Abstracts

RATIONALE: Substance P (SP) receptors are widely distributed in the cortex and epileptic seizures are known to cause increased SP expression. For this reason we examined the cellular mechanisms that underlie the modulation of cortical neurons by SP.
METHODS: Experiments were performed on male and female mice (P8[ndash]P13) that were deeply anesthetized with ether. The cortex was isolated in ice-cold artificial CSF (aCSF : 118 NaCl, 3 KCl, 1.5 CaCl[sub]2[/sub], 1 MgCl[sub]2[/sub], 25 NaHCO[sub]3[/sub], 1 NaH[sub]2[/sub]PO[sub]4[/sub], and 30 D-glucose, pH of 7.4 ) bubbled with carbogen (95% oxygen and 5% CO2). The cerebral hemispheres were separated at the midline. Slices (500 [mu]m thick) were sectioned 1500 [mu]m from the frontal pole (motor cortex), and were immediately transferred into a recording chamber maintained at 29[degree]C. After 30 min the K⁺ concentration was raised from 3 to 5 mM to obtain spontaneous rhythmic activity. Population activity recordings were obtained with suction electrodes positioned onto the surface of cortical layers 4 and 5. Intracellular whole-cell patch-clamp recordings were obtained from cortical neurons using the blind-patch technique. Cell layer and cell type were identified by staining each neuron with biocytin.
RESULTS: Intracellular recordings from cortical neurons were obtained simultaneously with extracellular recordings from populations of neurons located close to the intracellular recording site. The majority of slices exhibited population activity, which was characterized by slow ([lt] 0.5 Hz) recurrent and spontaneously generated oscillations. SP (0.1 [mu]M) had a biphasic effect: an initial increase in frequency was followed by a decrease in the frequency of these oscillations. Intracellular recordings revealed that 48% of the recorded neurons depolarized, 33% remained inactive and 19% exhibited a biphasic response to SP (n=21). SP caused in 33% of the recorded neurons a change in intrinsic membrane properties. Under control conditions depolarizing current injections evoked tonic regular spiking activity, which increased in frequency when increasing the amplitude of current injections. The same stimuli evoked rhythmic bursting activity in the presence of SP. This bursting activity was voltage-dependent. Increasing the amplitude of depolarizing current injections increased the frequency of bursting activity, while brief hyperpolarizing pulses reset the bursting activity, indicating that these bursts were intrinsic to the cortical neuron. The bursting properties persisted in the presence of Cd²⁺ at concentrations (200 [mu]M) known to block all Ca²⁺ currents. The bursting was blocked by TTX suggesting that the burst generating mechanism depend on the activation of a TTX-sensitive persistent Na⁺ current. In neurons that generated bursting activity in the presence of NMDA, SP caused a significant increase in the frequency of bursting and the duration of individual bursts.
CONCLUSIONS: Our data indicate that SP can enhance the excitability of cortical neurons by inducing Cd²⁺-insensitive, but TTX-sensitive bursting activity. This increased excitability is a possible mechanism that promotes epileptiform activity. We therefore hypothesize that antagonists for SP receptor might serve as anticonvulsants by inhibiting bursting properties in cortical neurons.
[Supported by: Falk Foundation (WvD, CJM, KEH)
Rett Syndrome Research Foundation (JMR)
NIH HL 60120 (JMR)]