Experimental microgyri disrupt the barrel field pattern in rat somatosensory cortex

Transcranial freeze lesions in neonatal rat pups produce microgyri and adjacent epileptogenic regions of neocortex that can be used to model human polymicrogyria. The hypothesis that the presence of microgyri is associated with abnormal cortical organization occurring within as well as adjacent to the microgyri was tested by creating microgyri within the face representation of somatosensory cortex. Microgyri were associated with a widespread disruption of the stereotypic whisker barrel field pattern delineated with cytochrome oxidase (CO) staining. CO-stained patches resembling barrel hollows were absent within the microgyrus, and were abnormally shaped and distributed outside of the microgyrus. Adjacent Nissl- or acetylcholinesterase-stained sections demonstrated that both cell clusters and thalamocortical afferents contributed to the abnormally organized paramicrogyral zone identified in CO-stained sections. Field potential recordings showed that this region of heavy CO staining corresponded to the epileptogenic zone adjacent to the microgyrus. Results support our hypothesis that the epileptogenic paramicrogyral zone develops an abnormal organization of cell clusters and thalamocortical projections that could contribute to epileptogenesis in the paramicrogyral zone.     

Growth of thalamic afferents into mouse barrel cortex.

We studied thalamocortical afferent (TCA) growth into somatosensory cortex as the whisker barrels emerge in postnatal mice. Ingrowing fibers from the ventrobasal (VB) thalamus were selectively labeled by two means. Under direct vision, individual axons and populations of axons were labeled in vitro with HRP, or in fixed tissue with Dil (1,1'-dioctodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), in pieces of brain containing both the source nucleus in the thalamus and its cortical target. Many simple thalamocortical afferents are already within the upper cortical plate at birth [postnatal day one (PND1)]. Initially, TCAs from each point in the thalamus distribute in the cortex as two-dimensional "Gaussians," which overlap laterally to constitute a uniform projection pattern. The projection is topographic, because adjacent focal injections within VB label adjacent cortical loci. Subsequent development of barreloids (thalamic representations of the whiskers) partitions the TCA projection into a set of whisker-related Gaussians, centered on cortical targets whose collective topography reflects that of the source pattern. After barreloids form on about PND3, but before barrels appear in cytoarchitecture on about PND5, the overlapping TCAs segregate into dense terminal clusters in layer IV, around which barrels later mature. Time series of single fibers traced with camera lucida explain this transformation that is so noticeable at the population level. As early as PND1, individual TCAs emit multiple ascending collaterals on their horizontal run through white matter and oblique ascent into upper cortex. Subsequently, by PND4, and proceeding at least through PND7, there is accelerated terminal arborization of selected appropriate collateral branches and pruning back of other inappropriate ones. The selection mechanism appears to result from within-group reinforcement events that are stronger for branches toward the center of each whisker-related Gaussian distribution.     

Cortical damping: analysis of thalamocortical response transformations in rodent barrel cortex

In the whisker-barrel system, layer IV excitatory neurons respond preferentially to high-velocity deflections of their principal whisker, and these responses are inhibited by deflections of adjacent whiskers. Thalamic input neurons are amplitude and velocity sensitive and have larger excitatory and weaker inhibitory receptive fields than cortical neurons. Computational models based on known features of barrel circuitry capture these and other differences between thalamic and cortical neuron response properties. The models' responses are highly sensitive to thalamic firing synchrony, a finding subsequently confirmed in real barrels by in vivo experiments. Here, we use dynamic systems analysis to examine how barrel circuitry attains its sensitivity to input timing, and how this sensitivity explains the transformation of receptive fields between thalamus and cortex. We find that strong inhibition renders the net effect of intracortical connections suppressive or damping, distinguishing it from previous amplifying models of cortical microcircuits. In damping circuits, recurrent excitation enhances response tuning not by amplifying responses to preferred inputs, but by enabling them to better withstand strong inhibitory influences. Dense interconnections among barrel neurons result in considerable response homogeneity. Neurons outside the barrel layer respond more heterogeneously, possibly reflecting diverse networks and multiple transformations within the cortical output layers.     

In vivo modulation of the activity of pyramidal neurons in the rat medial prefrontal cortex by 5-HT2A receptors: relationship to thalamocortical afferents

The activation of 5-HT(2A) receptors in medial prefrontal cortex (mPFC) by the hallucinogen DOI increases the firing activity of dorsal raphe (DR) 5-HT neurons and prefrontal 5-HT release. Here we show that the i.v. administration of DOI markedly affected the firing rate of identified pyramidal neurons recorded extracellularly. DOI excited (481%) 21/56 neurons, inhibited (11%) 17/56 neurons and left the rest unaffected (overall 2.4-fold increase in firing rate). Both effects were antagonized by 5-HT(2A) receptor blockade. 5-HT(2A)-mediated orthodromic excitations were recorded in pyramidal neurons projecting to DR after electrical stimulation of this nucleus. We also examined whether the effects of DOI in mPFC involve thalamic excitatory inputs. The disinhibition of the mediodorsal and centromedial nuclei of the thalamus by local bicuculline resembled the effects of DOI as it increased pyramidal cell firing and 5-HT release in mPFC. However, the selective activation of prefrontal micro -opioid and mGlu II receptors counteracted the effects of the thalamic disinhibition but not those of DOI. Moreover, extensive thalamic lesions did not alter the effect of DOI on pyramidal cell firing and 5-HT release. We conclude that DOI increases the activity of the mPFC-DR circuit by an action on postsynaptic 5-HT(2A) receptors unrelated to thalamocortical afferents.     

Serotonin depletion and barrel cortex development: impact of growth impairment vs. serotonin effects on thalamocortical endings

Converging evidence supports a role of serotonin (5-hydroxytryptamine; 5-HT) in barrel cortex development. Systemic administration of 5-HT-depleting drugs reduces cross-sectional whisker barrel areas in the somatosensory cortex (SSC) of neonatal rats. Here we assess the relative impact on barrel pattern formation of (i) 5-HT depletion and (ii) decreased brain growth, which is often associated with pharmacological 5-HT depletion, by comparing the effects of 5-HT-depleting drugs with those of reduced protein intake. Left hemisphere 5-HT levels in the SSC and right hemisphere whisker barrel areas were assessed at postnatal day 6 (P6) in the same animal following injection of p-chloroamphetamine (PCA) or p-chlorophenylalanine (PCPA) at P0. Both drugs significantly reduced cortical 5-HT content and mean barrel areas at P6, but also body and brain growth. Differences in brain weight accounted for 84.4% of the variance in barrel size, with negligible contributions by cortical 5-HT content. PCPA-treated animals sacrificed at P14 yielded similar trends, albeit less pronounced. Finally, reduced protein intake resulted in lower body weight and cortical 5-HT levels at P6, but yielded no change in brain weight or mean barrel area. Barrel formation therefore appears markedly less sensitive to 5-HT depletion per se than to drug-induced growth impairment.     

NMDA receptors pattern early activity in the developing barrel cortex in vivo

N-methyl-D-aspartate (NMDA) type of glutamate receptors play an important role in activity-dependent plasticity in the developing cortex. However, the physiological patterns of cortical activity that activate NMDA receptors in vivo remain largely unknown. We performed full-band recordings from the barrel cortex of neonatal rats in vivo and found that the dominant pattern of the early activity, network driven spindle bursts, are associated with large amplitude NMDA receptor-dependent delta waves. The major sink of delta waves was in the dense cortical plate, which coincided with the sinks of sensory-evoked responses as well as fast spindle-burst oscillations. Pharmacological analysis revealed major contributions from NMDA and alpha-aminopropionate (AMPA) type of glutamate receptors in the generation of delta waves, whereas fast oscillations primarily involved only AMPA receptors. Our results suggest that the 2 component spindle burst is generated by rhythmic, presumably thalamocortical, synaptic input which entrains an AMPA receptor-mediated fast oscillation and who's summation generates an NMDA and AMPA receptor mediated delta wave. The massive summation of thalamocortical activity during the spindle bursts thus provides a long time window for co-incident activation of cortical neurons by the thalamocortical cells which may contribute to the formation of thalamocortical synapses in the barrel cortex during the critical period of developmental plasticity.     

Heterosynaptic facilitation of in vivo thalamocortical long-term potentiation in the adult rat visual cortex by acetylcholine

Acetylcholine (ACh) plays a permissive role in developmental plasticity of fibers from the lateral geniculate nucleus (LGN) to the primary visual cortex (V1). These fibers remain plastic and express long-term potentiation (LTP) in adult rodents, but it is not known if ACh modulates this form of plasticity in the mature V1. We show that, in anesthetized rats, theta burst stimulation (TBS) of the LGN using 5 or 40 theta cycles produced moderate (approximately 20%) and stronger (approximately 40%) potentiation, respectively, of field postsynaptic potentials recorded in the ipsilateral V1. Basal forebrain stimulation (100 Hz) 5 min after TBS enhanced LTP induced by both weak (5 theta cycles) and strong (40 theta cycles) induction protocols. Both effects were reduced by systemic administration of the muscarinic receptor antagonist scopolamine. Basal forebrain stimulation did not enhance LTP when applied 30 min after or 5 min prior to TBS, suggesting that ACh affects early LTP induction mechanisms. Application of the cholinergic agonist carbachol in V1 by means of reverse microdialysis mimicked the effect of basal forebrain stimulation. We conclude that heterosynaptic facilitation of V1 plasticity by ACh extends beyond early postnatal maturation periods and acts to convert weak potentiation into pronounced, long-lasting increases in synaptic strength.     

Optical imaging and electrophysiology of rat barrel cortex. II. Responses to paired-vibrissa deflections

A study was undertaken to investigate the response of the rodent somatosensory barrel cortex to paired-whisker stimuli. Cortical responses to controlled whisker deflections were recorded by (i) conventional multi-unit extracellular recording within the cytochrome oxidase rich barrels centers, and (ii) intrinsic signal optical imaging, a technique that measures an optical correlate of neuronal activity thought to be related to the deoxygenation of hemoglobin in activated regions. Stimuli were applied to two whiskers in sequence, at temporal separations ranging from 0 to 60 ms. Over intervals of 10-40 ms, the primary effect of paired-whisker stimulation was suppressive. We suggest that paired-whisker inhibition results from the activation of layer IV fast-spike units within the principle whisker's barrel, by excitatory input arriving from a surround-whisker. Paired-whisker stimulation produces inhibition in intrinsic images, because it results in a net reduction in layer II/III and/or layer IV metabolism. Intra- cortical inhibition may serve to convert the sequence of inputs from the whisker array into a barrel cortex magnitude code that can be read by higher cortical areas.      

Optical imaging and electrophysiology of rat barrel cortex. I. Responses to small single-vibrissa deflections

A study was undertaken to investigate the response of the rodent somatosensory barrel cortex to single-whisker, near-threshold vibrissal stimuli. Cortical responses to controlled whisker deflections were recorded by (i) conventional multi-unit extracellular recording within the cytochrome oxidase rich barrels centers and the interbarrel septa, and (ii) intrinsic signal optical imaging, a technique that provides a spatial view of cortical activation thought to be related to the deoxygenation of hemoglobin in activated areas. Barrel cortex neurons responded weakly to whisker deflections of 0.04 degrees. Their response to a series of small stimuli of increasing amplitude was well-fitted by a logarithmic function. Responses to larger stimuli declined monotonically with distance from the center of the barrel column, and were characterized by greater onset and offset firing rates, by greater post-excitatory reduction of firing to below spontaneous levels, and by shorter response latency. In comparison to measurements taken previously from primary vibrissal afferent fibers, we conclude that cortical cells can respond to activity in a very small fraction of first-order sensory neurons.      

Cell type-specific three-dimensional structure of thalamocortical circuits in a column of rat vibrissal cortex

Soma location, dendrite morphology, and synaptic innervation may represent key determinants of functional responses of individual neurons, such as sensory-evoked spiking. Here, we reconstruct the 3D circuits formed by thalamocortical afferents from the lemniscal pathway and excitatory neurons of an anatomically defined cortical column in rat vibrissal cortex. We objectively classify 9 cortical cell types and estimate the number and distribution of their somata, dendrites, and thalamocortical synapses. Somata and dendrites of most cell types intermingle, while thalamocortical connectivity depends strongly upon the cell type and the 3D soma location of the postsynaptic neuron. Correlating dendrite morphology and thalamocortical connectivity to functional responses revealed that the lemniscal afferents can account for some of the cell type- and location-specific subthreshold and spiking responses after passive whisker touch (e.g., in layer 4, but not for other cell types, e.g., in layer 5). Our data provides a quantitative 3D prediction of the cell type-specific lemniscal synaptic wiring diagram and elucidates structure-function relationships of this physiologically relevant pathway at single-cell resolution.     

Pharmacological modulation of cortical plasticity following kainic acid lesion in rat barrel cortex

OBJECT: The brain shows remarkable capacity for plasticity in response to injury. To maximize the benefits of current neurological treatment and to minimize the impact of injury, the authors examined the ability of commonly administered drugs, dextroamphetamine (D-amphetamine) and phenytoin, to positively or negatively affect the functional recovery of the cerebral cortex following excitotoxic injury. METHODS: Previous work from the same laboratory has demonstrated reorganization of whisker functional responses (WFRs) in the rat barrel cortex after excitotoxic lesions were created with kainic acid (KA). In the present study, WFRs were mapped using intrinsic optical signal imaging before and 9 days after creation of the KA lesions. During the post-lesion survival period, animals were either treated with intraperitoneal D-amphetamine, phenytoin, or saline or received no treatment. Following the survival period, WFRs were again measured and compared with prelesion data. RESULTS: The findings suggest that KA lesions cause increases in WFR areas when compared with controls. Treatment with D-amphetamine further increased the WFR area (p < 0.05) while phenytoin-treated rats showed decreases in WFR areas. There was also a statistically significant difference (p < 0.05) between the D-amphetamine and phenytoin groups. CONCLUSIONS: These results show that 2 commonly used drugs, D-amphetamine and phenytoin, have opposite effects in the functional recovery/plasticity of injured cerebral cortex. The authors' findings emphasize the complex nature of the cortical response to injury and have implications for understanding the biology of the effects of different medications on eventual functional brain recovery.     

Information processing streams in rodent barrel cortex: the differential functions of barrel and septal circuits

Rodent somatosensory cortex contains an isomorphic map of the mystacial whiskers in which each whisker is represented by neuronal populations, or barrels, that are separated from each other by intervening septa. Separate afferent pathways convey somatosensory information to the barrels and septa that represent the input stages for 2 partially segregated circuits that extend throughout the other layers of barrel cortex. Whereas the barrel-related circuits process spatiotemporal information generated by whisker contact with external objects, the septa-related circuits encode the frequency and other kinetic features of active whisker movements. The projection patterns from barrel cortex indicate that information processed by the septa-related circuits is used both separately and in combination with information from the barrel-related circuits to mediate specific functions. According to this theory, outputs from the septal processing stream modulate the brain regions that regulate whisking behavior, whereas both processing streams cooperate with each other to identify external stimuli encountered by passive or active whisker movements. This theoretical view prompts several testable hypotheses about the coordination of neuronal activity during whisking behavior. Foremost among these, motor brain regions that control whisker movements are more strongly coordinated with the septa-related circuits than with the barrel-related circuits.     

Spatial patterns of excitation and inhibition evoked by lateral connectivity in layer 2/3 of rat barrel cortex

In the rat barrel cortex, neurons in layer 4 are topographically arranged in a precise columnar structure, and the excitatory feed-forward input from layer 4 to layer 2/3 projects almost exclusively within the home barrel column. Here we analyzed the lateral connectivity that links neighboring columns in layer 2/3, which is necessary for integrating information across whiskers. We examined the spatial distributions of three different functional types of lateral connections in layer 2/3 of the rat barrel cortex: glutamate receptor-mediated excitatory connections, GABA(A) receptor-mediated inhibitory connections and GABA(B) receptor-mediated inhibitory connections. Synaptic potentials of pyramidal neurons, which are measures of the strength of connections, were evoked by a horizontal array of stimulation electrodes. The synaptic potentials and their decrease with distance from the stimulation site were measured in two types of slices whose planes were parallel to or orthogonal to barrel rows. Excitatory and GABA(B) receptor-mediated inhibitory connections were stronger along barrel rows than across them, whereas GABA(A) receptor-mediated inhibitory connections did not show such a tendency. These results indicate that lateral connectivity in layer 2/3 varies on the basis of not only excitatory polarity but also receptor subtypes.     

Thalamo-cortical afferents control transient expression of the dopamine D(3) receptor in the rat somatosensory cortex.

The D(3) dopamine receptor (D(3)R) is selectively and transiently expressed in the barrel neurons of the somatosensory cortex (SI) between the first and second postnatal weeks. The D(3)R expression starts after the initial ingrowth of thalamocortical afferents (TCAs) into the barrel cortex and could be induced or controlled by them. We show that unilateral electrolytic lesion of the thalamic ventrobasal complex immediately after birth leads to a decrease in the D(3)R mRNA concentration in the lesioned SI 7 days after the lesion, whereas the D(3)R binding is little affected. Fourteen days after the neonatal thalamic lesion, the D(3)R binding and mRNA are drastically reduced and the barrel-like pattern of the D(3)R is absent. Elevation of the D(3) binding normally seen between the first and second postnatal weeks does not occur. Thalamic lesion on P6 differentially affects the D(3)R expression. One day after the lesion, the D(3) binding and mRNA are down-regulated, but the effect is transient. Five days after the lesion the concentration of D(3) mRNA in the lesioned hemisphere returns to the control level. The typical barrel-like pattern of D(3)R expression is evident in the lesioned SI, although TCAs are completely absent. Quantitative analysis demonstrated elevated cellular levels of the D(3) mRNA in barrel neurons 5 days after the lesion. These higher levels are needed, perhaps, to support the increased production of the D(3)R protein appropriate for this age. Age-related dynamics of the D(3)R binding is retained in the lesioned SI, although the concentration of D(3)R sites remains reduced. These data demonstrate that intact thalamic input is essential for the formation of mechanisms responsible for developmental regulation of the D(3)R expression in the SI.     

Temporal and spatial pattern of expression of cyclic nucleotide-gated channels in developing rat visual cortex.

Cyclic nucleotide-gated channels, ligand-gated and highly permeable to calcium, are good candidates for transducing signals received by migrating cells, growth cones and developing synapses. The level of calcium in growth cones is important for axon guidance. Further, cyclic nucleotides, whose levels can be altered by nitric oxide and other transmitters, are known to alter growth cone motility. We use rat visual cortex as a model in our semi-quantitative RT-PCR and in situ hybridization studies to determine the developmental time course and localization of all three CNG family members (rod, olfactory and cone/testis). We demonstrate that in the cortex, the three channel subtypes are each expressed in a distinct temporal and spatial pattern in only sensorimotor and occipital regions of the cortex. Specifically, the rod and olfactory subtypes are present at the time of migration and rapid dendritic outgrowth, and the cone/testis subtype is highly expressed after eye opening. These results suggest CNG channels may play a role in both early and late events in visual cortical development.     

Subcolumnar dendritic and axonal organization of spiny stellate and star pyramid neurons within a barrel in rat somatosensory cortex

Excitatory neurons at the level of cortical layer 4 in the rodent somatosensory barrel field often display a strong eccentricity in comparison with layer 4 neurons in other cortical regions. In rat, dendritic symmetry of the 2 main excitatory neuronal classes, spiny stellate and star pyramid neurons (SSNs and SPNs), was quantified by an asymmetry index, the dendrite-free angle. We carefully measured shrinkage and analyzed its influence on morphological parameters. SSNs had mostly eccentric morphology, whereas SPNs were nearly radially symmetric. Most asymmetric neurons were located near the barrel border. The axonal projections, analyzed at the level of layer 4, were mostly restricted to a single barrel except for those of 3 interbarrel projection neurons. Comparing voxel representations of dendrites and axon collaterals of the same neuron revealed a close overlap of dendritic and axonal fields, more pronounced in SSNs versus SPNs and considerably stronger in spiny L4 neurons versus extragranular pyramidal cells. These observations suggest that within a barrel dendrites and axons of individual excitatory cells are organized in subcolumns that may confer receptive field properties such as directional selectivity to higher layers, whereas the interbarrel projections challenge our view of barrels as completely independent processors of thalamic input.     

Cardiac afferents to the nucleus of the tractus solitarius: A WGA-HRP study in the rat.

Central distribution of the sensory fibers of the heart was investigated in the rat by the use of transganglionic transport of horseradish peroxidase (HRP). After the left intercostal thoracotomy was done under deep anesthesia and artificial respiration, wheat germ agglutinin-conjugated HRP (WGA-HRP) was injected into the left and right ventricular walls and the apex of the heart. HRP-labeled fibers were observed to be distributed to the dorsomedial portion of the medulla oblongata through the vagal nerve. The labeled fibers were present in various subnuclei of the nucleus of the tractus solitarius (NTS) bilaterally at the level of +0.36 to -1.74 mm to the obex. However, the most conspicuous feature in the present study was that the labeled fibers were exclusively confined to the medial, ventrolateral and commissural NTS with some distribution to the dorsolateral NTS. Although the labeling in the medial and ventrolateral NTS was observed to extend rostrocaudally, it was of interest that the labeling in the medial NTS was divided into the ventral and dorsal parts at the level around the obex. Accumulation of the labeled fibers in the commissural NTS was found at the level caudal to the obex and these fibers were traced to the caudal portion of its subnucleus with a gradual decrease in number. This pattern of distribution of cardiac afferents in the NTS was considered to be peculiar to the rat, because it was quite different from that reported previously in the cat.