Regional spread of activin βA mRNA induction
The relatively slow development of epileptogenesis in the amygdala-kindling model allows a detailed analysis of the progressive development of focal seizures with secondary generalization (Kalynchuk, 2000; Racine et al., 2002). Activin, as with BDNF, is a known neurotrophic factor, so cells showing elevated message levels may be implicated in roles underlying the enduring neuronal changes associated with epilepsy (Gall, 1993). The data show a spatiotemporal spread of gene induction across the amygdala and cortical areas affected by the seizures.
The 2 hours postseizure survival time in experiment 1 was selected because earlier reports showed that pharmacological intervention could be neuroprotective against status epilepticus (Kelly and McIntyre, 1994) at 2 hours and because kindling-induced up-regulation of mRNAs for cytokines such as TGFβ occurred at 2 hours (Plata-Salaman et al., 2000). At this time point, immediate-early genes (IEGs) such as c-fos mRNA are absent or no longer expressed, having peaked much earlier after the seizure (Burazin and Gundlach, 1996). Indeed, we performed parallel hybridizations with ribonucleotide probes for c-fos and zif268 (NGFI-A), and we saw only inconsistent, weak bilateral global cortical expression in the same animals (data not shown).
The functional markers c-fos and 2-deoxyglucose are responsive to most forms of excitatory neural activity and thereby show widespread elevations in cortical and subcortical labeling (Clark et al., 1991; Engel et al., 1978; Labiner et al., 1993), parts of which may not be relevant to the synaptic events responsible for enduring changes in kindled seizure susceptibility. Thus, it is not surprising that the activin βA mRNA expression pattern represents a discrete subportion of the early-appearing and transient IEG activation pattern. It is possible, of course, that the limited distribution of expressing cells is due to the inability of some neuronal groups to respond with elevated activin βA mRNA expression.
Results of this experiment can be compared with data based on functional markers used to map the spread of seizure effects following similar amygdala-kindling paradigms or status epilepticus. The previous studies were carried out with 2-deoxyglucose autoradiography (Engel et al., 1978; McIntyre et al., 1991), Fos immunohistochemistry (White and Price, 1993a), or IEG mRNA hybridization (Clark et al., 1991; Hosford et al., 1995; Sitcoske O'Shea et al., 2000). In these studies, brain areas responding early in the course of seizure development include the amygdala near the stimulation site and the adjacent piriform cortex. At later stages, widespread cortical and olfactory areas, hippocampus, and several subcortical structures—the ventral striatum (accumbens), basal forebrain and bed nucleus of the stria terminalis, thalamus (mediodorsal nucleus), and substantia nigra—show strong activation. Lesion studies have argued for the critical importance of the ipsilateral piriform cortex (Schwabe et al., 2000), insular cortex (Kodama et al., 2001), perirhinal cortex (Kelly and McIntyre, 1996; Kelly et al., 2002), and basal amygdala (White and Price, 1993b) in the seizure evolution process leading to convulsive behavior.
The induced activin βA mRNA expression pattern is strikingly discrete and anatomically maps several structures known to be important in seizure development and the epileptic state. However, some components of the temporal spread of activin labeling are not easily explained. Spread of activity-associated activin βA mRNA induction from amygdala to cortex could occur via direct axonal projections from the BL to layers II and V of the perirhinal, insular, and prelimbic areas (Krettek and Price, 1977). These are the areas (and layers) that showed strong activin βA mRNA induction with the first stage 2 (partial) seizures. Projections along this continuum are augmented by additional projections from the endopiriform nucleus (Behan and Haberly, 1999) and perirhinal cortex (Kelly and McIntyre, 1996; McIntyre et al., 1996) to insular and prelimbic cortices, all ipsilaterally. The first appearance of activin in the convexity cortex was bilateral (Figs. 1c,d, 2) and did not appear to require prior bilateral labeling along the sulcal cortex. Spread of activin βA message to the contralateral side occurred independently for amygdala and cortex. Thus, for example, in animals 4 (1s2) and 18 (1s5), neocortical labeling was stronger contralaterally than ipsilaterally, whereas amygdala labeling was predominantly ipsilateral. In another animal 19 (1s5), amygdala labeling was strictly and strongly ipsilateral, whereas cortical labeling was highly restricted bilaterally to the rostral insular and prelimbic areas and ipsilaterally to the caudal piriform cortex (data not shown).
The rapid switch to bilaterally symmetrical labeling in the amygdala does not closely resemble crossed amygdala projections, which spread beyond the homologous contralateral structure. For example, whereas the basolateral nuclei (notably the magnocellular basal nuclei) project to their contralateral homologous counterparts, they also project to nonhomologous structures, such as the central and anterior nuclei and the nucleus of the lateral olfactory tract (Savander et al., 1997), which did not show contralateral activin βA mRNA induction. It is noteworthy that the BL is necessary for spread of kindling, as determined by focal deactivation studies (White and Price, 1993b).
Corticocortical connections across the corpus callosum typically have a discontinuous, banded termination pattern (Cipolloni and Peters, 1979), which does not resemble the horizontally uniform pattern of mRNA for activin βA or other genes (see, e.g., Bengzon et al., 1993; Burazin and Gundlach, 1996) contralateral to the stimulation. Thus, the bilateral and rather symmetrical pattern of cortical activation is not easy to explain anatomically, which is analogous to the situation in the amygdala (see above). Horizontal spread of activity within the cortex might account for the uniformity of labeling strength.
Appearance of bilateral activin βA mRNA induction in the primary motor cortex (M1) at the first stage 5 seizure (1s5) shows early involvement of this area in the kindling phenomenon. The motor cortex is presumably the source of the behavioral responses (clonus) seen in the animals upon stimulation (Kelly et al., 1999). Synaptic changes here could underlie the lowered threshold for seizure recruitment and convulsive expression.
The distinctive pattern of activin βA mRNA expression overlaps with existing constitutive expression of activin receptors (ActR-II and ActR-IIB; Fig. 4; Cameron et al., 1994). These receptors are especially enriched in the endopiriform nucleus and the lateral, basal, and posterior cortical amygdaloid nuclei, which show elevated activin βA mRNA, but they are also strongly present in the central, medial, and anterior amygdaloid nuclei, which did not show activin βA mRNA elevations. This demonstrates that the discrete activin βA mRNA expression profile does coincide with some but not all areas of high densities of receptors.
Duration of mRNA induction after the seizure
Experiment 2 was designed to address the postseizure duration of activin βA mRNA expression in comparison with two other representative markers, c-fos, an immediate-early gene; and BDNF, a neurotrophic factor. After a full generalized (6s5) seizure, c-fos mRNA was strongly induced in the cerebral cortex bilaterally only at 1 hour, and its expression levels had returned to baseline by 2 hours. In contrast, both activin βA and BDNF showed coordinated bilateral induction in cortical layers II/III at 1, 2, and 6 hours, returning to baseline by 24 hours. The selectively extended period of elevated mRNA expression of activin βA and BDNF argues that these transcripts code for proteins that serve functions similar to those of neurotrophic factors regulating development and maintenance of the epileptic state and do not merely reflect transiently elevated neuronal activity. The subtle variations in laterality and time of peak expression between activin βA and BDNF mRNAs argue that they are under slightly different regulatory control.
Laminar and cellular analysis
Activin βA mRNA expression was largely confined to layer II neurons, which were typically pyramidal (e.g., Fig. 6c). These neurons presumably were not γ-aminobutyric acid (GABA)-ergic (being GAD67 mRNA negative); however, they did coexpress induced BDNF mRNA. Thus, both activin βA and BDNF mRNAs are selectively induced in presumably glutamatergic cortical projection neurons. Additional molecular markers of kindling-induced neocortical layer II neuronal mRNA expression include BDNF (present study; Ernfors et al., 1991; Isackson et al., 1991) and neuropsin (Okabe et al., 1996). BDNF is known to be neuroprotective and, in addition, may participate in synaptic changes that could support the epileptic state. Neuropsin is a serine protease that may affect synaptic architecture by proteolytic degradation of extracellular matrix proteins, such as fibronectin (Tomimatsu et al., 2002), and has been suggested to be neuroprotective to hyperexcitability (Davies et al., 2001). Activin is protective in some models of excitotoxic/ischemic brain injury (Hughes et al., 1999; Mattson, 2000; Tretter et al., 2000), and antiinflammatory actions, resembling those of TGFβ, have been shown (de Kretser et al., 1999). These molecules may play a role in the phenomenon of kindling-induced protection against kainate seizure-induced neuronal degeneration (Kelly and McIntyre, 1994).
Together the striking and distinctive induction pattern of activin βA mRNA, the selective localization to neocortex layer II pyramidal neurons, and the coincident expression of BDNF mRNA suggest that activin A may be an important molecule in the kindling process. We suggest that, as with BDNF and neuropsin, activin may play a neuroprotective role. Activin receptors are protein serine kinases (Mathews and Vale, 1993) that selectively activate Smad2/3 via activin receptor-like kinase-4 (ALK-4) signaling (Massague, 1998). The roles of these Smads in neuronal function are not well known, but Smads are involved in neuronal remodeling (Zheng et al., 2003), neuroprotection (Docagne et al., 2002), and inhibition of calcium influx (Williams et al., 2002). These Smads physically interact in the cell with many important molecules (Miyazawa et al., 2002), such as calmodulin (Zimmerman et al., 1998), nuclear factor-κB subunits (Lopez-Rovira et al., 2000), hormone receptors (Miyazawa et al., 2002), and the Jun family of AP-1 transcription factors (Liberati et al., 1999). In contrast, BDNF acts through transmembrane receptor tyrosine kinases (Trk), notably TrkB, leading to important intracellular signaling cascades such as MAPK-mediated signaling and CREB phosphorylation. Evidence suggests that BDNF contributes to the epileptic state (Binder et al., 2001), largely because BDNF+/– mice show suppressed development of kindling (Kokaia et al., 1995). Future work with regard to activin will focus on its actions through the ActR-II receptors and Smad pathways. As with BDNF, work showing the results of activin application and blockade in physiological studies will further reveal its roles in adult neuronal plasticity.