Release of nociceptin-like substances from the dorsal horn of rat spinal cords in situ was measured by the immobilized-antibody microprobe technique. Spinal cords removed from anesthetized 4-6 week-old rats were superfused with oxygenated Krebs solution at room temperature (21 ± 1°C). Glass microelectrodes, coated with antibodies to nociceptin, were inserted into the dorsal horn of the lumbar spinal cord (1.9 mm lateral to the midline to a depth 2.5 mm below the surface of the cord) for 15 rain periods before, during and after electrical stimulation applied to the dorsal root entry zone of the same segment. There was a basal release of immunoreactive nociceptin- like substance (irNC) from the dorsal horns during the pre-stimulation period. A significant increase in irNC release was detected during the period of electrical stimulation and this increase was maintained for at least 15 min following the cessation of electrical stimulation. These results provide the first evidence on the release of irNC, albeit non-quantitative, from the in situ rat spinal cord dorsal horn and an enhanced release upon electrical stimulation.

Antibody-coated microprobes have been demonstrated to be useful for detecting the release of neuropeptide transmitters from discrete sites in the central nervous system (CNS). This technique uses glass micropipettes taken through a series of chemical coatings, starting with a γ- aminopropyltriethoxysilane solution and ending with the antibody specific to the peptide transmitter of interest. The key to the reliability and repeatability of the technique is a uniform, even coating of the siloxane polymer to the glass micropipette. The microprobes, as they are called following the completion of the coating process, are inserted stereotaxically into a specific area of the CNS and the physiological intervention is performed. Tip diameters are around 5-10 μm and, depending on the length of the pipette inserted into the CNS, diameters of the pipette shaft will approach 40-50 μm. Once removed, the microprobe is then incubated with the radiolabeled peptide. Binding of the radiolabeled peptide will occur to the antibody sites not occupied by the endogenously released peptide. The images of the microprobes on sensitive autoradiographic film are analyzed for differences in the optical density along a specified length of probe. Areas of lighter density signify sites along the microprobe where endogenous peptide was biologically released during the physiological intervention. Knowing the exact location of the probe tip in vivo in the CNS permits identification of neurophysiological sites corresponding along the length of the microprobe where the peptide was released.

During myocardial ischemia, the cranial cervical spinal cord (C1-C2) modulates the central processing of the cardiac nociceptive signal. This study was done to determine 1) whether C2 SCS-induced release of an analgesic neuropeptide in the dorsal horn of the thoracic (T4) spinal cord; 2) if one of the sources of this analgesic peptide was cervical propriospinal neurons, and 3) if chemical inactivation of C2 neurons altered local T4 substance P (SP) release during concurrent C2 SCS and cardiac ischemia. Ischemia was induced by intermittent occlusion of the left anterior descending coronary artery (CoAO) in urethane-anesthetized Sprague-Dawley rats. Release of dynorphin A (1-13), (DYN) and SP was determined using antibody-coated microprobes inserted into T4. SCS alone induced DYN release from laminae I-V in T4, and this release was maintained during CoAO. C2 injection of the excitotoxin, ibotenic acid, prior to SCS, inhibited T4 DYN release during SCS and ischemia; it also reversed the inhibition of SP release from T4 dorsal laminae during C2 SCS and CoAO. Injection of the κ-opioid antagonist, nor-binaltorphimine, into T4 also allowed an increased SP release during SCS and CoAO. CoAO increased the number of Fos-positive neurons in T4 dorsal horns but not in the intermediolateral columns (IML), while SCS (either alone or during CoAO) minimized this dorsal horn response to CoAO alone, while inducing T4 IML neuronal recruitment. These results suggest that activation of cervical propriospinal pathways induces DYN release in the thoracic spinal cord, thereby modulating nociceptive signals from the ischemic heart.

Antibody coated microprobes, inserted into the spinal cord at the L4-5 level, were used to detect whether endomorphin-2 (Endo2) was released from spinal dorsal horns in anesthetized rats in response to formalin injected into the hindpaw footpads. Saline injections were used as a control and substance P (SP) was measured to verify activation of nociceptive afferent fibers. SP but not Endo2 was released during pre-stimulation periods. Saline injections did not cause the release of either Endo2 or SP from the spinal cord. Formalin injections caused an increase in Fos expression as well as a release of SP, but not Endo2 from the ipsilateral side dorsal horn in L4-5. We conclude that Endo2 does not play a role in mediating the in vivo responses to acute inflammatory nociceptive signals at the spinal level in the anesthetized rat model.

The in-vivo observation of the neural activities generated by a large number of closely located neurons is believed to be crucial for understanding the nervous system. Moreover, the functional electrical stimulation of the central nervous system is an effective method to restore physiological functions such as limb control, sound sensation, and light perception. The Deep Brain Stimulation (DBS) is being successfully used in the treatment of tremor and rigidity associated with advanced Parkinson's disease. Cochlear implants have also been employed as an effective treatment for sensorineural deafness by means of delivering the electrical stimulation directly to the auditory nerve. The most significant contribution of this PhD study is the development of next-generation microprobes for the simultaneous stimulation and recording of the cortex and deep brain structures. For intracortical applications, millimetre length multisite microprobes that are rigid enough to penetrate into the cortex while integrated with flexible interconnection cables are demanded. In chronic applications, the flexibility of the cable minimizes the tissue damage caused by the relative micro-motion between the brain and the microprobe. Although hybrid approaches have been reported to construct such neural microprobes, these devices are brittle and may impose severe complications if they break inside the tissue. In this project, MEMS fabrication processes were employed to produce non-breakable intracortical microprobes with an improved structural design. These 32 channel devices are integrated with flexible interconnection cables and provide enough mechanical strength for penetration into the tissue. Polyimide-based flexible implants were successfully fabricated and locally reinforced at the tip with embedded 15 µm-thick gold micro-needles. In DBS applications, centimetre long microprobes capable of stimulating and recording the neural activity are required. The currently available DBS probes, manufactured by Medtronic, provide only four cylindrical shaped electrode sites, each 1.5 mm in height and 1.27 mm in diameter. Although suitable for the stimulation of a large brain volume, to measure the activity of a single neuron but to avoid measuring the average response of adjacent cells, recording sites with dimensions in the range of 10 - 20 µm are required. In this work, novel Three Dimensional (3D) multi channel microprobes were fabricated offering 32 independent stimulation and recording electrodes around the shaft of the implant. These microprobes can control the spatial distribution of the charge injected into the tissue to enhance the efficacy and minimize the adverse effects of the DBS treatment. Furthermore, the device volume has been reduced to one third the volume of a conventional Medtronic DBS lead to significantly decrease the tissue damage induced by implantation of the microprobe. For both DBS and intracortical microprobes, the impedance characteristics of the electrodes were studied in acidic and saline solutions. To reduce the channel impedance and enhance the signal to noise ratio, iridium (Ir) was electroplated on gold electrode sites. Stable electrical characteristics were demonstrated for the Ir and gold electrodes over the course of a prolonged pulse stress test for 100 million cycles. The functionality and application potential of the fabricated microprobes were confirmed by the in-vitro measurements of the neural activity in the mouse hippocampus. In order to reduce the number of channels and simplify the signal processing circuitry, multiport electrostatic-actuated switch matrices were successfully developed, fabricated, and characterized for possible integration with neural microprobes to construct a site selection matrix. Magnetic-actuated switches have been also investigated to improve the operation reliability of the MEMS switching devices.

The in-vivo observation of the neural activities generated by a large number of closely located neurons is believed to be crucial for understanding the nervous system. Moreover, the functional electrical stimulation of the central nervous system is an effective method to restore physiological functions such as limb control, sound sensation, and light perception. The Deep Brain Stimulation (DBS) is being successfully used in the treatment of tremor and rigidity associated with advanced Parkinson's disease. Cochlear implants have also been employed as an effective treatment for sensorineural deafness by means of delivering the electrical stimulation directly to the auditory nerve. The most significant contribution of this PhD study is the development of next-generation microprobes for the simultaneous stimulation and recording of the cortex and deep brain structures. For intracortical applications, millimetre length multisite microprobes that are rigid enough to penetrate into the cortex while integrated with flexible interconnection cables are demanded. In chronic applications, the flexibility of the cable minimizes the tissue damage caused by the relative micro-motion between the brain and the microprobe. Although hybrid approaches have been reported to construct such neural microprobes, these devices are brittle and may impose severe complications if they break inside the tissue. In this project, MEMS fabrication processes were employed to produce non-breakable intracortical microprobes with an improved structural design. These 32 channel devices are integrated with flexible interconnection cables and provide enough mechanical strength for penetration into the tissue. Polyimide-based flexible implants were successfully fabricated and locally reinforced at the tip with embedded 15 µm-thick gold micro-needles. In DBS applications, centimetre long microprobes capable of stimulating and recording the neural activity are required. The currently available DBS probes, manufactured by Medtronic, provide only four cylindrical shaped electrode sites, each 1.5 mm in height and 1.27 mm in diameter. Although suitable for the stimulation of a large brain volume, to measure the activity of a single neuron but to avoid measuring the average response of adjacent cells, recording sites with dimensions in the range of 10 - 20 µm are required. In this work, novel Three Dimensional (3D) multi channel microprobes were fabricated offering 32 independent stimulation and recording electrodes around the shaft of the implant. These microprobes can control the spatial distribution of the charge injected into the tissue to enhance the efficacy and minimize the adverse effects of the DBS treatment. Furthermore, the device volume has been reduced to one third the volume of a conventional Medtronic DBS lead to significantly decrease the tissue damage induced by implantation of the microprobe. For both DBS and intracortical microprobes, the impedance characteristics of the electrodes were studied in acidic and saline solutions. To reduce the channel impedance and enhance the signal to noise ratio, iridium (Ir) was electroplated on gold electrode sites. Stable electrical characteristics were demonstrated for the Ir and gold electrodes over the course of a prolonged pulse stress test for 100 million cycles. The functionality and application potential of the fabricated microprobes were confirmed by the in-vitro measurements of the neural activity in the mouse hippocampus. In order to reduce the number of channels and simplify the signal processing circuitry, multiport electrostatic-actuated switch matrices were successfully developed, fabricated, and characterized for possible integration with neural microprobes to construct a site selection matrix. Magnetic-actuated switches have been also investigated to improve the operation reliability of the MEMS switching devices.

Release of endomorphin (ENDO)-2 like substances from the dorsal horn of the isolated rat spinal cord was measured by the immobilized-antibody microprobe technique. Spinal cords were removed from anesthetized 4-6 week old rats and superfused with oxygenated Krebs solution at room temperature. Glass microprobes coated with ENDO-2 antibodies were inserted into the dorsal horn of the lumbar spinal cord 1.5 mm lateral to the midline to a depth 2.5 mm below the dorsal surface of the cord. Each probe remained in situ for 10 min periods before, during and after electrical stimulation applied to the dorsal root entry zone of the same spinal segment. There was no detectable basal release of immunoreactive endomorphin-2 like substance (irENDO) from the dorsal horns during the pre-stimulation, nor following the stimulation period. A significant release of irENDO was measured during the electrical stimulation. These results provide the first evidence of a irEndo release that is correlated spatially with the dorsal horn laminae I and II where ENDO-2-immunoreactive fibers are concentrated in the dorsal horn in response to electrical activation of primary afferent fibers.

The upper cervical spinal region functions as an intraspinal controller of thoracic spinal reflexes and contributes to neuronal regulation of the ischemic myocardium. Our objective was to determine whether stimulation of the C2 cervical spinal cord (SCS) of rats modified the input signal at the thoracic spinal cord when cardiac ischemia-sensitive (sympathetic) afferents were activated by transient occlusion of the left anterior descending coronary artery (CoAO). Changes in c-Fos expression were used as an index of neuronal activation within the spinal cord and brain stem. The pattern of substance P (SP) release, a putative nociceptive transmitter, was measured using antibody-coated microprobes. Two SCS protocols were used: reactive SCS, applied concurrently with intermittent CoAO and preemptive, sustained SCS starting 15 min before and continuing during the repeated intermittent CoAO. CoAO increased SP release from laminae I and II in the T4 spinal cord above resting levels. Intermittent SCS with CoAO resulted in greater levels of SP release from deeper laminae IV-VII in T4 than CoAO alone. In contrast, SP release from laminae I and II was inhibited when CoAO was applied during preemptive, sustained SCS. Preemptive SCS likewise reduced c-Fos expression in the T4 spinal cord (laminae I-V) and nucleus tractus solitarius but increased expression in the intermediolateral cell column of T4 compared with CoAO alone. These results suggest that preemptive SCS from the high cervical region modulates sensory afferent signaling from the ischemic myocardium.

Antibody-coated microprobes were inserted into the thoracic (T3-4) spinal cord in urethane-anesthetized Sprague-Dawley rats to detect the differences in the release of immunoreactive substance P-like (irSP) substances in response to differential activation of cardiac nociceptive sensory neurons (CNAN). CNAN were stimulated either by intrapericardial infusion of an inflammatory ischemic exudate solution (IES) containing algogenic substances (i.e., 10 mM each of adenosine, bradykinin, prostaglandin E2, and 5-hydroxytryptamine), or by transient occlusion of the left anterior descending coronary artery (CoAO). There was widespread basal release of irSP from the thoracic spinal cord. Stimulation of the CNAN by IES did not alter the pattern of release of irSP. Conversely, CoAO augmented the release of irSP from T3-4 spinal segments from laminae I-VII. This CoAO-induced irSP release was eliminated after thoracic dorsal rhizotomy. These results indicate that heterogeneous activation of cardiac afferents, as with focal coronary artery occlusion, represents an optimum input for activation of the cardiac neuronal hierarchy and for the resultant perception of angina. Excessive stimulation of cardiac nociceptive afferent neurons elicited during regional coronary artery occlusion involves the release of SP in the thoracic spinal cord and suggests that local spinal cord release of SP may be involved in the neural signaling of angina.

Electrostimulatory forms of therapy can reduce angina that arises from activation of cardiac nociceptive afferent fibers during transient ischemia. This study sought to determine the effects of electrical stimulation of left thoracic vagal afferents (C8-T1 level) on the release of putative nociceptive [substance P (SP)] and analgesic [dynorphin (Dyn)] peptides in the dorsal horn at the T4 spinal level during coronary artery occlusion in urethane-anesthetized Sprague-Dawley rats. Release of Dyn and SP was measured by using antibody-coated microprobes. While Dyn and SP had a basal release, occlusion of the left anterior descending coronary artery only affected SP release, causing an increase from lamina I-VII. Left vagal stimulation increased Dyn release, inhibited basal SP release, and blunted the coronary artery occlusion-induced release of SP. Dyn release reflected activation of descending pathways in the thoracic spinal cord, because vagal afferent stimulation still increased the release of Dyn after bilateral dorsal rhizotomy of T2-T5. These results indicate that electrostimulatory therapy, using vagal afferent excitation, may induce analgesia, in part, via inhibition of the release of SP in the spinal cord, possibly through a Dyn-mediated neuronal interaction.