Karl J. Iremonger

Selective Attenuation of Afferent Synaptic Transmission as a Mechanism of Thalamic Deep Brain Stimulation-Induced Tremor Arrest

Deep brain stimulation (DBS) of the ventrolateral thalamus stops several forms of tremor. Microelectrode recordings in the human thalamus have revealed tremor cells that fire synchronous with electromyographic tremor. The efficacy of DBS likely depends on its ability to modify the activity of these tremor cells either synaptically by stopping afferent tremor signals or by directly altering the intrinsic membrane currents of the neurons. To test these possibilities, whole-cell patch-clamp recordings of ventral thalamic neurons were obtained from rat brain slices. DBS was simulated (sDBS) using extracellular constant current pulse trains (125 Hz, 60–80 μs, 0.25–5 mA, 1–30 s) applied through a bipolar electrode. Using a paired-pulse protocol, we first established that thalamocortical relay neurons receive converging input from multiple independent afferent fibers. Second, although sDBS induced homosynaptic depression of EPSPs along its own pathway, it did not alter the response from a second independent pathway. Third, in contrast to the subthalamic nucleus, sDBS in the thalamus failed to inhibit the rebound potential and the persistent Na+ current but did activate the Ih current. Finally, in eight patients undergoing thalamic DBS surgery for essential tremor, microstimulation was most effective in alleviating tremor when applied in close proximity to recorded tremor cells. However, stimulation could still suppress tremor at distances incapable of directly spreading to recorded tremor cells. These complementary data indicate that DBS may induce a “functional deafferentation” of afferent axons to thalamic tremor cells, thereby preventing tremor signal propagation in humans.

GnRH Neurons Elaborate a Long-Range Projection with Shared Axonal and Dendritic Functions

Information processing by neurons has been traditionally envisioned to occur in discrete neuronal compartments. Specifically, dendrites receive and integrate synaptic inputs while axons initiate and conduct spikes to distal neuronal targets. We report here in mice, using morphological reconstructions and electrophysiology, that the gonadotropin-releasing hormone (GnRH) neurons that control mammalian fertility do not conform to this stereotype and instead possess a single projection structure that functions simultaneously as an axon and dendrite. Specifically, we show that the GnRH neuron projection to the median eminence to control pituitary hormone secretion possesses a spike initiation site and conducts action potentials while also exhibiting spines and synaptic appositions along its entire length. Classical axonal or dendritic markers are not detectable in the projection process. Activation of ionotropic glutamate and/or GABA receptors along the GnRH neuron projection is capable of depolarizing the membrane potential and initiating action potentials. In addition, focal glutamate application to the projection is able to regulate the width of propagating spikes. These data demonstrate that GnRH neurons elaborate a previously uncharacterized neuronal projection that functions simultaneously as an axon and dendrite. This structure, termed a “dendron,” greatly expands the dynamic control of GnRH secretion into the pituitary portal system to regulate fertility.

Retrograde Opioid Signaling Regulates Glutamatergic Transmission in the Hypothalamus

Opioid signaling in the CNS is critical for controlling cellular excitability, yet the conditions under which endogenous opioid peptides are released and the precise mechanisms by which they affect synaptic transmission remain poorly understood. The opioid peptide dynorphin is present in the soma and dendrites of vasopressin neurons in the hypothalamus and dynamically controls the excitability of these cells in vivo. Here, we show that dynorphin is released from dendritic vesicles in response to postsynaptic activity and acts in a retrograde manner to inhibit excitatory synaptic transmission. This inhibition, which requires the activation of κ-opioid receptors, results from a reduction in presynaptic release of glutamate vesicles. The opioid inhibition is downstream of Ca2+ entry and is likely mediated by a direct modulation of presynaptic fusion machinery. These findings demonstrate that neurons may self-regulate their excitability through the dendritic release of opioids to inhibit excitatory synaptic transmission.

Glutamate regulation of GnRH neuron excitability

The gonadotropin-releasing hormone (GnRH) neuronal network is the master controller of the reproductive axis. It is widely accepted that the amino acid transmitters GABA and glutamate play important roles in controlling GnRH neuron excitability. However, remarkably few studies have examined the functional role of direct glutamate regulation of GnRH neurons. Dual-labeling investigations have shown that GnRH neurons express receptor subunits required for AMPA, NMDA and kainate signaling in a heterogeneous manner. Electrophysiological and calcium imaging studies have confirmed this heterogeneity and shown that while the majority of adult GnRH neurons express AMPA/kainate receptors, only small sub-populations have functional NMDA or metabotropic glutamate receptors. Accumulating evidence suggests that one important role of direct glutamate signaling at GnRH neurons is for their activation at the time of puberty. Whereas in vivo studies have indicated the importance of NMDA signaling within the whole of the GnRH neuronal network, including afferent neurons and glia, investigations at the level of the GnRH neuron suggest that peripubertal changes in AMPA receptor expression may be dominant in the mouse. The sources of glutamatergic inputs to the GnRH neurons are only just beginning to be examined and include the anteroventral periventricular nucleus as well as the possibility that GnRH neurons may use glutamate as a neurotransmitter in recurrent collateral innervation. It is expected that a full understanding of the glutamatergic regulation of GnRH neurons will provide significant insight into the mechanisms underlying their control of reproductive function.

Integration of Asynchronously Released Quanta Prolongs the Postsynaptic Spike Window

Classically, the release of glutamate in response to a presynaptic action potential causes a brief increase in postsynaptic excitability. Previous reports indicate that at some central synapses, a single action potential can elicit multiple, asynchronous release events. This raises the possibility that the temporal dynamics of neurotransmitter release may determine the duration of altered postsynaptic excitability. In response to physiological challenges, the magnocellular neurosecretory cells (MNCs) in the paraventricular nucleus of the hypothalamus (PVN) exhibit robust and prolonged increases in neuronal activity. Although the postsynaptic conductances that may facilitate this form of activity have been investigated thoroughly, the role of presynaptic release has been largely overlooked. Because the specific patterns of activity generated by MNCs require the activation of excitatory synaptic inputs, we sought to characterize the release dynamics at these synapses and determine whether they contribute to prolonged excitability in these cells. We obtained whole-cell recordings from MNCs in brain slices of postnatal day 21–44 rats. Stimulation of glutamatergic inputs elicited large and prolonged postsynaptic events that resulted from the summation of multiple, asynchronously released quanta. Asynchronous release was selectively inhibited by the slow calcium buffer EGTA-AM and potentiated by brief high-frequency stimulus trains. These trains caused a prolonged increase in postsynaptic spike activity that could also be eliminated by EGTA-AM. Our results demonstrate that glutamatergic terminals in PVN exhibit asynchronous release, which is important in generating large postsynaptic depolarizations and prolonged spiking in response to brief, high-frequency bursts of presynaptic activity.

In Vivo Recordings of GnRH Neuron Firing Reveal Heterogeneity and Dependence upon GABAA Receptor Signaling

The gonadotropin-releasing hormone (GnRH) neurons are the key cells regulating fertility in all mammalian species. The scattered distribution of these neurons has made investigation of their properties extremely difficult and the key goal of recording their electrical activity in vivo near impossible. The caudal-most extension of the GnRH neuron continuum brings some cells very close to the base of the brain at the level of the anterior hypothalamic area. Taking insight from this, we developed an experimental procedure in anesthetized GnRH-GFP mice that allows the electrical activity of these GnRH neurons to be recorded in vivo. On-cell recordings revealed that the majority of GnRH neurons (86%) were spontaneously active, exhibiting a range of firing patterns, although only a minority (15%) exhibited burst firing. Mean firing frequencies ranged from 0.06 to 3.65 Hz, with the most common interspike interval being ∼500 ms. All GnRH neurons tested were activated by AMPA and kisspeptin. Whereas the GABAA receptor agonist muscimol evoked excitatory, inhibitory, or mixed effects on GnRH neuron firing, the GABAA receptor antagonist picrotoxin resulted in a consistent suppression of firing. These observations represent the first electrical recordings of GnRH neurons in vivo. They reveal that GnRH neurons in vivo exhibit considerable heterogeneity in their firing patterns with both similarities and differences to firing in vitro. These variable patterns of firing in vivo are found to be critically dependent upon ongoing GABAA receptor signaling.

Initiation and Propagation of Action Potentials in Gonadotropin-Releasing Hormone Neuron Dendrites

Gonadotropin-releasing hormone (GnRH) neurons are the final output neurons in a complex neuronal network that regulates fertility. The morphology of GnRH neuron dendrites is very different to other central neurons in that they are very long, thin, and unbranched. To study the function of these dendrites, we have used Na+ and Ca2+ imaging in combination with dual soma and dendrite electrical recordings in brain slices from GnRH-GFP mice. Here, we show that GnRH neurons actively propagate Na+ spikes throughout their dendrites. Multisite dendritic recordings confirmed that these spikes were observed in one of the dendrites before the soma in the great majority of neurons tested. Na+ imaging experiments revealed that the initial 150 μm of dendrite has a higher density of functional Na+ channels than more distal regions, suggesting that this region of dendrite is highly excitable and may be the site of spike initiation. Finally, we show that the depolarization from dendritic spikes opens voltage-gated Ca2+ channels giving rise to dendritic Ca2+ transients. Together, these findings suggest that the proximal dendrites of GnRH neurons are highly excitable and are likely to be the site of action potential initiation in these neurons.

Definition of the hypothalamic GnRH pulse generator in mice

Significance Neural networks located in the hypothalamus are responsible for generating ultradian patterns of hormone secretion that control a wide variety of functions. How these neural networks generate pulsatile hormone secretion remains unknown. We report here that a population of hypothalamic kisspeptin neurons represents the gonadotropin-releasing hormone (GnRH) pulse generator. These cells have the remarkable ability to generate synchronized GnRH secretion every 9 min to drive pulsatile gonadotropin hormone secretion in the blood. These observations indicate the arcuate kisspeptin neurons as the origin of reproductive hormone pulsatility in mice and offer the prospect of better understanding and manipulating fertility in the clinic. The pulsatile release of luteinizing hormone (LH) is critical for mammalian fertility. However, despite several decades of investigation, the identity of the neuronal network generating pulsatile reproductive hormone secretion remains unproven. We use here a variety of optogenetic approaches in freely behaving mice to evaluate the role of the arcuate nucleus kisspeptin (ARNKISS) neurons in LH pulse generation. Using GCaMP6 fiber photometry, we find that the ARNKISS neuron population exhibits brief (∼1 min) synchronized episodes of calcium activity occurring as frequently as every 9 min in gonadectomized mice. These ARNKISS population events were found to be near-perfectly correlated with pulsatile LH secretion. The selective optogenetic activation of ARNKISS neurons for 1 min generated pulses of LH in freely behaving mice, whereas inhibition with archaerhodopsin for 30 min suppressed LH pulsatility. Experiments aimed at resetting the activity of the ARNKISS neuron population with halorhodopsin were found to reset ongoing LH pulsatility. These observations indicate the ARNKISS neurons as the long-elusive hypothalamic pulse generator driving fertility.

Dual regulation of anterograde and retrograde transmission by endocannabinoids.

Endocannabinoids (eCBs) are feedback messengers in the nervous system that act at the presynaptic nerve terminal to inhibit transmitter release. Here we report that in brain slices from rat, eCBs are released from vasopressin (VP) neurons in the paraventricular nucleus of the hypothalamus following coincident bursts of presynaptic and postsynaptic spiking. eCBs transiently depress glutamate release from excitatory terminals and, in doing so, prevent the synapses from undergoing long-term depression (LTD). Specifically, we show that blockade of CB1 receptors unmasks LTD following coincident presynaptic and postsynaptic activity. This LTD is presynaptic in nature, but requires the release of the opioid peptide dynorphin from the postsynaptic neuron. Dynorphin release and subsequent LTD require the activation of postsynaptic metabotropic glutamate receptors (mGluRs). Our findings indicate that eCBs, by transiently depressing glutamate release, limit mGluR activation and indirectly gate release of dynorphin from the postsynaptic neuron. We propose that eCBs, in addition to their well described role in the rapid modulation of transmitter release from the nerve terminal, also regulate the release of other retrograde transmitters and thus encode for multiple temporal windows of synaptic plasticity.

Glutamatergic synaptic transmission in neuroendocrine cells: Basic principles and mechanisms of plasticity.

Glutamate synapses drive the output of neuroendocrine cells in the hypothalamus, but until recently, relatively little was known about the fundamental properties of transmission at these synapses. Here we review recent advances in the understanding of glutamate signals in magnocellular neurosecretory cells (MNCs) in the paraventricular (PVN) and supraoptic nuclei (SON) of the hypothalamus that serve as the last step in synaptic integration before neurohormone release. While these synapses exhibit many similarities with other glutamate synapses described throughout the brain, they also exhibit a number of unique properties that are particularly well suited to the physiology of this system and will be discussed here. In addition, a number of recent studies begin to provide insights into new forms of synaptic plasticity that may be common in other brain regions, but in these cells, may serve important adaptive roles.