In recent years, it has become evident that the majority of cortical in-hibitory neurons in rats and mice originate in the lateral and medial ganglionic eminence and migrate tangentially into the cerebral cortex (for review see Corbin et al., 2001; Marin and Rubenstein, 2003). The same feature had not been specifically demonstrated in ferrets, but we investigated this process in organotypic cultures of normal and MAM-treated neonatal ferrets. To do this, we prepared organotypic cultures of normal ferret cortex at ages ranging from E27 to postnatal day 2 (P2).
The cultured slices were injected in the ganglionic eminence with DiI or other dyes taken up by cells migrating away from this site. The slices remained in culture for 2–5 days. We observed that large num-bers of neurons leave the ganglionic eminence in normal and treated neonatal ferrets. Because ferrets are altricial animals that mature slowly during development, large numbers of neurons are migrating from the ganglionic eminence at birth and continue to migrate up to P2 (probably even later, but these dates were not tested). At younger ages, the neu-rons leaving the ganglionic eminence follow overall trajectories similar to those described by others in rodents. There is a deep route of migra-tion that runs just above the cortical ventricular zone before turning to enter the neocortex and a more superficial route in which neurons travel in the subplate or in layer 1 before joining the cortical layers (Marin and Rubenstein, 2003). In rats and mice, the majority of these neurons express GABA and are interneurons in mature cortex. We tested this in our ferret
model by immunoreacting the slices containing migrating neurons with antibodies directed against GABA; 68% of the labeled neurons leaving the ganglionic eminence are GABAergic, showing similar properties to these neurons in rodents.
The same set of experiments in MAM treated cortex (examined at E38-P2) indicated that similar numbers of neurons leave the ganglionic eminence and migrate into the cerebral cortex (Figure 5). In MAM-treated animals, however, the neurons migrating from the ganglionic eminence appeared more disorganized on their route to the cerebral cor-tex. To verify this observation, we determined the angle of orientation of each leading process of a migrating cell for normal and MAM-treated organotypic cultures. The leading process was measured in relation to the pia. An angle was calculated and placed into a bin that contained the 90◦orientation (out of 360◦), i.e., either oriented radially (toward the pia or the ventricle) or oriented tangentially (medially or laterally). Ori-entations either radially toward the pia or tangentially in the dorsal di-rection were considered the “proper’’ didi-rections. After 2 days in culture, cells migrating from the ganglionic eminence of either normal or MAM treated brains were oriented properly, while after 5 days in culture, the migrating neurons were less likely oriented in the proper direction than those originating from the normal ganglionic eminence (Figure 5).
In addition, when the labeled neurons migrating from the ganglionic eminence were immunoreacted for GABA, fewer of them were double labeled (37%), suggesting that the environment in E33 MAM-treated cortex was not conducive to maintaining a GABAergic phenotype.
This led us to wonder whether the source of disorientation was in the migrating neurons themselves, or due to cues originating from the route of migration or target site. To evaluate these questions we prepared mixed organotypic co-cultures obtained at P0, which included explants of normal ganglionic eminence paired with MAM-treated cortical ex-plants and vice versa. DiI was injected into the ganglionic eminence and the paired explants remained in culture for 5 days. If the cortical ex-plant was normal, the neurons migrating from the ganglionic eminence were oriented in the designated “proper’’ directions, even if the gan-glionic eminence explant originated from a MAM-treated animal. This suggests that a property of E33 MAM treated cortex impaired proper migration of neurons leaving the ganglionic eminence, while features of normal cortex encouraged proper tangential migration (Figure 5).
Conclusion
MAM treatment on E33 leads to dramatic diminution of layer 4.
The relative absence of layer 4 leads to further changes that include improper termination of thalamic afferent fibers, widespread distribu-tion of GABAAαreceptors, and the failure of information transfer in cortical responses to sensory stimulation (Noctor et al., 2001; Palmer et al., 2001; Jablonska et al., 2004; McLaughlin and Juliano, 2005). Further
Figure 5 A.An example of cells leaving the ganglionic eminence after an injec-tion of DiI into a normal organotypic culture. The injecinjec-tion was made on P0 and the slice remained in culture for 2 days. The red line indicates the border of the pia. B. These pie charts indicate the percent of neurons that left the ganglionic eminence and their direction of migration as indicated by the angle of the lead-ing process after 2 days in culture (DIC) or after 5 days in culture. The “proper’’
orientation is indicated in yellow, either toward the pia or medially. The other orientations are indicated in gray. C. Examples of migrating neurons originat-ing from the ganglionic eminence and labeled with DiI in E33 MAM treated animals. Neurons oriented correctly and migrating for 2 days in culture are on the left, neurons that are more disoriented and migrating for 5 days in culture are indicated on the right. D. Pie charts indicating the orientation of migrat-ing neurons from cocultures of normal cortex and E33 MAM treated ganglionic eminence (LGE) or E33 MAM treated cortex and normal LGE. Indicated in the graphs on the left the orientations of the migrating neurons injected on E38 and maintained in culture for 5 days. On the right are graphs of the directions of neurons migrating after injection on P0 after 5 days in culture. In each case, more neurons are oriented in the “proper’’ direction (yellow) when the cortex is normal. Normal ganglionic eminence did not result in a normal pattern of migration.
analysis shows that cells expressing GABA do not migrate properly and neurons expressing distinct types of calcium binding proteins accumu-late in the lower and central layers, rather than reaching their proper sites in the upper layers. Presumed excitatory cells do not show abnor-mal distributions in MAM treated cortex. Further support for the failure of GABAergic cells to migrate properly is seen in the disorientation of cells leaving the ganglionic eminence and heading toward the cortex in E33 MAM treated animals. We suggest that the relative absence of layer 4 leads to a cascade of effects that result in the inability of cells originating in the ganglionic eminence (presumptive GABAergic cells) to migrate effectively. This leads to a mature cortex in which many GABAergic cells fail to reach their proper targets. As a result, the cortex is not able to appropriately respond to somatic stimulation, probably due to im-properly placed GABAergic cells and GABAAreceptors combined with widespread thalamic afferents, which results in an inability to trans-fer information through the cortical layers. Findings from our model of cortical dysplasia coincide with many observations for human cor-tical dysplasia, including the consistent finding of altered GABAergic systems (Baraban et al., 2000; Castro et al., 2002; Jablonska et al., 2004;
Luhmann et al., 1998).
References
Aizenman, C.D., Kirkwood, A., and Bear, M.F. (1996). A current source density analysis of evoked responses in slices of adult rat visual cortex:
Implications for the regulation of long-term potentiation. Cereb. Cortex 6:751–
758.
Baraban, S.C., Wenzel, H.J., Hochman, D.W., and Schwartzkroin, P.A. (2000).
Characterization of heterotopic cell clusters in the hippocampus of rats ex-posed to methylazoxymethanol in utero. Epilepsy Res. 39:87–102.
Benardete, E.A., and Kriegstein, A.R. (2002). Increased excitability and de-creased sensitivity to GABA in an animal model of dysplastic cortex. Epilepsia 43:970–982.
Benes, S.M. and Berretta, S. (2001). GABAergic interneurons: implications for understanding schizophrenia and bipolar disorder. Neuropsychopharmacology 25:1–27.
Bielas, S., Higginbotham, H., Koizumi, H., Tanaka, T., and Gleeson, J.G. (2004).
Cortical neuronal migration mutants suggest separate but intersecting path-ways. Annu. Rev. Cell Dev. Biol. 20:593–618.
Castro, P.A., Pleasure, S.J., and Baraban, S.C. (2002). Hippocampal heterotopia with molecular and electrophysiological properties of neocortical neurons.
Neuroscience 114:961–972.
Corbin, J.G., Nery, S., and Fishell, G. (2001). Telencephalic cells take a tangent:
non-radial migration in the mammalian forebrain. Nat. Neurosci. 4 Suppl:1177–
1182.
Crino, P. (2004). Malformations of cortical development: molecular pathogenesis and experimental strategies. Adv. Exp. Med. Biol. 548:175–191.
Di, S., Baumgartner, C., and Barth, D.S. (1990). Laminar analysis of extracellular field potentials in rat vibrissa/barrel cortex. J. Neurophysiol. 63:832–840.
Jablonska, B., Smith, A.L., Palmer, S.L., Noctor, S.C., and Juliano, S.L. (2004).
GABAA receptors reorganize when layer 4 in ferret somatosensory cortex is disrupted by methylazoxymethanol (MAM). Cereb. Cortex 14:432–440.
Jones, E.G., and Hendry, S.H. (1989). Differential calcium binding protein im-munoreactivity distinguishes classes of relay neurons in monkey thalamic nuclei. Eur. J. Neurosci. 3:222–246.
Kenan-Vaknin, G., and Teyler, T.J. (1994). Laminar pattern of synaptic activ-ity in rat primary visual cortex: Comparison of in vivo and in vitro studies employing the current source density analysis. Brain Res. 635: 37–48.
Luhmann, H.J., Karpuk, N., Qu, M., and Zilles, K. (1998). Characterization of neuronal migration disorders in neocortical structures. II. Intracellular in vitro recordings. J. Neurophysiol. 80:92–102.
Marin, O., and Rubenstein, J.L. (2003). Cell migration in the forebrain. Annu.
Rev. Neurosci. 26:441–483.
McLaughlin, D.F., and Juliano, S.L. (2005). Disruption of layer 4 development al-ters laminar processing in ferret somatosensory cortex. Cereb. Cortex, 15:1791–
1803.
Meier, J., Akyeli, J., Kirischuk, S., and Grantyn, R. (2003). GABA(A) receptor activity and PKC control inhibitory synaptogenesis in CNS tissue slices. Mol.
Cell Neurosci. 23:600–613.
Mitzdorf, U. (1988). Evoked Potentials and their physiological causes: an access to delocalized cortical activity. Springer Series in Brain Dymanics 1.
Noctor, S.C., Scholnicoff, N.J., and Juliano, S.L. (1997). Histogenesis of ferret somatosensory cortex. J. Comp. Neurol. 387:179–193.
Noctor, S.C., Palmer, S.L., McLaughlin, D.F., and Juliano, S.L. (2001). Disrup-tion of layers 3 and 4 during development results in altered thalamocortical projections in ferret somatosensory cortex. J. Neurosci. 21:3184–3195.
Palmer, S. L., Noctor, S.C., Jablonska, B., and Juliano, S.L. (2001). Laminar specific alterations of thalamocortical projections in organotypic cultures following layer 4 disruption in ferret somatosensory cortex. Eur. J. Neurosci. 13:1559–
1571.
Paysan, J., and Fritschy, J.M. (1998). GABAA-receptor subtypes in developing brain. Actors or spectators? Perspect. Dev. Neurobiol. 5:179–192.
Ross, M.E. (2002). Brain malformations, epilepsy, and infantile spasms. Int. Rev.
Neurobiol. 49:333–352.
Santhakumar, V., and Soltesz, I. (2004). Plasticity of interneuronal species diver-sity and parameter variance in neurological diseases. Trends Neurosci. 27:504–
510.
Schroeder, C.E., Seto, S., Arezzo, J.C., and Garraghty, P.E. (1995). Electrophysio-logical evidence for overlapping dominant and latent inputs to somatosensory cortex in squirrel monkeys. J. Neurophysiol. 74:722–732.
Studler, B., Fritschy, J.M., and Brunig, I. (2002). GABAergic and glutamatergic terminals differentially influence the organization of GABAergic synapses in rat cerebellar granule cells in vitro. Neuroscience 114:123–133.
8
Influence of Thalamocortical Activity on Sensory Cortical Development
and Plasticity
Sarah L. Pallas, Mei Xu, and Khaleel A. Razak
Abstract
The cerebral cortical hemispheres are organized into multiple struc-turally and functionally distinct areas. The positioning of these areas is nearly invariant across individuals within a species and even between closely related species. We are interested in determining how these cor-tical areas are specified during development. Another main area of in-terest is how one cortical area might be induced to take on the identity of, and thus substitute for, another cortical area. We have been taking several different approaches to this long-standing issue. This chapter will report on some of our most recent findings. A more complete sum-mary of our previous work can be found in several other review articles (Pallas, 2001, 2002, 2005, and in press).
Thalamocortical afferent (TCA) targeting is relatively specific during development, in that TCAs do not exhibit the level of exuberancy and subsequent pruning seen in corticofugal projections. The mechanism underlying TCA targeting, however, is unknown. We are exploring the relative roles of gene expression patterns and neuronal activity in direct-ing thalamocortical targetdirect-ing specificity and thus in specifydirect-ing cortical areas. We summarize data from three studies of cortical development and plasticity in ferrets, an altricial species with protracted postnatal development. In one study, we investigated the temporal relationship between the targeted ingrowth of TCAs and opposing expression gradi-ents of Pax6, Emx2, Cad6, and Cad8. Using real-time PCR coupled with tracing of TCA projections, we found that Pax6 and Emx2 expression gradients are declining during TCA ingrowth. They may orchestrate gradients of neurogenesis and/or provide regional patterning signals.
On the other hand, differential expression of Cad6 and Cad8 is maximal during TCA targeting and synapse formation, and could play a causal role. In the second set of studies described here, we examined the ef-fect of sensory deprivation on cortical specification. If normal levels or
sources of neural activity are necessary for targeting, then manipulations of activity during TCA ingrowth should disrupt targeting. Consistent with this prediction, we find that bilateral cochlear ablation in P14 fer-rets results in mistargeting of LGN axons to primary auditory cortex.
The third section discusses results from cross-modal plasticity stud-ies. In contrast to sensory deprivation, redirection of retinal axons into auditory thalamus does not cause TCA mistargeting, but does respec-ify several aspects of auditory cortical structure and function. Finally, we examine the relationship between gene expression and the changes seen in cross-modal plasticity. Together these experiments support the idea that early gene expression patterns provide positional information contributes to gradients of neurogenesis and regional identity, but ac-tivity plays an essential role in later patterning and plasticity at the level of TCA targeting to functionally defined cortical areas.