih was identified to be responsible for the depolarizing sag and was increased across OVX → diestrous → proestrous mice. Experiments in OVX mice given estradiol replacement identified an estradiol-dependent increase in Ih within RP3V kisspeptin neurons. Ih in these cells was found to contribute to their subthreshold membrane properties and the dynamics of rebound firing following hyperpolarizing stimuli in an estrous cycle-dependent manner. Only a minor role was found for Ih in modulating the spontaneous burst firing of RP3V kisspeptin neurons. These observations identify Ih as an ionic current that is regulated in a cyclical manner by circulating estradiol within the female brain, and suggest that such plasticity in the intrinsic properties of RP3V kisspeptin neurons may contribute to the generation of the preovulatory GnRH surge.

Modulation of GABAergic transmission by endogenous glutamate in the rat supraoptic nucleus

The presence of group III metabotropic glutamate receptors on GABAergic terminals in the supraoptic nucleus suggests that the level of glutamate in the extracellular space may regulate synaptic strength at inhibitory synapses. To test this hypothesis we examined the consequences of increasing ambient glutamate on GABA‐mediated synaptic activity in supraoptic neurons. The concentration of the excitatory amino acid in the extracellular space was increased pharmacologically by blocking glutamate transporters. Inhibition of the astrocyte‐specific GLT‐1 glutamate transporter led to a reversible decrease in evoked inhibitory postsynaptic current amplitude. This modulation had a presynaptic origin as revealed by analysis of paired‐pulse ratio and miniature inhibitory currents. Furthermore, blocking group III metabotropic glutamate receptors with the specific antagonist MAP4 prevented the depression of GABAergic transmission induced by glutamate transporter blockade. Thus, presynaptic metabotropic glutamate receptors located on inhibitory terminals in the supraoptic nucleus appear to sense changes in ambient glutamate and modify GABA release accordingly. However, it seems that such changes need to reach a certain magnitude because the discrete deficit in glutamate clearance which occurs in the supraoptic nucleus of lactating rats is not sufficient to modulate GABA‐mediated transmission. These results suggest that ambient glutamate contributes to the modulation of synaptic efficacy not only at glutamatergic synapses but also at inhibitory GABAergic synapses.

Differential Actions of Prolactin on Electrical Activity and Intracellular Signal Transduction in Hypothalamic Neurons

In many tissues, including brain, prolactin action is predominantly mediated by the Janus kinase/signal transducer and activator of transcription (STAT) signal transduction pathway, leading to changes in gene transcription. However, prolactin can also exert rapid actions on electrical activity of hypothalamic neurons. Here, we investigate whether both responses occur in a single cell type, focusing on three specific populations known to be influenced by prolactin: GnRH neurons, tuberoinfundibular dopamine (TIDA) neurons, and neurons in the anteroventral-periventricular nucleus in female mice. We performed phosphorylated STAT5 (pSTAT5) immunohistochemistry to identify prolactin-responsive neurons after in vivo prolactin treatment. In addition, we carried out in vitro electrophysiology in slices from transgenic mice expressing green fluorescent protein driven by the GnRH or tyrosine hydroxylase promoters as well as from C57BL/6J mice to assess acute electrical responses to prolactin. Approximately 88% of TIDA neurons expressed pSTAT5 in diestrous mice, rising to 97% after prolactin treatment. All TIDA neurons also showed a rapid increase in firing rate after prolactin treatment. In contrast, very few GnRH neurons (11%) showed pSTAT5 in response to prolactin, and none showed a change in electrical activity. Finally, in the anteroventral-periventricular nucleus, most neurons (69%) responded to prolactin treatment with an increase in pSTAT5, but only 2/38 (∼5%) showed changes in electrical activity in response to prolactin. These observations show that prolactin recruits different combinations of electrical and transcriptional responses in neurons depending upon their anatomical location and phenotype. This may be critical in establishing appropriate responses to prolactin under different physiological conditions.