Optogenetic Release of ACh Induces Rhythmic Bursts of Perisomatic IPSCs in Hippocampus

Acetylcholine (ACh) influences a vast array of phenomena in cortical systems. It alters many ionic conductances and neuronal firing behavior, often by regulating membrane potential oscillations in populations of cells. Synaptic inhibition has crucial roles in many forms of oscillation, and cholinergic mechanisms regulate both oscillations and synaptic inhibition. In vitro investigations using bath-application of cholinergic receptor agonists, or bulk tissue electrical stimulation to release endogenous ACh, have led to insights into cholinergic function, but questions remain because of the relative lack of selectivity of these forms of stimulation. To investigate the effects of selective release of ACh on interneurons and oscillations, we used an optogenetic approach in which the light-sensitive non-selective cation channel, Channelrhodopsin2 (ChR2), was virally delivered to cholinergic projection neurons in the medial septum/diagonal band of Broca (MS/DBB) of adult mice expressing Cre-recombinase under the control of the choline-acetyltransferase (ChAT) promoter. Acute hippocampal slices obtained from these animals weeks later revealed ChR2 expression in cholinergic axons. Brief trains of blue light pulses delivered to untreated slices initiated bursts of ACh-evoked, inhibitory post-synaptic currents (L-IPSCs) in CA1 pyramidal cells that lasted for 10's of seconds after the light stimulation ceased. L-IPSC occurred more reliably in slices treated with eserine and a very low concentration of 4-AP, which were therefore used in most experiments. The rhythmic, L-IPSCs were driven primarily by muscarinic ACh receptors (mAChRs), and could be suppressed by endocannabinoid release from pyramidal cells. Finally, low-frequency oscillations (LFOs) of local field potentials (LFPs) were significantly cross-correlated with the L-IPSCs, and reversal of the LFPs near s. pyramidale confirmed that the LFPs were driven by perisomatic inhibition. This optogenetic approach may be a useful complementary technique in future investigations of endogenous ACh effects

Abnormal Development of the Earliest Cortical Circuits in a Mouse Model of Autism Spectrum Disorder

Autism spectrum disorder (ASD) involves deficits in speech and sound processing. Cortical circuit changes during early development likely contribute to such deficits. Subplate neurons (SPNs) form the earliest cortical microcircuits and are required for normal development of thalamocortical and intracortical circuits. Prenatal valproic acid (VPA) increases ASD risk, especially when present during a critical time window coinciding with SPN genesis. Using optical circuit mapping in mouse auditory cortex, we find that VPA exposure on E12 altered the functional excitatory and inhibitory connectivity of SPNs. Circuit changes manifested as “patches” of mostly increased connection probability or strength in the first postnatal week and as general hyper-connectivity after P10, shortly after ear opening. These results suggest that prenatal VPA exposure severely affects the developmental trajectory of cortical circuits and that sensory-driven activity may exacerbate earlier, subtle connectivity deficits. Our findings identify the subplate as a possible common pathophysiological substrate of deficits in ASD

L- IPSCs are triggered primarily by mAChRs and reduced by endocannabinoids.

<p>A) Repeated light trains (5 Hz, 10 s, 5-ms pulses) elicited IPSC bursts (A1) that were nearly abolished by atropine (A2). A3) Group data of IPSCs in basal (Pre) conditions, and after light stimulation (Post). Values of amplitudes and frequency for all IPSCs (left and middle graphs), and frequency of large IPSCs (i.e., two s.d.s > the mean basal IPSC; right graph). B1) In atropine, light trains (10 Hz, 0.5 s) elicited small IPSC bursts (5 traces, mean in red) that were reduced by the non-selective nAChR antagonist, mecamylamine, 10 µM (B2, 2 traces, different cell than A1, A2). C1) L-IPSCs recorded in a CA1 pyramidal cell elicited by a 5 Hz, 10-s train of light pulses. DSI was induced by a 3-s voltage step (to +20 mV). C2) Control trials show IPSC bursts in the absence of DSI. C3, C4) DSI of L-IPSCs was blocked by the CB1R antagonist, AM251, 5 µM (different cell than C1, C2). C5) Group data (n = 4) of DSI in control conditions and after bath application of AM251; ^*  =  p<0.05; ^**  =  p<0.01.</p

ChR2- mCherry expression in cholinergic MS/DBB projection neurons and light-induced IPSCs in CA1 pyramidal cells.

<p>A) Tissue section through the MS/DBB showing ChR2-mCherry, immunostaining for ChAT, and the merged image. B) Section of the hippocampus showing ChR2-mCherry expression in axons. sr  =  <i>s. radiatum</i>; sp  =  <i>s. pyramidale</i>; so  =  <i>s. oriens</i>. C) Schematic drawing of the recording arrangement. D1) Continuous recording from a CA1 pyramidal cell in a ChR2-expressing slice. Repeated 10-s trains of 5-ms blue light flashes repeated at 2-min intervals (squares) elicited bursts of L-IPSCs (downward deflections). A single light pulse (downward triangle) had no obvious effect. The small upward deflections (truncated in the illustration) are capacitive transients produced by conductance pulses given to the cell. D2) Two trials (1 and 3 in D1) in the absence of iGluR antagonists were aligned at the time of the light train and overlapped. D3) Two trials (4 and 5 in D1) in the presence of iGluR antagonists, 5 µM NBQX plus 5 µM CGP37849, were aligned and overlapped. An expanded portion of the trace (below) reveals the occurrence of rhythmic L-IPSCs after the end of the light train. (The traces in D2 and D3 are shown at a larger amplitude scale than in D1, and the largest IPSCs are cut off.) The autocorrelation function (D4, left) and power spectrum (D4, right) illustrate the regularity (peak frequency ∼3 Hz) of L-IPSC activity from this cell.</p

L- IPSCs oscillate rhythmically at low frequency in eserine and 4-AP.

<p>Results from a typical pyramidal cell recorded in the presence of the cholinesterase inhibitor, eserine, 1 µM, and a low concentration of the voltage-gated K⁺ channel blocker 4-AP, 20 µM, in the extracellular solution. A train (5-ms light pulses, 5 Hz, 5 s) was delivered during the horizontal blue line. A) After a delay of several seconds a burst of large L-IPSCs began and persisted for >1 min. The IPSCs occurred rhythmically, with a peak frequency of ∼3 Hz (power spectrum in B1, autocorrelogram in B2). Eserine and 4-AP were present in all subsequent experiments.</p

ACh-release-induced rhythmic LFPs are well correlated with L- IPSCs.

<p>A1) Extracellular recording in <i>s. pyramidale.</i> Light trains (5 Hz, 10 s) elicited bursts of LFPs. A2) Group data showing increase in total LFP power obtained by integrating the spectral analysis over the range of 2–12 Hz, before and after atropine application (n = 5, *p<0.05). B) As in (A) except that two extracellular electrodes were placed ∼200 µm apart. The plot shows that the two L-LFPs were temporally cross-correlated. C) Simultaneous recordings of L-IPSCs and LFPs recorded ∼200 µm away. C1) Cross-correlation plot indicates L-IPSC peak occurs very near the LFP peak. C2) Sample traces of simultaneous L-IPSC, L-LFP recordings. D) Sink-source analysis of L-LFPs. D1) Dots indicate recording locations. D2) Two extracellular electrodes recorded L-LFPs at locations 1 – 5; traces show LFP cross-correlations versus the simultaneous LFP recorded in <i>s. pyramidale</i> (4). The records suggest an LFP current source near 4.</p