Presynaptic Interactions between Trigeminal and Cervical Nociceptive Afferents Supplying Upper Cervical Lamina I Neurons

Abstract

Cervical and trigeminal afferents innervate neighboring cranial territories, and their convergence on upper cervical dorsal horn neurons provides a potential substrate for pain referral in primary headache syndromes. Lamina I neurons are central to this mechanism, as they relay convergent nociceptive input to supraspinal pain centers. Unfortunately, little is known about the interactions between trigeminal and cervical afferents supplying Lamina I neurons. Here, we used rats of both sexes to show that cervical and trigeminal afferents interact via presynaptic inhibition, where monosynaptic inputs to Lamina I neurons undergo unidirectional as well as reciprocal presynaptic control. This means that afferent-driven presynaptic inhibition shapes the way trigeminal and cervical Aδ-fiber and C-fiber input reaches Lamina I projection neurons (PNs) and local-circuit neurons (LCNs). We propose that this inhibition provides a feedforward control of excitatory drive to Lamina I neurons that regulates their convergent and cervical-specific or trigeminal-specific processing modes. As a consequence, disruption of the trigeminal and cervical afferent-driven presynaptic inhibition may contribute to development of primary headache syndromes.SIGNIFICANCE STATEMENT Cervical and trigeminal afferents innervate neighboring cranial territories, and their convergence on upper cervical dorsal horn neurons provides a potential substrate for pain referral in primary headache syndromes. Lamina I neurons are central to this mechanism as they relay convergent nociceptive input to supraspinal pain centers. Here, we show that cervical and trigeminal afferents interact via presynaptic inhibition, where inputs to Lamina I neurons undergo unidirectional as well as reciprocal control. The afferent-driven presynaptic inhibition shapes the trigeminocervical Aδ-fiber and C-fiber input to Lamina I neurons. This inhibition provides control of excitatory drive to Lamina I neurons that regulates their convergent and cervical-specific or trigeminal-specific processing modes. Disruption of this control may contribute to development of primary headache syndromes.

Keywords: C2 spinal nerve; dorsal root potentials; nociceptive afferents; presynaptic inhibition; trigeminal nerve; trigeminocervical complex.

Figure 1.

Recordings in the ex vivo brainstem-cervical cord preparation. A, Left panel, Schematic of the preparation used for recording TDRPs. The TDRPs were evoked by stimulating the C2 SN. The recording electrode was positioned on the ipsilateral trigeminal dorsal root close to where it entered the brainstem. Note, the C2 SN has been reflected toward the contralateral side. Right, Effect of picrotoxin on the TDRP. B, C2 DRPs were evoked by stimulating the TN. The recording electrode was positioned where the C2 root entered the spinal cord. Right, Effect of picrotoxin on the DRP. Both TDRPs and DRPs were studied in the hemisected preparation. C, Recording of ipsilateral cervical and trigeminal afferent inputs to the upper cervical Lamina I neurons in the isolated brainstem-cervical cord preparation. Note, the C2 SN has been reflected toward the contralateral side to gain access to Lamina I neurons. Left panel, Example responses (5 traces with their average) in a convergent Lamina I (LI) neuron (cell 267). In this neuron, C2 SN conditioning did not affect the monosynaptic C-fiber input evoked by stimulation of the TN. Holding potential, −80 mV. Averaged superimposed traces are shown below. The schematic at the bottom of the panel illustrates two monosynaptic inputs, which are not affected by presynaptic inhibition. Filled arrowheads indicate monosynaptic components for the EPSCs evoked by the test stimuli. Right panel, Example responses in a cervical-specific neuron (cell 33). In this neuron, TN conditioning did not affect the monosynaptic Aδ-fiber EPSC evoked by stimulation of the C2 SN. Note that the TN contacts the recorded neuron via a polysynaptic excitatory input. Holding potential, −85 mV. R, rostral; C, caudal.

Figure 2.

Responses modulated by presynaptic inhibition. Aa, Ab show afferent-driven presynaptic inhibition of the components of monosynaptic input (filled arrowheads). Aa, A full block of a monosynaptic trigeminal C-fiber EPSC by C2 SN conditioning (cell 178 from Table 1, note that the monosynaptic input to this convergent PN from the C2 SN is not indicated). Schematic shows how presynaptic inhibition affects the parent branch of the central axon. In, inhibitory interneuron. Holding potential, −85 mV. Ab, A partial block of the monosynaptic cervical C-fiber EPSC by TN conditioning (cell 129 from Table 1). Schematic illustrates how presynaptic inhibition affects the terminal branch of the afferent. Holding potential, −80 mV. Note the TN did not show monosynaptic or polysynaptic inputs to this cervical-specific neuron, but induced presynaptic inhibition of its monosynaptic cervical input. Ba, Bb, Inhibitory effects on the neuronal network. Ba, An overall reduction of the C2 SN-evoked test response after the TN conditioning (cell 198 from Table 1, the monosynaptic input to this convergent neuron from the TN is not indicated). Note the lack of the effect on monosynaptic Aδ-fiber-mediated and C-fiber-mediated EPSCs but a substantial reduction of the polysynaptic component. Schematic shows presynaptic inhibition at the cervical afferent branch supplying an intercalated excitatory neuron (Ex) or/and at the axon terminal of the intercalated neuron. Holding potential, −85 mV. Bb, A complete inhibition of the polysynaptic component in a neuron lacking monosynaptic input. C2 SN conditioning abolishes polysynaptic TN-mediated EPSCs in a cervical-specific Lamina I neuron (cell 132 from Table 1). Schematic shows presynaptic inhibition at the trigeminal afferent branch supplying an intercalated excitatory neuron (Ex) or/and at the axon terminal of the intercalated neuron. Holding potential, −70 mV. For each type of response, five traces are shown with their average. Averaged responses to the test stimuli are shown superimposed. Filled arrowheads indicate monosynaptic components only for the EPSCs evoked by the test stimuli. Ca, Numbers of Lamina I neurons affected by different types of presynaptic inhibition (left) and by the full or partial suppression of Aδ-fiber and C-fiber components of input (right). Data are shown for C2 SN (red) and TN (blue) conditioning. Cb, Numbers of convergent, cervical-specific and trigeminal-specific Lamina I neurons with inputs controlled by different forms of afferent-driven inhibition.

Figure 3.

Unidirectional presynaptic inhibition. A, Example of unidirectional inhibition of monosynaptic trigeminal C-fiber input to a convergent Lamina I neuron by C2 SN conditioning (cell 110 from Table 1). Note, the TN conditioning had no effect on the monosynaptic C2 SN input. Holding potential, −80 mV. B, unidirectional inhibition of the polysynaptic C2 SN input to a trigeminal-specific Lamina I neuron by TN conditioning (cell 52 from Table 1). Note, the C2 SN conditioning had no effect on monosynaptic trigeminal Aδ-fiber and C-fiber inputs. Holding potential, −80 mV. Schematic showing that presynaptic inhibition occurs at the cervical afferent terminal supplying an intercalated excitatory neuron (Ex) or/and at the axon terminal of the intercalated neuron. Five traces are shown with averaged traces superimposed. Averaged responses to the test stimuli are shown superimposed below. Filled arrowheads indicate monosynaptic components only for the EPSCs evoked by the test stimuli.

Figure 4.

Reciprocal presynaptic inhibition. An example of reciprocal afferent-driven presynaptic inhibition in a convergent Lamina I neuron (cell 256 from Table 1). C2 SN conditioning abolished the monosynaptic C-fiber-mediated EPSC component of the TN input, and TN conditioning completely abolished the monosynaptic C-fiber EPSC component of the C2 SN input. Note, both conditioning stimuli reduced the polysynaptic component activated by the test stimuli. Holding potential, −80 mV. Schematic depicting how an inhibitory neuron (In) can mediate presynaptic inhibition of afferents from both nerves. Five traces are shown for each response and the averaged responses are superimposed. Filled arrowheads indicate monosynaptic components only for the EPSCs evoked by the test stimuli.

Figure 5.

Inputs evoked by simultaneous stimulation of C2 SN and TN. The effect is illustrated for two convergent Lamina I neurons differently affected by presynaptic inhibition. Upper part, A PN (cell 185; Table 1) showing a partial (19%) unidirectional presynaptic inhibition of its monosynaptic Aδ-input from the TN. Top traces, EPSCs evoked by stimulating the C2 SN (left), TN (middle), and both nerves at the same time (right). Filled arrowheads indicate monosynaptic components. Five traces (gray) are shown for each type of stimulation and their averages are given in red, blue, and magenta, respectively. The averaged traces are shown superimposed below (left). C2 SN and TN, averaged response evoked by simultaneously stimulating both nerves. Right, The EPSC evoked by simultaneously stimulating both nerves is superimposed with the sum of the EPSCs evoked by the C2 SN and TN stimulations (C2 SN + TN, black). Holding potential, −80 mV. Bottom part, A convergent Lamina I neuron (cell 266; Table 1) showing a reciprocal inhibition of monosynaptic C components and a substantial effect on the network. Left, The averaged EPSCs are compared. Filled arrowheads indicate monosynaptic components. Right, The EPSC evoked by simultaneously stimulating both nerves is superimposed with the sum of the EPSCs evoked by the C2 SN and TN stimulations. Holding potential, −85 mV. The histogram shows the area under the EPSC (i.e., integral) evoked by stimulating the C2 SN, TN and both nerves simultaneously (C2 SN and TN), compared with the calculated sum of the C2 SN and TN inputs (C2 SN + TN). Pooled data from 14 neurons. Note, the integrals for simultaneously evoked inputs and the sum of two individual inputs are similar. N.s., nonsignificant.

Figure 6.

Presynaptic interactions between trigeminal and cervical afferents supplying upper cervical Lamina I. Schematic illustrating the major forms of presynaptic interactions between cervical and trigeminal Aδ-afferents and C-afferents supplying Lamina I neurons. Presynaptic inhibition of monosynaptic inputs can be reciprocal or unidirectional and affect both the parent or terminal branches of the afferent fiber. Presynaptic inhibition of the neuronal network regulates polysynaptic input to Lamina I neurons. This can be evoked by inhibiting the afferent axon, which supplies an intercalated excitatory neuron (Ex), or the axon terminal of the intercalated neuron. In some Lamina I neurons, no interaction between cervical and trigeminal afferent fibers was detected (no effect). PN, projection neuron; LCN, local-circuit neuron; In, inhibitory interneuron. Note, the precise laminar location of the inhibitory interneurons and excitatory intercalated neurons is not known.