Combining Optogenetics and Electrophysiology to Analyze Projection Neuron Circuits.

A set of methods is described for channelrhodopsin-2 (ChR2)-based synaptic circuit analysis that combines photostimulation of virally transfected presynaptic neurons' axons with whole-cell electrophysiological recordings from retrogradely labeled postsynaptic neurons. The approach exploits the preserved photoexcitability of ChR2-expressing axons in brain slices and can be used to assess either local or long-range functional connections. Stereotaxic injections are used both to express ChR2 selectively in presynaptic axons of interest (using rabies virus [RV] or adeno-associated virus [AAV]) and to label two types of postsynaptic projection neurons of interest with fluorescent retrograde tracers. In brain slices, tracer-labeled postsynaptic neurons are targeted for whole-cell electrophysiological recordings, and synaptic connections are assessed by sampling voltage or current responses to light-emitting diode (LED) photostimulation of ChR2-expressing axons. The data are analyzed to estimate the relative amplitude of synaptic input and other connectivity parameters. Pharmacological and electrophysiological manipulations extend the versatility of the basic approach, allowing the dissection of monosynaptic versus disynaptic responses, excitatory versus inhibitory responses, and more. The method enables rapid, quantitative characterization of synaptic connectivity between defined pre- and postsynaptic classes of neurons.

Reciprocal interareal connections to corticospinal neurons in mouse M1 and S2.

Primary motor (M1) and secondary somatosensory (S2) cortices, although anatomically and functionally distinct, share an intriguing cellular component: corticospinal neurons (CSP) in layer 5B. Here, we investigated the long-range circuits of CSPs in mouse forelimb-M1 and S2. We found that interareal projections (S2 → M1 and M1 → S2) monosynaptically excited pyramidal neurons across multiple layers, including CSPs. Area-specific differences were observed in the relative strengths of inputs to subsets of CSPs and other cell types, but the general patterns were similar. Furthermore, subcellular mapping of the dendritic distributions of these corticocortical excitatory synapses onto CSPs in both areas also showed similar patterns. Because layer 5B is particularly thick in M1, but not S2, we studied M1-CSPs at different cortical depths, quantifying their dendritic morphology and mapping inputs from additional cortical (M2, contralateral M1, and local layer 2/3) and thalamic (VL nucleus) sources. These results indicated that CSPs exhibit area-specific modifications on an otherwise conserved synaptic organization, and that different afferents innervate M1-CSP dendritic domains in a source-specific manner. In the cervical spinal cord, CSP axons from S2 and M1 partly converged on middle layers, but S2-CSP axons extended further dorsally, and M1-CSP axons ventrally. Thus, our findings identify many shared features in the circuits of M1 and S2 and show that these areas communicate via mutual projections that give each area monosynaptic access to the other area's CSPs. These interareally yoked CSP circuits may enable M1 and S2 to operate in a coordinated yet differentiated manner in the service of sensorimotor integration.

A genuine layer 4 in motor cortex with prototypical synaptic circuit connectivity.

The motor cortex (M1) is classically considered an agranular area, lacking a distinct layer 4 (L4). Here, we tested the idea that M1, despite lacking a cytoarchitecturally visible L4, nevertheless possesses its equivalent in the form of excitatory neurons with input-output circuits like those of the L4 neurons in sensory areas. Consistent with this idea, we found that neurons located in a thin laminar zone at the L3/5A border in the forelimb area of mouse M1 have multiple L4-like synaptic connections: excitatory input from thalamus, largely unidirectional excitatory outputs to L2/3 pyramidal neurons, and relatively weak long-range corticocortical inputs and outputs. M1-L4 neurons were electrophysiologically diverse but morphologically uniform, with pyramidal-type dendritic arbors and locally ramifying axons, including branches extending into L2/3. Our findings therefore identify pyramidal neurons in M1 with the expected prototypical circuit properties of excitatory L4 neurons, and question the traditional assumption that motor cortex lacks this layer.

Synaptic microcircuit dysfunction in genetic models of neurodevelopmental disorders: focus on Mecp2 and Met.

Recent findings in the genetics of neurodevelopmental syndromes have ushered in an exciting era of discovery in which substrates of neurologic dysfunction are being identified at the synaptic and microcircuit levels in mouse models of these disorders. We review recent progress in this area, focusing on two examples of mouse models of autism spectrum disorders (ASDs): Mecp2 models of Rett syndrome, and a Met-knockout model of non-syndromic forms of autism. In both cases, a dominant theme is changes in synaptic strength, associated with hyper-connectivity or hypo-connectivity in specific microcircuits. Alterations in intrinsic neuronal excitability are also found, but do not appear to be as common. The microcircuit-specific nature of synaptic changes observed in these ASD models indicates that it will be necessary to define mechanisms of circuit dysfunction on a case-by-case basis, not only in neocortex but also in brainstem and other sub-cortical areas. Thus, functional microcircuit analysis is emerging as an important line of investigation, highly complementary to neurogenetic and molecular strategies, and holds promise for generating models of the underlying pathophysiology and for guiding development of novel therapeutic strategies.

Circuit-specific intracortical hyperconnectivity in mice with deletion of the autism-associated Met receptor tyrosine kinase.

Local hyperconnectivity in the neocortex is a hypothesized pathophysiological state in autism spectrum disorder (ASD). MET, a receptor tyrosine kinase that regulates dendrite and spine morphogenesis, has been established as a risk gene for ASD. Here, we analyzed the synaptic circuit organization of identified pyramidal neurons in the anterior frontal cortex of mice with a dorsal pallium-derived, conditional knock-out (cKO) of Met. Synaptic mapping by glutamate uncaging identified layer 2/3 as the main source of local excitatory input to layer 5 projection neurons in controls. In both cKO and heterozygotes, this pathway was stronger by a factor of approximately 2. This increase was both sublayer and projection-class specific, restricted to corticostriatal neurons in upper layer 5B and not neighboring corticopontine neurons. Paired recordings in cKO slices demonstrated increased unitary connectivity. We propose that excitatory hyperconnectivity in specific neocortical microcircuits constitutes a physiological basis for Met-mediated ASD risk.