A transgenic mouse model reveals fast nicotinic transmission in hippocampal pyramidal neurons

Abstract

The relative contribution to brain cholinergic signaling by synaptic- and diffusion-based mechanisms remains to be elucidated. In this study, we examined the prevalence of fast nicotinic signaling in the hippocampus. We describe a mouse model where cholinergic axons are labeled with the tauGFP fusion protein driven by the choline acetyltransferase promoter. The model provides for the visualization of individual cholinergic axons at greater resolution than other available models and techniques, even in thick, live, slices. Combining calcium imaging and electrophysiology, we demonstrate that local stimulation of visualized cholinergic fibers results in rapid excitatory postsynaptic currents mediated by the activation of α7-subunit-containing nicotinic acetylcholine receptors (α7-nAChRs) on CA3 pyramidal neurons. These responses were blocked by the α7-nAChR antagonist methyllycaconitine and potentiated by the receptor-specific allosteric modulator 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxanol-3-yl)-urea (PNU-120596). Our results suggest, for the first time, that synaptic nAChRs can modulate pyramidal cell plasticity and development. Fast nicotinic transmission might play a greater role in cholinergic signaling than previously assumed. We provide a model for the examination of synaptic properties of basal forebrain cholinergic innervation in the brain.

Figure 1. BAC transgenic vector for generation of the ChAT-tauGFP mouse

A tauGFP-polyA cassette was introduced precisely at the first codon of the ChAT gene coding exon 3 of the BAC clone RP23-431D9, by Red-mediated homologous recombination in E. coli. The modified BAC was injected to make transgenic mice.

Figure 2. Expression of tauGFP in the brain and peripheral nervous system

A. Thick (250 μm transverse section through the diencephalon showing the bright fluorescence of the habenulo-interpeduncular tract (HabInttr). B. Ventral view of the forebrain of a ChAT-GFP mouse. Bright GFP fluorescence is evident in the interpeduncular nucleus (nIp). Endogenous GFP fluorescence also is visible in linear arrays associated with the Islands of Calleja within olfactory tubercle (Olf Tub) (See also Fig. 4 A, B). The cholinergic fibers of the parabigeminal-collicular tract (PbC) are visible within the optic tracts. C. Transverse section through the interpeduncular nucleus showing the bright fluorescence of the terminals of the habenolinterpeduncular tract. Fascicles of the oculomotor nerve (N.III) appear lateral to the interpeduncular nucleus. D. Transverse section through the medial habenular nucleus (mHab) and the habenulointerpeduncular tract. E. GFP fluorescence in the enteric nervous system of the gut. F. Fluorescent motor endplate on straite muscle of the tongue. G. Section through the vallate paplilla showing GFP fluorescence in a subset of taste cells (Type III cells) of the taste buds.

Figure 3. tauGFP expression in the hindbrain revealed by GFP immunofluorescence

A. Transverse section through the rostral medulla showing the hindbrain motor nuclei and nerve roots. Scattered cholinergic cells also are present within the nucleus of the solitary tract (nTS). DMNX, dorsal motor nucleus of the vagus, nA, nucleus ambiguus, nTS, nucleus of the solitary tract; N X, vagus nerve root; N XII, hypoglossal nerve root; nXII, hypoglossal nucleus. V = 4^th ventricle. B. Transverse section through the level of the pons showing the trochlear nerve root (N IV) and raphe nuclei (nR). Some of the large neurons of the pedunculopontine nucleus (PPn) exhibit immunoreactivity for GFP (green) but most show only ChAT protein immunoreactivity (magenta). C. Transverse section through the rostral medulla at the level of entrance of the facila nerve (N VII). The motor nucleus (n VI) and root fibers (N VI) of the abducens nerve also are visible at this level. Large mossy fiber terminals are visible within the cerebellar vermis (CbV). Scale same as in A. D. Higher magnification of the vermis slightly anterior to the level of panel C showing large cholinergic mossy fiber terminals.

Figure 4. Transgene expression in the basal forebrain

Immunofluorescence as revealed by double label with anti-GFP (green) and anti-ChAT (red). Nissl counterstain is shown in blue. A. Low magnification section through the anterior telencephalon at the level of the septal nuclei. Numerous cells double labeled for ChAT and GFP are visible in the nuc. diagonal band (nDB) and caudoputamen (CP) (shown at higher magnification in panel D). In addition, a dense cholinergic plexus is associated with the Islands of Calleja (ICj) within the ventral pallidum and olfactory tubercle (Olf Tu). AC, root of the anterior commissure. B. Higher magnification of the ventromedial portion of the telencephalon showing dense cholinergic innervation of the ventral pallidum and olfactory tubercle. C. High magnification of a section through frontal cortex, pia is shown at the top. Small cholinergic interneurons of the cortex (magenta arrows) show immunoreactivity for ChAT protein, but lack reactivity for GFP. In contrast the dense fiber plexus of layer I shows robust GFP and ChAT immunoreactivity. D. High magnification image of the large cholinergic cells of the caudoputamen. The dense plexus of cholinergic fibers within this structure is readily apparent in these tauGFP transgenic animals.

Figure 5. tauGFP positive fiber distribution in the hippocampus

A. Low magnification image of a hippocampus from an adult mouse. Left panel: an image of the entire hippocampus, stained with anti-GFP antibody, showing the distribution of tauGFP. Scale Bar 250 μm Right panel: Green- Cholinergic fibers stained with anti-GFP antibody. Red- staining with anti-ChAT antibody. Yellow – ChAT- and GFP-positive structures. Blue- DAPI staining to label the cellular layers. There is a widespread distribution of cholinergic fibers throughout the hippocampus. Higher density of staining can be observed adjoining the CA3 pyramidal and granule cell layers. Scale Bar- 200 μm B. Higher magnification images of the CA3 region. Green- Anti-GFP Ab staining. Blue- Nissl staining. Left panel shows the fiber distribution in the stratum lucidum (SL). A cholinergic interneuron is visible in this image. Right panel; the pyramidal cell layer showing the highly intercalated, punctate cholinergic fibers suggesting the existence of somatic synapses (SO - stratum oriens). Scale Bar. 20 μm C. Left panel: The dentate granule cell layer (GCL) showing a similar diffuse arrangement of cholinergic fibers in the cellular layer. Right panel: The hilus (H) and the molecular layer (ML) showing relatively high expression of fibers. Scale Bar. 20 μm

Figure 6. Imaging calcium changes in CA3 pyramidal neurons to electrical stimuli

A. Left panel: Pseudocolor image of fura 2 stained CA 3 pyramidal neurons (Red) from an acute hippocampal slice. Middle panel: GFP fluorescence (Green) from the same field. Note the highly branched nature of the processes. Right panel: A merged image showing the fibers and the fura 2 loaded pyramidal neurons. B. Same merged image as in A. showing the position of the stimulating pipette and cells that responded to the stimulus (yellow) and some of the neurons that did not respond (white). C. Stimulus evoked calcium transients from the responding neurons (Left traces) and from neurons that did not respond (Right traces). Responding neurons showed rapid calcium transients in response to axonal stimulation. D. Two panels (under 20 × magnification) showing the position of the stimulation pipette placedeither in the stratum oriens (Left panel) or in stratum lucidum (Middle panel). Red arrows indicate positions of the responding pyramidal neurons. Right panel: Data showing the distance (in μm) of neurons from the stimulating pipette. Y-axis represents the responders (1) and non-responders (0). Of a total of 146 neurons analyzed from the two fields, only six responded, and these were scattered across the field. Two of the responding neurons had identical distances from the pipette.

Figure 7. Properties of stimulus evoked calcium transients in CA3 pyramidal neurons

A. Calcium transients, from two cells (Left and Right panel), evoked by single 100 μs pulses given 10 s apart. While a single stimulus gave a rapid time locked (within 1 s) signal, there were distinct failures and the signals were also accompanied by spontaneous calcium oscillations. B. Left panel: Calcium transients averaged from 21 neurons (bars show SEM), in response to 100 pulses given at 100 Hz. The stimulus paradigm reliably elicited time locked signals. Right panel: The calcium transients were rapid and decayed in a biphasic manner with τ_fast of 1.7 s and a τ_slow of 10 s. C. The small numbers of responding neurons were not due to lack of nAChRs in the others. Top pair; response from a pyramidal neuron to an electrical stimulus and to a 5 s application of 5 μM nicotine. Bottom pair: a different neuron, not responding to an electrical stimulus but nonetheless showing a robust transient in response to nicotine application. D. Left panel- Response of a single neuron in the absence (Black), or presence (Red) of 200 μM mecamylamine. There was a significant attenuation of the response in the presence of the general nAChR antagonist. Right panel- Compilation of mecamylamine inhibition. Normalized data from 19 cells-Control (open bars), mecamylamine (hatched bars). The responses were integrated from onset to 5 s (Fast), from 5 s to 40 s (Slow ). All components were significantly blocked by the antagonist (***- p< 0.001).

Figure 8. Synaptic EPSCs evoked by stimulation of cholinergic fibers

A. Left: Evoked EPSCs on CA3 pyramidal neurons. A series of 10 responses to focal stimulation (100 μs). Right: Amplitude distribution of 236 individual EPSCs. B. Left trace: Calcium signals from individual excitation wavelengths (340 nm & 380 nm) showing changes in the opposite direction. Middle trace: Change in Ratio from the same pyramidal neuron. Right trace: the same neuron under voltage clamp reveals a large and fast EPSC in response to a single 100 μs pulse at 80 μA intensity. C. Position specificity of the evoked responses. An evoked response was elicited by placing the stimulation pipette on a cholinergic axon (position 1 in the trace). The stimulation pipette was then moved from its original place to a position approximately equi-distant from the recorded neuron (position 2 in the trace). The response was abolished. Returning the pipette to its original position (position 3 in the trace) restored the current.

Figure 9. Pharmacology of the evoked responses

A. Evoked responses from a CA3 pyramidal neuron and a hilar interneuron to a single 100 μs stimulus. Stim- the first stimulated response. +MLA- stimulation immediately following a 20 s application of 50 nM MLA using a puffer pipette aimed at the cell soma. Wash–responses after 1-2 minutes of washout. B. Inhibition by MLA in single neurons. In both pyramidal neurons (n = 4) and interneurons (n = 5) MLA completely attenuated the responses. In two neurons from each cell type, partial to complete recovery was observed upon washout of the antagonist. C. Left traces: Responses from a pyramidal neuron in the absence (Control) and the presence (+ PNU) of 10 μM PNU 120596. The neuron was pre-incubated with the drug for 15 minutes. PNU significantly increased the amplitude of the responses. Right traces: Normalized responses from the two conditions showing no significant changes in the decay kinetics.