Effects of sensory learning on intracortical synaptic transmission in the barrel cortex of mice

Pairing tactile stimulation of a row of whiskers with a tail shock results in an expansion of the functional representation of the stimulated whiskers within the primary somatosensory cortex of mice. Using the same paradigm, the present study examined field potentials evoked in ex vivo slices of the barrel cortex. The amplitude of responses, evoked by single and repetitive stimuli in layer IV-layer II/III pathway contained within the barrel column corresponding to the whisker stimulated during training, was unchanged. In contrast, in a transcolumnar pathway from the "trained" barrel to layer II/III of the neighboring, "untrained" column, the amplitude of responses was reduced and responses to trains of stimuli applied at 40 Hz, but not at lower frequencies, depressed faster. These data are suggestive of a selective weakening of excitatory transmission and/or enhancement of inhibitory transmission in transcolumnar pathways, which accompany associative learning-induced cortical plasticity.     

Excitatory signal flow and connectivity in a cortical column: focus on barrel cortex

A basic feature of the neocortex is its organization in functional, vertically oriented columns, recurring modules of signal processing and a system of transcolumnar long-range horizontal connections. These columns, together with their network of neurons, present in all sensory cortices, are the cellular substrate for sensory perception in the brain. Cortical columns contain thousands of neurons and span all cortical layers. They receive input from other cortical areas and subcortical brain regions and in turn their neurons provide output to various areas of the brain. The modular concept presumes that the neuronal network in a cortical column performs basic signal transformations, which are then integrated with the activity in other networks and more extended brain areas. To understand how sensory signals from the periphery are transformed into electrical activity in the neocortex it is essential to elucidate the spatial-temporal dynamics of cortical signal processing and the underlying neuronal ‘microcircuits’. In the last decade the ‘barrel’ field in the rodent somatosensory cortex, which processes sensory information arriving from the mysticial vibrissae, has become a quite attractive model system because here the columnar structure is clearly visible. In the neocortex and in particular the barrel cortex, numerous neuronal connections within or between cortical layers have been studied both at the functional and structural level. Besides similarities, clear differences with respect to both physiology and morphology of synaptic transmission and connectivity were found. It is therefore necessary to investigate each neuronal connection individually, in order to develop a realistic model of neuronal connectivity and organization of a cortical column. This review attempts to summarize recent advances in the study of individual microcircuits and their functional relevance within the framework of a cortical column, with emphasis on excitatory signal flow.     

Local circuits targeting parvalbumin-containing interneurons in layer IV of rat barrel cortex

Interactions between inhibitory interneurons and excitatory spiny neurons and also other inhibitory cells represent fundamental network properties which cause the so-called thalamo-cortical response transformation and account for the well-known receptive field differences of cortical layer IV versus thalamic neurons. We investigated the currently largely unknown morphological basis of these interactions utilizing acute slice preparations of barrel cortex in P19-21 rats. Layer IV spiny (spiny stellate, star pyramidal and pyramidal) neurons or inhibitory (basket and bitufted) interneurons were electrophysiologically characterized and intracellularly biocytin-labeled. In the same slice, we stained parvalbumin-immunoreactive (PV-ir) interneurons as putative target cells after which the tissue was subjected to confocal image acquisition. Parallel experiments confirmed the existence of synaptic contacts in these types of connection by correlated light and electron microscopy. The axons of the filled neurons differentially targeted barrel PV-ir interneurons: (1) The relative number of all contacted PV-ir cells within the axonal sphere was 5–17% for spiny (n = 10), 32 and 58% for basket (n = 2) and 12 and 13% for bitufted (n = 2) cells. (2) The preferential subcellular site which was contacted on PV-ir target cells was somatic for four and dendritic for five spiny cells; for basket cells, there was a somatic and for bitufted cells a dendritic preference in each examined case. (3) The highest number of contacts on a single PV-ir cell was 9 (4 somatic and 5 dendritic) for spiny neurons, 15 (10 somatic and 5 dendritic) for basket cells and 4 (1 somatic and 3 dendritic) for bitufted cells. These patterns suggest a cell type-dependent communication within layer IV microcircuits in which PV-ir interneurons provide not only feed-forward but also feedback inhibition thus triggering the thalamo-cortical response transformation.     

Cortical columnar processing in the rat whisker-to-barrel system.

Controlled whisker stimulation and single-unit recordings were used to elucidate response transformations that occur during the processing of tactile information from ventral posterior medial thalamus (VPM) through cortical columns in the rat whisker/barrel cortex. Whiskers were either deflected alone, using punctate ramp-and-hold stimuli, or in combination with a random noise vibration applied simultaneously to two or more neighboring whiskers. Quantitative data were obtained from five anatomically defined groups of neurons based on their being located in: VPM, layer IV barrels, layer IV septa, supragranular laminae, and infragranular laminae. Neurons in each of these populations displayed characteristic properties related to their response latency and time course, relative magnitudes of responses evoked by stimulus onset versus offset, strength of excitatory responses evoked by the noise stimulus, and/or the degree to which the noise stimulus, when applied to neighboring whiskers, suppressed or facilitated responses evoked by the columnar whisker. Results indicate that within layer IV itself there are at least two anatomically distinct networks, barrel and septum, that independently process afferent information, transforming thalamic input in similar but quantitatively distinguishable ways. Transformed signals are passed on to circuits in supragranular and infragranular laminae. In the case of supragranular neurons, evidence suggests that circuits there function in a qualitatively different fashion from those in layer IV, diminishing response differentials between weak and strong inputs, rather than enhancing them. Compared to layer IV, the greater heterogeneity of receptive field properties in nongranular layers suggests the existence of multiple, operationally distinct local circuits in the output layers of the cortical column.     

Effects of Early Sensory Experience on the Formation of the Functional Properties of Neurons in the Whisker Projection Area in the Rat Neocortex

The effects of specific afferentation on the formation of the functional properties of neurons in the projection representation of the whiskers in the neocortex were studied. Vibrotactile sensitivity was restricted by daily clipping of the whiskers during the first three weeks of life. The qualitative and quantitative characteristics of the responses of neurons in the “lemniscal” layers (IV and Vb) and the “paralemniscal” layer (Va) to stimulation of the whiskers in young (27–40 days) and older (41–57 days) were studied. These experiments showed that 1) excessive excitatory responses and deficient inhibition were produced as a direct consequence of restricting the specific sensory input in young animals; 2) atypical responses were present in the “lemniscal” and “paralemniscal” layers in vibrissectomized animals, which may result from impairment of the distribution of thalamic afferents to the neocortex; 3) there was excessive inhibition in older vibrissectomized animals, i.e., actualization of inhibitory processes was a delayed consequence of vibrissectomy.     

Whisker trimming begun at birth or on postnatal day 12 affects excitatory and inhibitory receptive fields of layer IV barrel neurons

In rats, whisker trimming during development leads to persistent alterations in the function of cortical barrel circuits and to behavioral deficits later in life. Here we examined how whisker trimming begun either at birth (P0) or on postnatal day 12 (P12), around the onset of whisking behavior, affects receptive fields of layer IV barrel neurons. All whiskers on the left face were trimmed for 40-45 days and then allowed to regrow fully. Extracellular single-unit recordings and controlled deflections of principal and adjacent whiskers (PW and AW, respectively), individually or in paired combinations, were used to assess excitatory and suppressive effects of neighboring whiskers on barrel neurons. Results indicate that whisker trimming both from P0 and P12 leads to enlarged excitatory and weakened inhibitory receptive fields in layer IV neurons. PW- and AW-evoked responses are larger in magnitude in trimmed than in control animals; AW-evoked responses are disproportionately affected, decreasing the spatial focus of barrel neurons. Deprivation after P12 accounts for approximately 50% of the total effect observed in P0 trimmed animals. Suppressive interactions, evoked by two whiskers deflected in succession, are weaker in trimmed than in control animals. Suppressive caudal/rostral and ventral/dorsal gradients, however, seem unaffected by sensory deprivation. Thus the developmental period during which experience persistently modifies maturing barrel circuitry extends up to and likely beyond the onset of whisking behavior. Sensory deprivation during this time affects development of both excitatory and inhibitory receptive fields of barrel neurons and likely impairs cortical integration of sensory information from multiple whiskers.     

Microstructure of the neocortex: comparative aspects

The appearance of the neocortex, its expansion, and its differentiation in mammals, represents one of the principal episodes in the evolution of the vertebrate brain. One of the fundamental questions in neuroscience is what is special about the neocortex of humans and how does it differ from that of other species? It is clear that distinct cortical areas show important differences within both the same and different species, and this has led to some researchers emphasizing the similarities whereas others focus on the differences. In general, despite of the large number of different elements that contribute to neocortical circuits, it is thought that neocortical neurons are organized into multiple, small repeating microcircuits, based around pyramidal cells and their input-output connections. These inputs originate from extrinsic afferent systems, excitatory glutamatergic spiny cells (which include other pyramidal cells and spiny stellate cells), and inhibitory GABAergic interneurons. The problem is that the neuronal elements that make up the basic microcircuit are differentiated into subtypes, some of which are lacking or highly modified in different cortical areas or species. Furthermore, the number of neurons contained in a discrete vertical cylinder of cortical tissue varies across species. Additionally, it has been shown that the neuropil in different cortical areas of the human, rat and mouse has a characteristic layer specific synaptology. These variations most likely reflect functional differences in the specific cortical circuits. The laminar specific similarities between cortical areas and between species, with respect to the percentage, length and density of excitatory and inhibitory synapses, and to the number of synapses per neuron, might be considered as the basic cortical building bricks. In turn, the differences probably indicate the evolutionary adaptation of excitatory and inhibitory circuits to particular functions.     

Inter- and intra-laminar connections of pyramidal cells in the neocortex

The flow of excitation through cortical columns has long since been predicted by studying the axonal projection patterns of excitatory neurones situated within different laminae. In grossly simplified terms and assuming random connectivity, such studies predict that input from the thalamus terminates primarily in layer 4, is relayed 'forward' to layer 3, then to layers 5 and 6 from where the modified signal may exit the cortex. Projection patterns also indicate 'back' projections from layer 5 to 3 and layer 6 to 4. More recently it has become clear that the interconnections between these layers are not random; forward projections primarily contact specific pyramidal subclasses and intracortical back projections innervate interneurones. This indicates that presynaptic axons or postsynaptic dendrites are capable of selecting their synaptic partners and that this selectivity is layer dependent. For the past decade, we and others have studied pyramidal cell targeting in circuits both within, and between laminae using paired intracellular recordings with biocytin filling and have begun to identify further levels of selectivity through the preferential targeting of electrophysiologically and/or morphologically distinct pyramidal subtypes. Presented here, therefore, is a brief overview of current thinking on the layer and subclass specific connectivity of neocortical principle excitatory cells.     

Microcircuits of excitatory and inhibitory neurons in layer 2/3 of mouse barrel cortex

Synaptic interactions between nearby excitatory and inhibitory neurons in the neocortex are thought to play fundamental roles in sensory processing. Here, we have combined optogenetic stimulation, whole cell recordings, and computational modeling to define key functional microcircuits within layer 2/3 of mouse primary somatosensory barrel cortex. In vitro optogenetic stimulation of excitatory layer 2/3 neurons expressing channelrhodopsin-2 evoked a rapid sequence of excitation followed by inhibition. Fast-spiking (FS) GABAergic neurons received large-amplitude, fast-rising depolarizing postsynaptic potentials, often driving action potentials. In contrast, the same optogenetic stimulus evoked small-amplitude, subthreshold postsynaptic potentials in excitatory and non-fast-spiking (NFS) GABAergic neurons. To understand the synaptic mechanisms underlying this network activity, we investigated unitary synaptic connectivity through multiple simultaneous whole cell recordings. FS GABAergic neurons received unitary excitatory postsynaptic potentials with higher probability, larger amplitudes, and faster kinetics compared with NFS GABAergic neurons and other excitatory neurons. Both FS and NFS GABAergic neurons evoked robust inhibition on postsynaptic layer 2/3 neurons. A simple computational model based on the experimentally determined electrophysiological properties of the different classes of layer 2/3 neurons and their unitary synaptic connectivity accounted for key aspects of the network activity evoked by optogenetic stimulation, including the strong recruitment of FS GABAergic neurons acting to suppress firing of excitatory neurons. We conclude that FS GABAergic neurons play an important role in neocortical microcircuit function through their strong local synaptic connectivity, which might contribute to driving sparse coding in excitatory layer 2/3 neurons of mouse barrel cortex in vivo.     

Geometric and functional organization of cortical circuits

Can neuronal morphology predict functional synaptic circuits? In the rat barrel cortex, 'barrels' and 'septa' delineate an orderly matrix of cortical columns. Using quantitative laser scanning photostimulation we measured the strength of excitatory projections from layer 4 (L4) and L5A to L2/3 pyramidal cells in barrel- and septum-related columns. From morphological reconstructions of excitatory neurons we computed the geometric circuit predicted by axodendritic overlap. Within most individual projections, functional inputs were predicted by geometry and a single scale factor, the synaptic strength per potential synapse. This factor, however, varied between projections and, in one case, even within a projection, up to 20-fold. Relationships between geometric overlap and synaptic strength thus depend on the laminar and columnar locations of both the pre- and postsynaptic neurons, even for neurons of the same type. A large plasticity potential appears to be incorporated into these circuits, allowing for functional 'tuning' with fixed axonal and dendritic arbor geometry.     

Modified sensory processing in the barrel cortex of the adult mouse after chronic whisker stimulation

Chronic stimulation of a mystacial whisker follicle for 24 h induces structural and functional changes in layer IV of the corresponding barrel, with an insertion of new inhibitory synapses on spines and a depression of neuronal responses to the stimulated whisker. Under urethane anesthesia, we analyzed how sensory responses of single units are affected in layer IV and layers II & III of the stimulated barrel column as well as in adjacent columns. In the stimulated column, spatiotemporal characteristics of the activation evoked by the stimulated whisker are not altered, although spontaneous activity and response magnitude to the stimulated whisker are decreased. The sensitivity of neurons for the deflection of this whisker is not altered but the dynamic range of the response is reduced as tested by varying the amplitude and repetition rate of the deflection. Responses to deflection of nonstimulated whiskers remain unaltered with the exception of in-row whisker responses that are depressed in the column corresponding to the stimulated whisker. In adjacent nonstimulated columns, neuronal activity remains unaltered except for a diminished response of units in layer II/III to deflection of the stimulated whisker. From these results we propose that an increased inhibition within the stimulated barrel reduced the magnitude of its excitatory output and accordingly the flow of excitation toward layers II & III and the subsequent spread into adjacent columns. In addition, the period of uncorrelated activity between pathways from the stimulated and nonstimulated whiskers weakens synaptic inputs from in-row whiskers in the stimulated barrel column.     

REORGANIZATION OF BARREL CIRCUITS LEADS TO THALAMICALLY-EVOKED CORTICAL EPILEPTIFORM ACTIVITY

We studied circuit activities in layer IV of rat somatosensory barrel cortex containing microgyri induced by neonatal freeze lesions. Structural abnormalities in GABAergic interneurons are present in the epileptogenic paramicrogyral area (PMG) and we therefore tested the hypothesis that decreased postsynaptic inhibition within barrel microcircuits occurs in the PMG and contributes to epileptogenesis when thalamocortical afferents are activated. In thalamocortical (TC) slices from na?ve animals, single electrical stimuli within the thalamic ventrobasal (VB) nucleus evoked transient cortical multi-unit activity lasting 65±42 ms. Similar stimuli in TC slices from lesioned barrel cortex elicited prolonged 850 ±100 ms paroxysmal discharges that originated in the PMG and propagated laterally over several mm. Paroxysmal discharges were shortened in duration by ~70 % when APV was applied, and were totally abolished by CNQX. The cortical paroxysmal discharges did not evoke thalamic oscillations. Whole cell patch clamp recordings showed that there was a shift in the balance of TC evoked responses in the PMG that favored excitation over inhibition. Dual whole-cell recordings in layer IV of the PMG indicated that there was selective loss of inhibition from fast-spiking interneurons to spiny neurons in the barrel circuits that likely contributed to unconstrained cortical recurrent excitation with generation and spread of paroxysmal discharges.     

The columnar and laminar organization of inhibitory connections to neocortical excitatory cells

The cytoarchitectonic similarities of different neocortical regions have given rise to the idea of 'canonical' connectivity between excitatory neurons of different layers within a column. It is unclear whether similarly general organizational principles also exist for inhibitory neocortical circuits. Here we delineate and compare local inhibitory-to-excitatory wiring patterns in all principal layers of primary motor (M1), somatosensory (S1) and visual (V1) cortex, using genetically targeted photostimulation in a mouse knock-in line that conditionally expresses channelrhodopsin-2 in GABAergic neurons. Inhibitory inputs to excitatory neurons derived largely from the same cortical layer within a three-column diameter. However, subsets of pyramidal cells in layers 2/3 and 5B received extensive translaminar inhibition. These neurons were prominent in V1, where they might correspond to complex cells, less numerous in barrel cortex and absent in M1. Although inhibitory connection patterns were stereotypical, the abundance of individual motifs varied between regions and cells, potentially reflecting functional specializations.     

Anatomical correlates of representational map reorganization induced by partial vibrissectomy in the barrel cortex of adult mice.

We examined the potential for changes in cortical connectivity to accompany long-term plastic changes in functional cortical representations of mystacial vibrissae. Plasticity in the barrel cortex of young adult mice was evoked by vibrissectomy that spared row C of whiskers. We found that 2-deoxyglucose brain mapping causes a progressive expansion of cortical representation of the spared vibrissae. Two months after vibrissectomy, when the width of the cortical map of the spared row of vibrissae doubled, living cortical slices of the barrel cortex were injected with fluorescent dextrans. The injections were centered on spared, deprived and control vibrissal columns. The injections labeled three intracortical projection systems: (i) local connections from one vibrissal column to neighboring columns; (ii) long-range projections running in the septa and walls of the barrels and spanning several barrels; and (iii) very-long-range fibers running horizontally in the lower part of layer V. The local, short-range projection system was analysed following small injections into the centers of columns in layers III and IV. We found that injections into spared barrels labeled axons extending for significantly greater distances in all layers (except layer V), and labeled cell bodies situated significantly further, than after injections into deprived or control barrels. Also, the total axonal density labeled by injections into the spared barrel was higher by 70% than for the deprived or control barrels. Alterations of topographical maps in adult somatosensory cortex may occur immediately after functional denervation, but may also increase with time, as in the case of our experimental situation. Our results indicate that persistent, long-term plastic change can remodel connectivity in the barrel cortex.     

Functional connectivity in layer IV local excitatory circuits of rat somatosensory cortex

There are two types of excitatory neurons within layer IV of rat somatosensory cortex: star pyramidal (SP) and spiny stellate cells (SS). We examined the intrinsic properties and connectivity between these neurons to determine differences in function. Eighty-four whole cell recordings of pairs of neurons were examined in slices of rat barrel cortex at 36 +/- 1 degrees C. Only minimal differences in intrinsic properties were found; however, differences in synaptic strength could clearly be shown. Connections between homonymous pairs (SS-SS or SP-SP) had a higher efficacy than heteronymous connections. This difference was mainly a result of quantal content. In 42 pairs, synaptic dynamics were examined. Sequences of action potentials (3-20 Hz) in the presynaptic neuron consistently caused synaptic depression (E2/E1=0.53+/-0.18). The dominant component of depression was release-independent; this depression occurred even when preceding action potentials had failed to cause a response. The release-dependence of depression was target specific; in addition, release-independence was greater for postsynaptic SPs. In a subset of connections formed only between SP and any other cell type (43%), synaptic efficacy was dependent on the presynaptic membrane potential (Vm); at -55 mV, the connections were almost silent, whereas at -85 mV, transmission was very reliable. We suggest that, within layer IV, there is stronger efficacy between homonymous than between heteronymous excitatory connections. Under dynamic conditions, the functional connectivity is shaped by synaptic efficacy at individual connections, by Vm, and by the specificity in the types of synaptic depression.     

Intracortical pathways mediate nonlinear fast oscillation (>200 Hz) interactions within rat barrel cortex

Whisker evoked fast oscillations (FOs; >200 Hz) within the rodent posteromedial barrel subfield are thought to reflect very rapid integration of multiwhisker stimuli, yet the pathways mediating FO interactions remain unclear and may involve interactions within thalamus and/or cortex. In the present study using anesthetized rats, a cortical incision was made between sites representing the stimulated whiskers to determine how intracortical networks contributed to patterns of FOs. With cortex intact, simultaneous stimulation of a pair of whiskers aligned in a row evoked supralinear responses between sites separated by several millimeters. In contrast, stimulation of a nonadjacent pair of whiskers within an arc evoked FOs with no evidence for nonlinear interactions. However, stimulation of an adjacent pair of whiskers in an arc did evoke supralinear responses. After a cortical cut, supralinear interactions associated with FOs within a row were lost. These data indicate a distinct bias for stronger long-range connectivity that extends along barrel rows and that horizontal intracortical pathways exclusively mediate FO-related integration of tactile information.     

Functional independence of layer IV barrels in rodent somatosensory cortex.

Layer IV of rodent primary somatosensory cortex is characterized by an array of whisker-related groups of neurons, known as "barrels." Neurons within each barrel respond best to a particular whisker on the contralateral face, and, on deflection of adjacent whiskers, display relatively weak excitation followed by strong inhibition. A prominent hypothesis for the processing of vibrissal information within layer IV is that the multiwhisker receptive fields of barrel neurons reflect interconnections among neighboring barrels. An alternative view is that the receptive field properties of barrel neurons are derived from operations performed on multiwhisker, thalamic inputs by local circuitry within each barrel, independently of neighboring barrels. Here we report that adjacent whisker-evoked excitation and inhibition within a barrel are unaffected by ablation of the corresponding adjacent barrel. In supragranular neurons, on the other hand, excitatory responses to the ablated barrel's associated whisker are substantially reduced. We conclude that the layer IV barrels function as an array of independent parallel processors, each of which individually transforms thalamic afferent input for subsequent processing by horizontally interconnected circuits in other layers.     

Primary visual cortex shows laminar‐specific and balanced circuit organization of excitatory and inhibitory synaptic connectivity

Key points

• Using functional mapping assays, we conducted a quantitative assessment of both excitatory and inhibitory synaptic laminar connections to excitatory neurons in layers 2/3–6 of the mouse visual cortex (V1).
• Laminar‐specific synaptic wiring diagrams of excitatory neurons were constructed on the basis of circuit mapping.
• The present study reveals that that excitatory and inhibitory synaptic connectivity is spatially balanced across excitatory neuronal networks in V1.

Abstract

In the mammalian neocortex, excitatory neurons provide excitation in both columnar and laminar dimensions, which is modulated further by inhibitory neurons. However, our understanding of intracortical excitatory and inhibitory synaptic inputs in relation to principal excitatory neurons remains incomplete, and it is unclear how local excitatory and inhibitory synaptic connections to excitatory neurons are spatially organized on a layer‐by‐layer basis. In the present study, we combined whole cell recordings with laser scanning photostimulation via glutamate uncaging to map excitatory and inhibitory synaptic inputs to single excitatory neurons throughout cortical layers 2/3–6 in the mouse primary visual cortex (V1). We find that synaptic input sources of excitatory neurons span the radial columns of laminar microcircuits, and excitatory neurons in different V1 laminae exhibit distinct patterns of layer‐specific organization of excitatory inputs. Remarkably, the spatial extent of inhibitory inputs of excitatory neurons for a given layer closely mirrors that of their excitatory input sources, indicating that excitatory and inhibitory synaptic connectivity is spatially balanced across excitatory neuronal networks. Strong interlaminar inhibitory inputs are found, particularly for excitatory neurons in layers 2/3 and 5. This differs from earlier studies reporting that inhibitory cortical connections to excitatory neurons are generally localized within the same cortical layer. On the basis of the functional mapping assays, we conducted a quantitative assessment of both excitatory and inhibitory synaptic laminar connections to excitatory cells at single cell resolution, establishing precise layer‐by‐layer synaptic wiring diagrams of excitatory neurons in the visual cortex.

Abbreviations

aCSF

artificial cerebrospinal fluid

DAPI

4′‐6‐diamidino‐2‐phenylindole

LSPS

laser scanning photostimulation

V1

primary visual cortex