Forelimb amputation-induced reorganization in the ventral posterior lateral nucleus (VPL) provides a substrate for large-scale cortical reorganization in rat forepaw barrel subfield (FBS).

In this study, we examined the role of the ventral posterior lateral nucleus (VPL) as a possible substrate for large-scale cortical reorganization in the forepaw barrel subfield (FBS) of primary somatosensory cortex (SI) that follows forelimb amputation. Previously, we reported that, 6 weeks after forelimb amputation in young adult rats, new input from the shoulder becomes expressed throughout the FBS that quite likely has a subcortical origin. Subsequent examination of the cuneate nucleus (CN) 1 to 30 weeks following forelimb amputation showed that CN played an insignificant role in cortical reorganization and led to the present investigation of VPL. As a first step, we used electrophysiological recordings in forelimb intact adult rats (n=8) to map the body representation in VPL with particular emphasis on the forepaw and shoulder representations and showed that VPL was somatotopically organized. We next used stimulation and recording techniques in forelimb intact rats (n=5) and examined the pattern of projection (a) from the forelimb and shoulder to SI, (b) from the forepaw and shoulder to VPL, and (c) from sites in the forepaw and shoulder representation in VPL to forelimb and shoulder sites in SI. The results showed that the projections were narrowly focused and homotopic. Electrophysiological recordings were then used to map the former forepaw representation in forelimb amputated young adult rats (n=5) at 7 to 24 weeks after amputation. At each time period, new input from the shoulder was observed in the deafferented forepaw region in VPL. To determine whether the new shoulder input in the deafferented forepaw VPL projected to a new shoulder site in the deafferented FBS, we examined the thalamocortical pathway in 2 forelimb-amputated rats. Stimulation of a new shoulder site in deafferented FBS antidromically-activated a cell in the former forepaw territory in VPL; however, similar stimulation from a site in the original shoulder representation, outside the deafferented region, in SI did not activate cells in the former forepaw VPL. These results suggest that the new shoulder input in deafferented FBS is relayed from cells in the former forepaw region in VPL. In the last step, we used anatomical tracing and stimulation and recording techniques in forelimb intact rats (n=9) to examine the cuneothalamic pathway from shoulder and forepaw receptive field zones in CN to determine whether projections from the shoulder zone might provide a possible source of shoulder input to forepaw VPL. Injection of biotinylated dextran amine (BDA) into physiologically identified shoulder responsive sites in CN densely labeled axon terminals in the shoulder representation in VPL, but also gave off small collateral branches into forepaw VPL. In addition, microstimulation delivered to forepaw VPL antidromically-activated cells in shoulder receptive field sites in CN. These results suggest that forepaw VPL also receives input from shoulder receptive sites in CN that are latent or subthreshold in forelimb intact rats. However, we speculate that following amputation these latent shoulder inputs become expressed, possibly as a down-regulation of GABA inhibition from the reticular nucleus (RTN). These results, taken together, suggest that VPL provides a substrate for large-scale cortical reorganization that follows forelimb amputation.

Prenatal alcohol exposure reduces the size of the forelimb representation in motor cortex in rat: an intracortical microstimulation (ICMS) mapping study.

Children with fetal alcohol spectrum disorder (FASD) often exhibit sensorimotor dysfunctions that include deficits in motor coordination and fine motor control. Although the underlying causes for these motor abnormalities are unknown, they likely involve interactions between sensory and motor systems. Rodent animal models have been used to study the effects of prenatal alcohol exposure (PAE) on skilled reaching and on the development and organization of somatosensory barrel field cortex. To this end, PAE delayed the development of somatosensory cortex, reduced the size of whisker and forelimb representations in somatosensory barrel field cortex, and delayed acquisition time to learn a skilled reaching task. However, whether PAE also affects the motor cortex (MI) remains to be determined. In the present study, we investigated the effect of PAE on the size of the forelimb representation in rat MI, thresholds for activation, and the overlap between motor and sensory cortical forelimb maps in sensorimotor cortex. Pregnant Sprague-Dawley rats were assigned to alcohol (Alc), pair-fed (PF), and chow-fed (CF) groups on gestation day 1 (GD1). Rats in the Alc group (n=4) were chronically intubated daily with binge doses of alcohol (6g/kg body weight) from GD1 to GD20 that resulted in averaged blood alcohol levels measured on GD10 (mean=191.5+/-41.9mg/dL) and on GD17 (mean=247.0+/-72.4mg/dL). PF (n=2) and CF (n=3) groups of pregnant rats served as controls. The effect of PAE on the various dependent measures was obtained from multiple male offspring from each dam within treatment groups, and litter means were compared between the groups from alcohol-treated and control (Ct: CF and PF) dams. At approximately 8 weeks of age, rats were anesthetized with ketamine/xylazine and the skull opened over sensorimotor cortex. A tungsten microelectrode was then inserted into the depths of layer V and intracortical microstimulation was used to deliver trains of pulses to evoke muscle contractions and/or movements; maximum stimulating < or =100microA. When a motor response was observed, the threshold for movement was measured and the motor receptive field projected to the cortical surface to serve as representative point for that location. A motor map for the forelimb representation was generated by systematically stimulating at adjacent sites until current thresholds reached the maximum and/or motor responses were no longer evoked. The major findings in this study were as follows: (1) PAE significantly reduced the area of the forelimb representation in the Alc offspring (6.01mm(2), standard error of the mean=+/-0.278) compared with the Ct offspring (8.03mm(2)+/-0.586), (2) PAE did not significantly reduce the averaged threshold for activation of movements between groups, (3) PAE significantly reduced the percent overlap (Alc=31.1%, Ct=55.4%) between the forelimb representation in sensory and motor cortices, and (4) no significant differences were observed in averaged body weight, hemisphere weight, or age of animal between treatment groups. These findings suggest that the effects of PAE are not restricted to somatosensory barrel field cortex but also involve the MI and may underlie deficits in motor control and sensorimotor integration observed among children with FASD.

Long-term effects of prenatal alcohol exposure on the size of the whisker representation in juvenile and adult rat barrel cortex.

Children of mothers who abused alcohol during pregnancy are often reported to suffer from growth retardation and central nervous system (CNS) abnormalities. The use of prenatal alcohol exposed (PAE) animal models has revealed reductions in body and brain weights as well as regional specific brain deficits in neonatal pups. Recently, we and others reported reductions in the size of the posteromedial barrel subfield (PMBSF) in first somatosensory cortex (SI) associated with the representation of the large mystacial vibrissae in neonatal rats and mice that were exposed to alcohol at various times during gestation. While these reductions in barrel field size were reported in neonates, it was unclear whether similar reductions persisted later in life or whether some catch-up might take place in older animals. In the present study, we examined the effect of PAE on measures of barrel field size in juvenile (6 weeks of age) and adult (7 months of age) rats; body and brain weights were also measured. Pregnant rats (Sprague-Dawley) were intragastrically gavaged during gestational days 1-20 with alcohol (6 g/kg) to simulate a binge-like pattern of alcohol consumption (Alc); 6 g/kg alcohol produced blood alcohol levels ranging between 207.4 and 478.6 mg/dl. Chow-fed (CF), pair-fed (PF), and cross-foster (XF) groups served as normal, nutritional/stress, and maternal controls, respectively, for juvenile rats; an XF group was not included for adult rats. The major findings in the present study are (i) PAE significantly reduced the size of the total barrel field in Alc juvenile rats (13%) and adult rats (9%) compared to CF controls, (ii) PAE significantly reduced the total averaged sizes of individual PMBSF barrels in juvenile (14%) and adult (13%) rats, (iii) PAE did not significantly alter the septal area between barrels or the barrel pattern, (iv) PAE significantly reduced body weight of juvenile rats but only in comparison to PF controls (18%), (v) PAE significantly reduced whole brain (8%) and forebrain (7%) weights of juvenile rats but not adult rats, (vi) no differences were observed in forebrain/PMBSF body ratios nor was forebrain weight correlated with PMBSF area, and (vii) PAE resulted in a greater reduction in anterior barrels compared to posterior barrels. These results suggest that the effects of PAE previously reported in neonate PMBSF areas persist into adulthood.

Early postnatal alcohol exposure reduced the size of vibrissal barrel field in rat somatosensory cortex (SI) but did not disrupt barrel field organization.

Prenatal alcohol exposure (PAE) has been shown to alter the somatosensory cortex in both human and animal studies. In rodents, PAE reduced the size, but not the pattern of the posteromedial barrel subfield (PMBSF) associated with the representation of the whiskers, in newborn, juvenile, and adult rats. However, the PMBSF is not present at birth, but rather first appears in the middle of the first postnatal week during the brain-growth spurt period. These findings raise questions whether early postnatal alcohol exposure might disrupt both barrel field pattern and size, questions that were investigated in the present study. Newborn Sprague-Dawley rats were assigned into alcohol (Alc), nutritional gastric control (GC), and suckle control (SC) groups on postnatal day 4 (P4). Rat pups in Alc and GC were artificially fed with alcohol and maltose-dextrin dissolved in milk, respectively, via an implant gastrostomy tube, from P4 to P9. Pups in the Alc group received alcohol (6.0 g/kg) in milk, while the GC controls received isocaloric equivalent maltose-dextrin dissolved in milk. Pups in the SC group remained with their mothers and breast fed throughout the experimental period. On P10, pups in each group were weighed, sacrificed, and their brains removed and weighed. Cortical hemispheres were separated, weighed, flattened, sectioned tangentially, stained with cytochrome oxidase, and PMBSF measured. The sizes of barrels and the interbarrel septal region within PMBSF, as well as body and brain weights were compared between the three groups. The sizes of PMSBF barrel and septal areas were significantly smaller (P<.01) in Alc group compared to controls, while the PMBSF barrel pattern remained unaltered. Body, whole-brain, forebrain, and hemisphere weights were significantly reduced (P<.01) in Alc pups compared to control groups. GC and SC groups did not differ significantly in all dependent variables, except body weight at P9 and P10 (P<.01). These results suggest that postnatal alcohol exposure, like prenatal exposure, significantly influenced the size of the barrel field, but not barrel field pattern formation, indicating that barrel field pattern formation consolidated prior to P4. These results are important for understanding sensorimotor deficits reported in children suffering from fetal alcohol spectrum disorder (FASD).

Prenatal alcohol exposure delays the development of the cortical barrel field in neonatal rats.

In-utero alcohol exposure produces sensorimotor developmental abnormalities that often persist into adulthood. The rodent cortical barrel field associated with the representation of the body surface was used as our model system to examine the effect of prenatal alcohol exposure (PAE) on early somatosensory cortical development. In this study, pregnant female rats were intragastrically gavaged daily with high doses of alcohol (6 gm/kg body weight) throughout the first 20 days of pregnancy. Blood alcohol levels were measured in the pregnant dams on gestational days 13 (G13) and G20. The ethanol treated group (EtOH) was compared to the normal control chowfed (CF) group, nutritionally matched pairfed (PF) group, and cross-foster (XF) group. Cortical barrel development was examined in pups across all treatment groups from G25, corresponding to postnatal day 2 (P2), to G32 corresponding to P9. The EtOH and control group pups were weighed, anesthetized, and perfused. Brains were removed and weighed with, and without cerebellum and olfactory bulbs, and neocortex was removed and weighed. Cortices were then flattened, sectioned tangentially, and stained with a metabolic marker, cytochrome oxidase (CO) to reveal the barrel field. Progression of barrel development was distinguished into three categories: (a) absent, (b) cloudy barrel-like pattern, and (c) well-formed barrels with intervening septae. The major findings are: (1) PAE delayed barrel field development by one or more days, (2) the barrel field first appeared as a cloudy pattern that gave way on subsequent days to an adult-like pattern with clearly demarcated intervening septal regions, (3) the barrel field developed differentially in a lateral-to-medial gradient in both alcohol and control groups, (4) PAE delayed birth by one or more days in 53% of the pups, (5) regardless of whether pups were born on G23 (normal expected birth date for non-alcohol controls) or as in the case for the alcohol-delayed pups born as late as G27, the barrel field was never present at birth suggesting the importance of postnatal experience on barrel field development, and (6) PAE did not disrupt the normal barrel field pattern, although both total body and brain weights were compromised. These findings suggest that PAE delays the development of the somatosensory cortex (SI); such delays may interfere with timing and formation of cortical circuits. It is unknown whether other nuclei along the somatosensory pathway undergo similar delays in development or if PAE selectively disrupts cortical circuitry.

Prenatal alcohol exposure (PAE) reduces the size of the forepaw representation in forepaw barrel subfield (FBS) cortex in neonatal rats: relationship between periphery and central representation.

Prenatal alcohol exposure (PAE) alters limb development that may lead to structural and functional abnormalities of the limb reported in children diagnosed with Fetal Alcohol Spectrum Disorder. To determine whether PAE alters the central representation of the forelimb we used the rodent barrel cortex as our model system where it was possible to visualize and quantitatively measure the size of the forepaw representation in the forepaw barrel subfield (FBS) in first somatosensory cortex. In the present study, we examined the effects of PAE on pattern and size of the forepaw and forepaw representation in FBS in neonatal rats at gestational day 32 that corresponds to postnatal day 9. Pregnant Sprague-Dawley rats were chronically intubated with binge doses of ethanol (6 g/kg) from gestational day 1 through gestational day 20. The offspring of the ethanol treated dams comprised the ethanol (EtOH) group. The effect of PAE on the EtOH group was compared with a nutritional-controlled pairfed (PF) group and a normal chowfed (CF) group. The ventral (glabrous) surface area of the forepaw digits, length of digit 2 through digit 5, and the corresponding glabrous forepaw digit representations in the FBS were measured and compared between treatment groups. In rats exposed to in utero alcohol, the sizes of the overall glabrous forepaw and forepaw digits were significantly reduced in EtOH pups compared to CF and PF pups; overall glabrous forepaw area was 11% smaller than CF controls. Glabrous digit lengths were also smaller in EtOH rats compared to CF controls and significantly smaller in digit 2 through digit 4. The glabrous digit representation in FBS was 18% smaller in the EtOH group when compared to the CF treatment. However, PAE did not produce malformations in the forepaw or alter the pattern of the forepaw representation in FBS; instead, PAE significantly reduced both body and brain weights compared to controls. Unexpectedly, little or no correlation was observed between the size of the glabrous forepaw compared to the size of the glabrous forepaw representation in the FBS for any of the treatment groups. The present findings of PAE-related alterations in sensory periphery and the central cortical representation may underlie deficits in sensorimotor integration reported among children with Fetal Alcohol Spectrum Disorder.

Prenatal alcohol exposure alters the size, but not the pattern, of the whisker representation in neonatal rat barrel cortex.

Maternal alcohol exposure results in a variety of neurodevelopmental abnormalities that include cognitive and sensorimotor dysfunctions that often persist into adulthood. Many reports of central nervous system disturbances associated within a clinical diagnosis of fetal alcohol syndrome point toward disturbances in central information processing. In this study, we used the rat barrel field cortex as a model system to examine the effects of prenatal alcohol exposure (PAE) on the organization and size of the large whisker representation in layer IV of the posteromedial barrel subfield (PMBSF) in somatosensory cortex. Pregnant rats (Sprague-Dawley) were intragastrically gavaged daily with alcohol doses (6 gm/kg body weight) from gestational day 1 to day 20 in a chronic binge pattern which produced blood alcohol levels ranging between 260 mg/dl and 324 mg/dl. Chow-fed (CF), pair-fed (PF), and cross-foster (XF) groups served as normal, nutritionally matched, and maternal controls, respectively, for the ethanol-exposed (EtOH) treatment group. All pups were examined on gestational day 32 corresponding approximately to postnatal day 9. EtOH and control group pups were weighed, anesthetized, and perfused. Brains were removed and weighed, with and without cerebellum and olfactory bulbs, and the neocortex was removed and weighed. Cortices were then flattened, sectioned tangentially, and stained with a metabolic marker-cytochrome oxidase-to reveal the barrel field. A subset of 27 cortical barrels, associated with the representation of the large whisker pad, was selected to examine in detail. The major results were: (i) the total barrel field area comprising the PMBSF was significantly reduced in EtOH (by 17%) and XF (by 16%) pups compared with CF pups, (ii) the sizes of individual barrels within the PMBSF were also significantly reduced in EtOH (16%) and XF (18%) pups, (iii) the septal region between barrels was also significantly reduced in EtOH (18%) and XF (12%) pups, (iv) anteriorly located barrels underwent greater reduction in size relative to the posteriorly located barrels, (v) body weights were also significantly reduced in EtOH (21%) and XF (27%) pups, (vi) total brain weight [with and without (forebrain) cerebellum/olfactory bulbs] and cortical weights were also significantly reduced in EtOH (total brain weight 15%, forebrain weight 16%, cortical weight 15%) and XF (18%, 19%, 20%) pups, and in contrast (vi) neither the overall barrel field pattern nor the pattern of individual barrels in the PMBSF was altered. These findings suggest that PAE reduces body and brain weight as well as the central cortical representation of the whisker pad, while leaving the overall barrel field pattern unperturbed. While these results might appear to support a miniaturization hypothesis (smaller PMBSF, smaller brain, smaller body weight), PAE also shows regional vulnerability within the PMBSF whereby anteriorly located barrels are most affected.

Delayed reorganization of the shoulder representation in forepaw barrel subfield (FBS) in first somatosensory cortex (SI) following forelimb deafferentation in adult rats.

We previously reported that 6-16 weeks after forelimb amputation in adult rats, neurons in layer IV of rat first somatosensory cortex (SI) in the forepaw barrel subfield (FBS) associated with the representation of the forepaw became responsive to new input from the shoulder (Pearson et al. 1999). These new shoulder-responsive sites in deafferented FBS had longer evoked response latencies than did sites in the shoulder representation located in the posterior part of the trunk subfield, hereafter referred to as the original shoulder representation. Furthermore, projection neurons in the original shoulder representation in both intact and deafferented adults did not extend their axons into the FBS, and ablation of the original shoulder representation cortex and/or the second somatosensory cortex (SII) failed to eliminate new shoulder input in the deafferented FBS (Pearson et al. 2001). These results led us to conclude that large-scale reorganization in FBS quite likely involved a subcortical substrate. In addition, the time course for large-scale cortical reorganization following forelimb amputation was unknown, and this information could shed light on potential mechanisms for large-scale cortical reorganization. In the present study, we extended our previous findings of large-scale cortical reorganization in the FBS by investigating the time course for reorganization following forelimb amputation. The major findings are: a) deafferented forelimb cortex remained unresponsive to shoulder stimulation during the 1st week following forelimb amputation; b) new responses to shoulder stimulation were first observed in deafferented forelimb cortex 2-3 weeks after forelimb amputation; however, the new shoulder input was restricted to locations in the former forearm cortex; c) islet(s) of new shoulder representation were first observed in deafferented FBS 4 weeks after amputation; these islets occupied a larger percentage of FBS in subsequent weeks; d) portions of FBS remained unresponsive as many as 4 months after deafferentation (maximum time examined between amputation and recording); and e) the increase in total size of the shoulder representation appeared to result from the establishment of new shoulder representations that were often discontinuous from the original shoulder representation. These findings provide evidence that forelimb amputation results in delayed reorganization of the FBS and we describe possible mechanisms and substrates underlying the reorganization.