An abrupt developmental shift in callosal modulation of sleep-related spindle bursts coincides with the emergence of excitatory-inhibitory balance and a reduction of somatosensory cortical plasticity

Abstract

Transecting the corpus callosum of postnatal day (P)1-6 rats disinhibits the production of spindle bursts (SBs) within primary somatosensory cortex (S1), most notably during periods of sleep-related myoclonic twitching. Here we investigated developmental changes in this callosally mediated disinhibition and its association with cortical plasticity. Recordings in P2-15 subjects revealed that callosotomy-induced disinhibition is a transient feature of early development that disappears abruptly after P6. This abrupt switch was accompanied by sharp decreases in myoclonic twitching and equally sharp increases in spontaneous SBs and in the number of GABAergic and glutamatergic presynaptic terminals in S1. Expression of the K+Cl- cotransporter 2 (KCC2) also increased across these ages. To determine whether these developmental changes are associated with alterations in cortical plasticity, pups were callosotomized at P1, P6, or P8, and tested over the subsequent week. Regardless of age, callosotomy immediately disrupted SBs evoked by forepaw stimulation. Over the next week, the P1 and P6 callosotomy groups exhibited full recovery of function; in contrast, the P8 group did not exhibit recovery of function, thus indicating an abrupt decrease in cortical plasticity between P6 and P8. Together, our data demonstrate that callosotomy-induced disinhibition is a transient phenomenon whose disappearance coincides with the onset of increased intrinsic connectivity, establishment of excitatory-inhibitory balance, and diminished plasticity in S1. Accordingly, our findings indicate that callosotomy-induced disinhibition of twitch-related SBs is a bioassay of somatosensory cortical plasticity and, in addition, support the hypothesis that myoclonic twitches, like retinal waves, actively contribute to cortical development and plasticity.

Figure 1

Changes in SB activity in sham and callosotomized (CCx) P2–15 rats. Top Panel: The rate of spontaneous SBs doubles after CCx at or before P6 but not thereafter. Middle Panel: CCx-induced disinhibition is accompanied by decreased interhemispheric SB latencies. Inset: Illustration of the method used to score SB latencies; only mean L⁻ values are presented in the figure. * significant difference between groups.

Figure 2

Developmental changes in percentage of time asleep and rates of active sleep-related twitching in sham and callosotomized (CCx) P2–15 rats. Top: In relation to shams, CCx had no effect on the percentage of time asleep across the 15-min recording period at any age. Bottom: Although the quantity of nuchal muscle twitches per 15 min decreased with age, especially after P6, CCx again had no effect on twitching at any age. Because twitches are produced within the brainstem, we anticipated that CCx would not influence their occurrence. Means ± sem.

Figure 3

(A) Effect of unilateral infusion of ACSF, muscimol, or bicuculline into S1 on spontaneous SB activity in sham and CCx P4 rats. Note that in CCx subjects, but not sham subjects, muscimol and bicuculline have equal and opposite effects on SB production. * significant difference from ACSF. Means ± sem. (B) The distribution of fluorescently labeled muscimol (0.01 M) after a 1 μl infusion into the left S1 of a P4 rat. Top: Coronal section of the left hemisphere showing the site of muscimol infusion (green box). Bottom: The boxed area above is magnified.

Figure 4

Cortical KCC2 protein levels increase at the end of the first postnatal week. (A) Western blot analysis of KCC2 expression in rat cortex at P5, P6, P7, P8, and P9. kDa, kilodalton. (B) The intensity of KCC2 bands normalized to P5. KCC2 levels are stable between P5 and P7, but increase sharply thereafter.

Figure 5

Changes in GABAergic and glutamatergic presynaptic terminals in P6–9 rats. (A) Number of S1 glutamatergic (VGLUT-1) and GABAergic (GAD) presynaptic terminals in layers 2–3 (left) and 4–6 (right), normalized to the number of cell nuclei, increase significantly at P7 in relation to P6. * significant difference from value at P6. Means ± sem. (B) Sections through S1 cortex in P6, P7, P8, or P9 sham-operated animals were stained with antibodies against inhibitory (GAD, green) and excitatory (VGlut-1, red) presynaptic terminals; nuclei were counterstained with DAPI (blue). The density of both GABAergic and glutamatergic terminals in layers 2–3 (top row) and layers 4–6 (bottom row) increases sharply between P6 and P7, with little subsequent change over the next two days.

Figure 6

Callosotomy (CCx) disrupts the SB response to peripheral stimulation of the contralateral forepaw in P2–15 rats. In sham subjects the response to stimulation of the forepaw reliably evokes an SB in contralateral S1 in P2–P15 infant rats; however, CCx immediately decreases the response to forepaw stimulation at all ages. * significant difference between groups. Means ± sem.

Figure 7

Effect of callosotomy (CCx) on evoked spindle burst (SB) responses to contralateral forepaw stimulation and recovery of function across different ages. Subjects received sham (squares) or CCx (triangles) surgery at (A) P1, (B) P6, or (C) P8 with subsequent testing over the ensuing week. In subjects that received CCx at P1, P6, and P8, evoked responding was immediately disrupted. Importantly, recovery of function was observed after 7 days in pups that received CCx at P1 or P6 (shaded boxes) but not in pups that received CCx at P8. * significant difference between groups. Means ± sem.

Figure 8

Model depicting hypothesized mechanisms underlying the development of excitatory-inhibitory balance in S1 between P6 and P10. The model depicts an S1 network comprising GABAergic interneurons (red circles) and glutamatergic pyramidal cells (green triangles) combined with callosal modulation of the intrinsic circuit. Left: At P6, CCx disinhibits SB activity in S1, suggesting net excitation (+) in the intrinsic cortical circuit and a net inhibitory influence (−) by the corpus callosum. Although GABA is typically depolarizing early in development, GABA-mediated inhibition can still occur as a result of shunting of glutamatergic excitatory postsynaptic currents (dashed line). According to this model, net callosal inhibition is achieved by preferential excitation of GABAergic interneurons as well as the presence of transient GABAergic callosal projections (most likely by inhibiting pyramidal cells, although such connectivity is not specified in the figure). Right: At P10 and after KCC2 upregulation, the hyperpolarizing effects of GABA predominate and the intrinsic cortical circuit has achieved excitatory-inhibitory balance. In addition, callosal inputs from the corpus callosum now exert equal effects on excitatory and inhibitory cortical neurons and GABAergic callosal projections have disappeared. As a consequence, CCx at this age has no discernible effect on spontaneous activity (although evoked responses are still affected).