TGF-beta1 in Aplysia: role in long-term changes in the excitability of sensory neurons and distribution of TbetaR-II-like immunoreactivity

Abstract

Exogenous recombinant human transforming growth factor beta-1 (TGF-beta1) induced long-term facilitation of Aplysia sensory-motor synapses. In addition, 5-HT-induced facilitation was blocked by application of a soluble fragment of the extracellular portion of the TGF-beta1 type II receptor (TbetaR-II), which presumably acted by scavenging an endogenous TGF-beta1-like molecule. Because TbetaR-II is essential for transmembrane signaling by TGF-beta, we sought to determine whether Aplysia tissues contained TbetaR-II and specifically, whether neurons expressed the receptor. Western blot analysis of Aplysia tissue extracts demonstrated the presence of a TbetaR-II-immunoreactive protein in several tissue types. The expression and distribution of TbetaR-II-immunoreactive proteins in the central nervous system was examined by immunohistochemistry to elucidate sites that may be responsive to TGF-beta1 and thus may play a role in synaptic plasticity. Sensory neurons in the ventral-caudal cluster of the pleural ganglion were immunoreactive for TbetaR-II, as well as many neurons in the pedal, abdominal, buccal, and cerebral ganglia. Sensory neurons cultured in isolation and cocultured sensory and motor neurons were also immunoreactive. TGF-beta1 affected the biophysical properties of cultured sensory neurons, inducing an increase of excitability that persisted for at least 48 hr. Furthermore, exposure to TGF-beta1 resulted in a reduction in the firing threshold of sensory neurons. These results provide further support for the hypothesis that TGF-beta1 plays a role in long-term synaptic plasticity in Aplysia.

Figure 1

Distribution of TβR-II in Aplysia tissues. Tissue samples were frozen on dry ice, crushed, and subsequently homogenized in an extraction buffer containing EDTA, EGTA, and protease inhibitors. A total of 50 μg of protein from each sample was loaded and resolved by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was probed with an affinity-purified polyclonal goat IgG raised against the extracellular domain of the human TGF-β type-II receptor and exposed to a HRP-conjugated rabbit–anti-goat IgG. Immunoreactive bands were visualized with a chemiluminescent substrate. (A) Two bands of protein (60 and 68 kD) are found in almost all Aplysia tissues examined, which may represent precursor and mature forms of the receptor. (B) One band of protein is detected in a membrane fraction.

Figure 2

TβR-II immunoreactivity in sections of pleural and pedal ganglia. (A) Staining is present in neuronal cell bodies in the pleural ganglia as well as in the neuropil, which may represent staining along neuronal processes. The cluster of mechanoafferent sensory neurons in the ventral–caudal cluster also exhibit immunoreactivity and can be identified by size and position (area between arrows). (S) Sheath; (N) neuropil. (B) Control section adjacent to that in A shows little staining. (C) Higher magnification view of mechanoafferent sensory neurons from a different section than that shown in A. (D) Immunoreactivity is also present in many cells of the pedal ganglion, particularly in the caudal region, shown in this section. Scale bars, 100 μm (A,B,D) and 50 μm (inset).

Figure 3

TβR-II immunoreactivity in sections of the abdominal ganglion. (A) Low-magnification view of the abdominal ganglion showing distribution of immunoreactivity in the ganglion. Some neurons in the caudal region are heavily stained (arrows), whereas other neurons show little immunoreactivity (solid arrowheads). Bag cells are also darkly stained (open arrowhead). (S) Sheath; (N) neuropil. (B) Higher magnification view of the neurons in the caudal region of the section shown in A. Arrow shows a positively stained neuron and arrowhead marks an unstained neuron. (C) Higher magnification view of the bag cell cluster from a different section than that in A. Scale bars, 200 μm in A and 100 μm in B and C.

Figure 4

TβR-II immunoreactivity in sections of the buccal ganglion. (A) Many neurons in the S1/S2 cluster exhibit immunoreactivity (arrow) as well as putative B8 neurons (arrowheads). Immunoreactivity is also present along tracts in the neuropil. (S) Sheath; (N) neuropil; (NV) nerve; (BC) buccal commissure. (B) Section from a more caudal region of the buccal ganglion than that in A showing more immunoreactive neurons. Scale bar, 100 μm.

Figure 5

Cultured neurons exhibit TβR-II immunoreactivity. Neurons were isolated and allowed to grow for 5 days before fixation. Immunoreactivity was visualized with a rhodamine-conjugated rabbit-anti-goat IgG. (A) Sensory neurons from the ventral–caudal cluster of the pleural ganglion grown in culture with no postsynaptic target exhibit immunoreactivity along the cell body and neurites. (B) Control cultures show little staining. (C) Not all sensory neurons exhibit the same intensity of immunoreactivity. Note that these sensory neurons exhibit higher levels of immunoreactivity than that in A. (D) Cocultured sensory neuron and L7, an identified motor neuron. The synaptic connection was verified by extracellularly stimulating the sensory neuron and recording an EPSP in the motor neuron (not shown). Both cell bodies are immunostained as well as the major axons of each neuron and finer neuronal processes. Scale bars, 30 μm.

Figure 6

TGF-β1 induces a long-term increase in sensory neuron excitability. (A) Examples of the spike trains evoked by current injection before, 24- and 48-hr after treatment with TGF-β1 or BSA (control) for 6 hr. Calibration bar, 100 msec, 20 mV. (B) Summary data of excitability changes at the 24- and 48-hr time points. Bars, means ± s.e.m. of the normalized excitability. Two-way ANOVA with repeated measures revealed a significant difference between the treatments of TGF-β1 (solid bars) and the control (open bars) (F_1,8 = 5.47, P < 0.05). (C) Summary data showing that TGF-β1 induces a long-term decrease in sensory neuron firing threshold. Bars, means ± s.e.m. of the normalized current necessary to evoke one action potential (F_1,8 = 8.59, P < 0.05).

Figure 6

TGF-β1 induces a long-term increase in sensory neuron excitability. (A) Examples of the spike trains evoked by current injection before, 24- and 48-hr after treatment with TGF-β1 or BSA (control) for 6 hr. Calibration bar, 100 msec, 20 mV. (B) Summary data of excitability changes at the 24- and 48-hr time points. Bars, means ± s.e.m. of the normalized excitability. Two-way ANOVA with repeated measures revealed a significant difference between the treatments of TGF-β1 (solid bars) and the control (open bars) (F_1,8 = 5.47, P < 0.05). (C) Summary data showing that TGF-β1 induces a long-term decrease in sensory neuron firing threshold. Bars, means ± s.e.m. of the normalized current necessary to evoke one action potential (F_1,8 = 8.59, P < 0.05).

Figure 6

TGF-β1 induces a long-term increase in sensory neuron excitability. (A) Examples of the spike trains evoked by current injection before, 24- and 48-hr after treatment with TGF-β1 or BSA (control) for 6 hr. Calibration bar, 100 msec, 20 mV. (B) Summary data of excitability changes at the 24- and 48-hr time points. Bars, means ± s.e.m. of the normalized excitability. Two-way ANOVA with repeated measures revealed a significant difference between the treatments of TGF-β1 (solid bars) and the control (open bars) (F_1,8 = 5.47, P < 0.05). (C) Summary data showing that TGF-β1 induces a long-term decrease in sensory neuron firing threshold. Bars, means ± s.e.m. of the normalized current necessary to evoke one action potential (F_1,8 = 8.59, P < 0.05).