Excitatory Effects of Calcitonin Gene-Related Peptide (CGRP) on Superficial Sp5C Neurons in Mouse Medullary Slices.

The neuromodulator calcitonin gene-related peptide (CGRP) is known to facilitate nociceptive transmission in the superficial laminae of the spinal trigeminal nucleus caudalis (Sp5C). The central effects of CGRP in the Sp5C are very likely to contribute to the activation of central nociceptive pathways leading to attacks of severe headaches like migraine. To examine the potential impacts of CGRP on laminae I/II neurons at cellular and synaptic levels, we performed whole-cell patch-clamp recordings in juvenile mouse brainstem slices. First, we tested the effect of CGRP on cell excitability, focusing on neurons with tonically firing action potentials upon depolarizing current injection. CGRP (100 nM) enhanced tonic discharges together with membrane depolarization, an excitatory effect that was significantly reduced when the fast synaptic transmissions were pharmacologically blocked. However, CGRP at 500 nM was capable of exciting the functionally isolated cells, in a nifedipine-sensitive manner, indicating its direct effect on membrane intrinsic properties. In voltage-clamped cells, 100 nM CGRP effectively increased the frequency of excitatory synaptic inputs, suggesting its preferential presynaptic effect. Both CGRP-induced changes in cell excitability and synaptic drives were prevented by the CGRP receptor inhibitor BIBN 4096BS. Our data provide evidence that CGRP increases neuronal activity in Sp5C superficial laminae by dose-dependently promoting excitatory synaptic drive and directly enhancing cell intrinsic properties. We propose that the combination of such pre- and postsynaptic actions of CGRP might underlie its facilitation in nociceptive transmission in situations like migraine with elevated CGRP levels.

Muscarinic Modulation of Morphologically Identified Glycinergic Neurons in the Mouse PreBötzinger Complex.

The cholinergic system plays an essential role in central respiratory control, but the underlying mechanisms remain elusive. We used whole-cell recordings in brainstem slices from juvenile mice expressing enhanced green fluorescent protein (EGFP) under the control of the glycine transporter type 2 (GlyT2) promoter, to examine muscarinic modulation of morphologically identified glycinergic neurons in the preBötzinger complex (preBötC), an area critical for central inspiratory rhythm generation. Biocytin-filled reconstruction of glycinergic neurons revealed that the majority of them had few primary dendrites and had axons arborized within their own dendritic field. Few glycinergic neurons had axon collaterals extended towards the premotor/motor areas or ran towards the contralateral preBötC, and had more primary dendrites and more compact dendritic trees. Spontaneously active glycinergic neurons fired regular spikes, or less frequently in a "burst-like" pattern at physiological potassium concentration. Muscarine suppressed firing in the majority of regular spiking neurons via M2 receptor activation while enhancing the remaining neurons through M1 receptors. Interestingly, rhythmic bursting was augmented by muscarine in a small group of glycinergic neurons. In contrast to its heterogeneous modulation of glycinergic neuronal excitability, muscarine generally depressed inhibitory and excitatory synaptic inputs onto both glycinergic and non-glycinergic preBötC neurons, with a stronger effect on inhibitory input. Notably, presynaptic muscarinic attenuation of excitatory synaptic input was dependent on M1 receptors in glycinergic neurons and on M2 receptors in non-glycinergic neurons. Additional field potential recordings of excitatory synaptic potentials in the M2 receptor knockout mice indicate that glycinergic and non-glycinergic neurons contribute equally to the general suppression by muscarine of excitatory activity in preBötC circuits. In conclusion, our data show that preBötC glycinergic neurons are morphologically heterogeneous, and differ in the properties of synaptic transmission and muscarinic modulation in comparison to non-glycinergic neurons. The dominant and cell-type-specific muscarinic inhibition of synaptic neurotransmission and spiking may contribute to central respiratory disturbances in high cholinergic states.

Release of CGRP from mouse brainstem slices indicates central inhibitory effect of triptans and kynurenate.

BACKGROUND: CGRP is contained in a substantial proportion of unmyelinated trigeminal neurons innervating intracranial tissues. Previously, we have described a hemisected rodent scull preparation and later the intact trigeminal ganglion to measure stimulated CGRP release from trigeminal afferents. METHODS: Here, we establish a preparation for examining CGRP release from central trigeminal terminals using single fresh slices of the mouse medullary brainstem. RESULTS: Basal and stimulated amount of CGRP substantially exceeded the detection level. Experiments were designed as matched pairs of at least six brainstem slices per animal. Stimulation with high potassium induced calcium-dependent and reversible CGRP release. Capsaicin stimulation of TRPV1 provoked concentration-dependent CGRP release. The anti-migraine drug naratriptan did not inhibit capsaicin-induced CGRP release from peripheral terminals but inhibited the release from brainstem slices. The glutamate antagonist kynurenate showed a similar pattern of site-specific inhibition of CGRP release. CONCLUSIONS: As observed earlier for other drugs used in the treatment of migraine this indicates that the central terminals in the spinal trigeminal nucleus may be the main site of action. The preparation allows evaluating the trigeminal brainstem as a pharmacological site of action.

Axonal failure during high frequency stimulation of rat subthalamic nucleus.

Deep brain stimulation (DBS) has been established as an effective surgical therapy for advanced Parkinson's disease (PD) and gains increasing acceptance for otherwise intractable neuropsychiatric diseases such as major depression or obsessive­compulsive disorders. In PD, DBS targets predominantly the subthalamic nucleus (STN) and relieves motor deficits only at high frequency (>100 Hz). In contrast to the well-documented clinical efficacy of DBS, its underlying principle remains enigmatic spawning a broad and, in part, contradictory spectrum of suggested synaptic and non-synaptic mechanisms within and outside STN. Here we focused on a crucial, but largely neglected issue in this controversy, namely the axonal propagation of DBS within and away from STN. In rat brain slices preserving STN projections to substantia nigra (SN) and entopeduncular nucleus (EP, the rodent equivalent of internal globus pallidus), STN-DBS disrupted synaptic excitation onto target neurons through an unexpected failure of axonal signalling. The rapid onset and, upon termination of DBS, recovery of this effect was highly reminiscent of the time course of DBS in the clinical setting. We propose that DBS-induced suppression of axonal projections from and to STN serves to shield basal ganglia circuitry from pathological activity arising in or amplified by this nucleus.

Layer-specific processing of excitatory signals in CA1 interneurons depends on postsynaptic M2 muscarinic receptors.

The hippocampus receives a diffuse cholinergic innervation which acts on pre- and postsynaptic sites to modulate neurotransmission and excitability of pyramidal cells and interneurons in an intricate fashion. As one missing piece in this puzzle, we explored how muscarinic receptor activation modulates the somatodendritic processing of glutamatergic input in CA1 interneurons. We performed whole-cell recordings from visually identified interneurons of stratum radiatum (SR) and stratum oriens (SO) and examined the effects of the cholinergic agonist carbachol (CCh) on EPSP-like waveforms evoked by brief glutamate pulses onto their proximal dendrites. In SO interneurons, CCh consistently reduced glutamate-induced postsynaptic potentials (GPSPs) in control rat and mice, but not in M2 muscarinic receptor knockout mice. By contrast, the overwhelming majority of interneurons recorded in SR of control and M2 receptor-deficient hippocampi exhibited muscarinic enhancement of GPSPs. Interestingly, the non-responding interneurons were strictly confined to the SR subfield closest to the subiculum. Our data suggest that postsynaptic modulation by acetylcholine of excitatory input onto CA1 interneurons occurs in a stratum-specific fashion, which is determined by the absence or presence of M2 receptors in their (somato-)dendritic compartments. Thus cholinergic projections might be capable of recalibrating synaptic weights in different inhibitory circuits of the CA1 region.

Evidence for a cortical-basal ganglia projection pathway in female zebra finches.

The anterior forebrain pathway in songbirds is a specialization of the avian basal ganglia pathway and is prominent in males that sing, but seem to be absent or incomplete in females that do not sing. We studied the connectivity in females in the in vitro slice preparation by applying the tracer Fluoro Ruby, biotinylated dextran amine, and cholera toxin B. We identified (1) retrograde labeled neurons in the lateral magnocellular nucleus of the anterior nidopallium (LMAN) projecting to the medial striatum (MSt), and (2) we identified fibers in the MSt labeled by anterograde transport after tracer injection into LMAN. Our data clearly demonstrate the existence of a cortico-basal ganglia pathway in female birds.