Gordon M. G. Shepherd

Transient and Persistent Dendritic Spines in the Neocortex In Vivo

Dendritic spines were imaged over days to months in the apical tufts of neocortical pyramidal neurons (layers 5 and 2/3) in vivo. A fraction of thin spines appeared and disappeared over a few days, while most thick spines persisted for months. In the somatosensory cortex, from postnatal day (PND) 16 to PND 25 spine retractions exceeded additions, resulting in a net loss of spines. The fraction of persistent spines (lifetime > or = 8 days) grew gradually during development and into adulthood (PND 16-25, 35%; PND 35-80, 54%; PND 80-120, 66%; PND 175-225, 73%), providing evidence that synaptic circuits continue to stabilize even in the adult brain, long after the closure of known critical periods. In 6-month-old mice, spines turn over more slowly in visual compared to somatosensory cortex, possibly reflecting differences in the capacity for experience-dependent plasticity in these brain regions.

Circuit analysis of experience-dependent plasticity in the developing rat barrel cortex

Sensory deprivation during a critical period reduces spine motility and disrupts receptive field structure of layer 2/3 neurons in rat barrel cortex. To determine the locus of plasticity, we used laser scanning photostimulation, allowing us to rapidly map intracortical synaptic connectivity in brain slices. Layer 2/3 neurons differed in their spatial distributions of presynaptic partners: neurons directly above barrels received, on average, significantly more layer 4 input than those above the septa separating barrels. Complementary connectivity was found in deprived cortex: neurons above septa were now strongly coupled to septal regions, while connectivity between barrel regions and layer 2/3 was reduced. These results reveal competitive interactions between barrel and septal circuits in the establishment of precise intracortical circuits.

Corticostriatal connectivity and its role in disease

Corticostriatal projections are essential components of forebrain circuits and are widely involved in motivated behaviour. These axonal projections are formed by two distinct classes of cortical neurons, intratelencephalic (IT) and pyramidal tract (PT) neurons. Convergent evidence points to IT versus PT differentiation of the corticostriatal system at all levels of functional organization, from cellular signalling mechanisms to circuit topology. There is also growing evidence for IT/PT imbalance as an aetiological factor in neurodevelopmental, neuropsychiatric and movement disorders — autism, amyotrophic lateral sclerosis, obsessive-compulsive disorder, schizophrenia, Huntingtons and Parkinsons diseases and major depression are highlighted here.

Topographic mapping of VMH → arcuate nucleus microcircuits and their reorganization by fasting

In the hypothalamic arcuate nucleus (ARC), pro-opiomelanocortin (POMC) neurons inhibit feeding and neuropeptide-Y (NPY) neurons stimulate feeding. We tested whether neurons in the ventromedial hypothalamic nucleus (VMH), a known satiety center, activate anorexigenic neuronal pathways in the ARC by projecting either excitatory synaptic inputs to POMC neurons and/or inhibitory inputs to NPY neurons. Using laser scanning photostimulation in brain slices from transgenic mice, we found that POMC and NPY neurons, which are interspersed in the ARC, are nevertheless regulated by anatomically distinct synaptic inputs. POMC neurons received strong excitatory input from the medial VMH (mVMH), whereas NPY neurons did not and, instead, received weak inhibitory input only from within the ARC. The strength of the excitatory input from the mVMH to POMC neurons was diminished by fasting. These data identify a new molecularly defined circuit that is dynamically regulated by nutritional state in a manner consistent with the known role of the VMH as a satiety center.

Top-down laminar organization of the excitatory network in motor cortex

Cortical layering is a hallmark of the mammalian neocortex and a major determinant of local synaptic circuit organization in sensory systems. In motor cortex, the laminar organization of cortical circuits has not been resolved, although their input-output operations are crucial for motor control. Here, we developed a general approach for estimating layer-specific connectivity in cortical circuits and applied it to mouse motor cortex. From these data we computed a laminar presynaptic → postsynaptic connectivity matrix, Wpost,pre, revealing a complement of stereotypic pathways dominated by layer 2 outflow to deeper layers. Network modeling predicted, and experiments with disinhibited slices confirmed, that stimuli targeting upper, but not lower, cortical layers effectively evoked network-wide events. Thus, in motor cortex, descending excitation from a preamplifier-like network of upper-layer neurons drives output neurons in lower layers. Our analysis provides a quantitative wiring-diagram framework for further investigation of the excitatory networks mediating cortical mechanisms of motor control.

Laminar and Columnar Organization of Ascending Excitatory Projections to Layer 2/3 Pyramidal Neurons in Rat Barrel Cortex

Excitatory synaptic projections to layer 2/3 (L2/3) pyramidal neurons in brain slices from the rat barrel cortex were measured using quantitative laser-scanning photostimulation (LSPS) mapping. In the barrel cortex, cytoarchitectonic “barrels” and “septa” in L4 define a stereotypical array of landmarks, allowing alignment and averaging of LSPS maps from multiple cells in different slices. We distinguished inputs to L2 and L3 neurons above barrels and septa. Average input maps revealed that barrel-related ascending projections (L4→2/3barrel) interdigitated with a novel septum-related projection (L5A→2septum). We also explored the functional organization of these projections by comparing the input maps of multiple cells in individual slices. L2/3 cells sharing the same barrel-related column showed strong correlations in their input maps, independent of their precise locations within the column; otherwise, correlations fell rapidly as a function of intersomatic separation. Our data indicate that barrel-related and septum-related columns are associated with distinct functional circuits. These projections are likely to mediate parallel processing of somatosensory signals within the barrel cortex, with L4→2/3barrel and L5A→2septum representing the intracortical continuations of, respectively, the subcortical lemniscal and paralemniscal systems conveying somatosensory information to the barrel cortex.

Induction of Spine Growth and Synapse Formation by Regulation of the Spine Actin Cytoskeleton

We explored the relationship between regulation of the spine actin cytoskeleton, spine morphogenesis, and synapse formation by manipulating expression of the actin binding protein NrbI and its deletion mutants. In pyramidal neurons of cultured rat hippocampal slices, NrbI is concentrated in dendritic spines by binding to the actin cytoskeleton. Expression of one NrbI deletion mutant, containing the actin binding domain, dramatically increased the density and length of dendritic spines with synapses. This hyperspinogenesis was accompanied by enhanced actin polymerization and spine motility. Synaptic strengths were reduced to compensate for extra synapses, keeping total synaptic input per neuron constant. Our data support a model in which synapse formation is promoted by actin-powered motility.

Sublayer-specific microcircuits of corticospinal and corticostriatal neurons in motor cortex

The mammalian motor system is organized around distinct subcortical subsystems, suggesting that the intracortical circuits immediately upstream of spinal cord and basal ganglia might be functionally differentiated as well. We found that the main excitatory pathway in mouse motor cortex, layer 2/3→5, is fractionated into distinct pathways targeting corticospinal and corticostriatal neurons, which are involved in motor control. However, connections were selective for neurons in certain sublayers: corticospinal neurons in upper layer 5B and corticostriatal neurons in lower 5A. A simple structural combinatorial principle accounts for this highly specific functional circuit architecture: potential connectivity is established by neuronal sublayer positioning and actual connectivity in this framework is determined by long-range axonal projection targets. Thus, intracortical circuits of these pyramidal neurons are specified not only by their long-range axonal targets or their layer or sublayer positions, but by both, in specific combinations.

Mice with behavioral evidence of tinnitus exhibit dorsal cochlear nucleus hyperactivity because of decreased GABAergic inhibition

Tinnitus has been associated with increased spontaneous and evoked activity, increased neural synchrony, and reorganization of tonotopic maps of auditory nuclei. However, the neurotransmitter systems mediating these changes are poorly understood. Here, we developed an in vitro assay that allows us to evaluate the roles of excitation and inhibition in determining the neural correlates of tinnitus. To measure the magnitude and spatial spread of evoked circuit activity, we used flavoprotein autofluorescence (FA) imaging, a metabolic indicator of neuronal activity. We measured FA responses after electrical stimulation of glutamatergic axons in slices containing the dorsal cochlear nucleus, an auditory brainstem nucleus hypothesized to be crucial in the triggering and modulation of tinnitus. FA imaging in dorsal cochlear nucleus brain slices from mice with behavioral evidence of tinnitus (tinnitus mice) revealed enhanced evoked FA response at the site of stimulation and enhanced spatial propagation of FA response to surrounding sites. Blockers of GABAergic inhibition enhanced FA response to a greater extent in control mice than in tinnitus mice. Blockers of excitation decreased FA response to a similar extent in tinnitus and control mice. These findings indicate that auditory circuits in mice with behavioral evidence of tinnitus respond to stimuli in a more robust and spatially distributed manner because of a decrease in GABAergic inhibition.

Circuit and plasticity defects in the developing somatosensory cortex of FMR1 knock-out mice

Silencing of the Fmr1 gene causes fragile X syndrome. Although defects in synaptic plasticity in the cerebral cortex have been linked to cognitive impairments in Fmr1 knock-out (ko) mice, the specific cortical circuits affected in the syndrome are unknown. Here, we investigated the development of excitatory projections in the barrel cortex of Fmr1 ko mice. In 2-week-old Fmr1 ko mice, a major ascending projection connecting layer 4 (L4) to L3 (L4→L3), was defective in multiple and independent ways: its strength was reduced, caused by a lower connection probability; the axonal arbors of L4 cells were spatially diffuse in L2/3; the L4→L3 projection did not show experience-dependent plasticity. By 3 weeks, the strength of the L4→L3 projection was similar to that of wild type. Our data indicate that Fmr1 shapes sensory cortical circuits during a developmental critical period.