Mapping and identification of GABAergic neurons in transgenic mice projecting to cardiac vagal neurons in the nucleus ambiguus using photo-uncaging

Abstract

The neural control of heart rate is determined primarily by the activity of preganglionic parasympathetic cardiac vagal neurons (CVNs) originating in the nucleus ambiguus (NA) in the brain stem. GABAergic inputs to CVNs play an essential role in determining the activity of these neurons including a robust inhibition during each inspiratory burst. The origin of GABAergic innervation has yet to be determined however. A transgenic mouse line expressing green florescent protein (GFP) in GABAergic cells was used in conjunction with caged glutamate to identify both clusters and individual GABAergic neurons that evoke inhibitory GABAergic synaptic responses in CVNs. Transverse slices were taken with CVNs patch-clamped in the whole cell configuration. Sections containing both the pre-Botzinger complex as well as the calamus scriptorius were divided into approximately 90 quadrants, each 200 x 200 microm and were sequentially photostimulated. Inhibitory post synaptic currents (IPSCs) were recorded in CVNs after a 5-ms photostimulation of 50 microM caged glutamate. The four areas that contained GABAergic cells projecting to CVNs were 200 microm medial, 400 microm medial, 200 microm ventral, and 1,200 microm dorsal and 1,000 microm medial to patched CVNs. Once foci of GABAergic cells projecting to CVNs were determined, photostimulation of individual GABAergic neurons was conducted. The results from this study suggest that GABAergic cells located in four specific areas project to CVNs, and that these cells can be individually identified and stimulated using photouncaging to recruit GABAergic neurotransmission to CVNs.

FIG. 1.

The spatially limited release of glutamate was accomplished by focal photostimulation of 50 μM caged glutamate using a ×10 objective for 5 ms. A, left: the results from a typical experiment. The inward currents evoked by the photorelease of glutamate were typically 100–1,000 pA at the surface of the neuron and decreased with increased distance between the patched cell and the uncaging location. The responses at distances >100 μm were negligible. Middle: illustration shows the experimental paradigm of examining the responses on uncaging glutamate at varying distances from the patched neuron. Data from 9 experiments are summarized in B.

FIG. 2.

The brain slice was divided into 200 × 200 μm areas with each foci being stimulated with a 5-ms pulse of 50 μM glutamate. Of 90–100 areas tested, only 4 areas were found to elicit an increase in inhibitory events in the patched cardiac vagal neuron (P < 0.05). The results shown are the typical evoked responses in a single GABAergic neuron (top) and the average results in 8–10 neurons (bottom) from each of these localized areas. The bar graphs are plotted in 500-ms bins, with the 1st 4 bars representing the 2 s prior to the photouncaging, and the last 5 bars representing the 0.5 ms associated with the photostimulation, and the 2-s poststimulation, respectively.

FIG. 3.

The spatially limited release of glutamate on an individual GABAergic neuron was accomplished by 5 ms focal photostimulation of 10 μM caged glutamate using a ×40 objective. As shown in A, the inward inhibitory currents evoked by the photorelease of glutamate were typically 50–150 pA at the surface of the neuron, which decreased with increased distance between the patched cell and the uncaging location. B: the responses at distances >50 μm were negligible. Left: traces are the responses from a typical experiment; middle: illustration depicts the experimental procedure of uncaging glutamate at increasing distances from the patched cell. Right: the average results from 10 neurons.

FIG. 4.

Depicted are the changes in action potential firing on release of caged glutamate (10 μM) using a ×40 objective. Glutamate was uncaged for a duration of 5 ms on the patched GABAergic cell, which fired multiple action potentials in response to the uncaging of caged glutamate. Cells were patched in the current clamp configuration (n = 8). GABAergic cells fired an average of 4 ± 0.8 Hz before stimulation with glutamate and then an average of 22.4 ± 5.2 Hz in the 1 s after the UV uncaging. All cells responded with action potentials to the uncaging within 33.25 ± 1.8 ms. The cells returned to baseline by 600 ms post stimulation. Right: illustration depicts the experimental paradigm.

FIG. 5.

Stimulation of an individual GABAergic cell projecting to a patched cardiac vagal neuron (CVN) was accomplished by the spatially limited release of glutamate (10 μM) onto the GABAergic cell using a ×40 objective (n = 17; A. and B. Top: the results from a typical experiment. Glutamate uncaged on the cell body of a GABAergic neuron evokes an increase in inhibitory events in a CVN from 2.8 ± 0.2 Hz prestimulation to 4.3 ± 0.3 Hz (P < 0.05). C: the application of GABAzine to the bath solution blocked the increase in inhibitory events. Right: illustration depicts the experimental design.