Michiko Hamba

Effects of lesion and stimulation of rat hypothalamic arcuate nucleus on the pain system

Electrolytic lesion and stimulation of the arcuate nucleus of the hypothalamus (ARH) were investigated in Wistar albino rats. 1) Discrete lesion of the ARH is followed by hyperalgesia as manifested by significantly high values of pain rating in the formalin test and by depressed analgesic effect of electroacupuncture stimulation on digastric electromyogram (dEMG) activity. 2) ARH stimulation appreciably suppressed both the responses of neurons in the lateral hypothalamus (LHA) and dEMG activity induced by tooth pulp stimulation. The suppressive influence of ARH stimulation on the responses of LHA neurons was antagonized by intraperitoneally injected and electrophoretically applied naloxone. It is thus suggested that the ARH is involved in induction or modulation of opioid-mediated analgesia.

Expression of c-fos-like protein in the rat brain after injection of interleukin-1-beta into the gingiva

Expression of the c-fos proto-oncogene in the rat brain was examined by immunostaining for fos, the nuclear protein product of the c-fos gene, after injection of interleukin-1-beta (IL-1 beta) into the gingiva of an incisor. The distribution pattern of labelled cells was compared with that induced by tooth pulp stimulation. Neurons that express fos-immunoreactivity (fos-IR) appeared in several regions in the neuraxis 1.5 h after IL-1 beta injection, peaked at 2 h, and then declined. Labelled cells were found bilaterally in regions that contribute to pain-relay and pain-inhibition. The distribution of labelled cells almost matched the pattern induced by noxious tooth pulp stimulation. In indomethacin-pretreated animals, no neurons expressing fos-IR were found in nuclei associated with relay of nociception nor in nuclei contributing to inhibition of nociception. The results suggest that a small amount of IL-1 beta at the site of periodontal disease can induce fos-IR in brain neurons through increased prostaglandin production.

Wind-up of tooth pulp-evoked responses and its suppression in rat trigeminal caudal neurons.

Induction and suppression of wind-up were studied in 97 tooth pulp-driven neurons in the trigeminal subnucleus caudalis, using Wistar albino rats anesthetized with urethane and alpha-chloralose. Tooth pulp stimulation applied to an ipsilateral lower incisor evoked early discharges, indicating excitatory inputs from A-delta fibers and subsequent late discharges from C-fiber volleys in caudal neurons. Wind-up was efficiently evoked by stimulation delivered at 0.3-1 Hz, with current intensity sufficient to evoke late discharges. Conditioning stimulation of the arcuate nucleus of the hypothalamus (ARH) suppressed late discharges, including wind-up, without affecting the A-fiber response. Focal cooling of the periaqueductal gray (PAG) abolished the suppression by the ARH and further enhanced the wind-up of the caudal neurons. These results suggest: 1) Temporal summation of depolarization evoked by C-fiber volleys builds wind-up in caudal neurons; 2) ARH stimulation suppresses late discharges by blocking synaptic transmission from C-fiber inputs, and this interrupts prolonged facilitation of the neurons; 3) the ARH is involved in induction of inhibitory controls descending from the PAG to the trigeminal caudalis.

Stimulation-induced responses of the trigeminal caudal neurons in the brainstem preparation isolated from newborn rats.

The brainstem preparation with the trigeminal mandibular nerve attached was isolated from rats postnatal day 0-6 (P0-P6) to test if the potentiation could be induced in neonatal neurons in the trigeminal subnucleus caudalis by stimulation of the primary afferents. The stimulation-induced potentials in 92 neurons recorded extracellularly, and the synaptic potentials in 16 neurons recorded by the whole-cell patch clamp technique were examined. The extracellularly recorded neurons responded to stimulation (0.5 Hz) with either an increase, a decrease, or little change in spike numbers, and were classified as Type 1, Type 2, and Type 3, respectively. Type 1 neurons at P4 and older responded in a low Mg2+ solution with a progressive increase in spike number lasting for several minutes after the cessation of stimulation, i.e., short-term potentiation, STP. This potentiation was antagonized by 20 microM of (+)-MK-801 hydrogen maleate (MK-801) or 25 microM of 2-amino-5-phosphonovaleric acid. In contrast, Type 1 at P3 and younger did not exhibit STP. The age-related distinct response properties were observed between Type 1 neurons at P4-P6 and at P0-P3. The percentage of Type 1 in studied neurons increased from 24% at P0-P3 to 53% at P4-P6. In the intracellular experiment, the mean latency of excitatory postsynaptic potential (EPSP) of recorded neurons indicated that the conduction velocity of the convergent afferents was 0.37 m/s, in the range of C-fiber. Neurons were classified as Type E and Type I. Type E responded with EPSP only, or with both EPSP and inhibitory postsynaptic potentials (IPSP), while Type I responded with IPSP only. In Type E at P4 and older, a single stimulation produced a burst of spike discharges that lasted for several seconds. Stimulation at a hyperpolarized membrane potential showed that aggregated slow EPSPs lay under a burst of spike discharges, and that slow EPSPs, but not a short-latency EPSP, were completely blocked by MK-801. In contrast, Type E at P3 and younger did not evoke a burst of spikes. Morphological examination of recorded neurons showed that the formation of networks was sparse at P1 and rapidly developed up to P4. The results suggest that: (1) short-term potentiation is induced with the development of synaptic network formation in the caudal nucleus at P4 and older; (2) the summation of N-methyl-d-aspartate (NMDA)-mediated slow EPSPs build up a prolonged depolarization; and (3) the brainstem preparation is applicable for neurophysiological studies on the trigeminal pain system.

Nociceptive projection from tooth pulp to the lateral hypothalamus in rats

The response of the rat lateral hypothalamic (LHA) neurons to tooth pulp electrical stimulation and the sensory projection pathway from the incisor pulp to the LHA were studied by electrophysiology and histology. 1) LHA neurons that responded to contralateral lower incisor pulp stimulation were found in the lateral part of the LHA. These neurons also responded to intensive tail pinch, but not to innocuous stimuli nor to applied glucose. 2) Histological study after injection of WGA-HRP into the lateral part of the LHA revealed many retrogradely labeled neurons in the ventral part of the periventricular gray (PVG) in the mesencephalon. 3) The PVG neurons responded antidromically to LHA stimulation and to contralateral lower incisor pulp stimulation. 4) After injection of WGA-HRP into the ventral portion of the PVG, many labeled cells were found in the contralateral subnucleus caudalis in the spinal tract of the trigeminal nucleus (NTST) where termination of the pulpal afferent was previously reported. It is thus suggested that the PVG is the most likely site of transmission relay of nociceptive inputs from incisors to the LHA.

Effects of electroacupuncture on the neuronal activity of the arcuate nucleus of the rat hypothalamus.

Electroacupuncture (EA) effects on the spontaneous unit activities of 143 neurons in the hypothalamic arcuate nucleus (ARH) which were electrophysiologically identified to project to the median eminence were investigated using anesthetized rats weighing about 300 g. Stimuli were delivered unilaterally to a meridian point of Ho-Ku in the forepaw as rectangular pulses of 5 ms duration, intensity 300 to 500 microA, for 15 min at 3 and 45 Hz. Unit activities of ARH cells were extracellularly recorded and the mean firing rates were compared before and after EA stimulation. Stimulation at 3 or 45 Hz induced a long-lasting and naloxone-reversible suppression of the magnitude of the digastric electromyogram (dEMG) in the jaw opening reflex to 48 to 56% of the control value. Based upon the EA effects on the spontaneous firing rate, the ARH cells were classified into three types: the rate either increased (type I), decreased (type II), or did not change (type III). Type I, II, and III neurons composed 56, 40, and 4% of the recorded neurons (N = 45) when EA stimulation was applied at 3 Hz, and 27, 70, and 3% (N = 37) at 45 Hz, respectively. The distribution of the three types of ARH neurons after EA stimulation at 3 Hz was significantly different (P less than 0.05, chi-square test) from that at 45 Hz.

Newborn rat brainstem preparation with the trigeminal nerve attached for pain study

Studies using a brainstem-spinal cord preparation isolated from newborn rats have provided substantial information on neuro-physiology, -pharmacology and -anatomy of the respiratory center, such as mechanisms of respiratory rhythm generation, development of a respiratory center or respiratory reflex [T. Murakoshi, T. Suzue, S. Tamai, A pharmacological study on respiratory rhythm in the isolated brainstem-spinal cord preparation from newborn rat, Br. J. Pharmac. 86 (1985) 95-104 [5]; H. Onimaru, A. Arata, I. Homma, Primary respiratory rhythm generator in the medulla of brainstem-spinal cord preparations from newborn rats, Brain Res. 445 (1988) 314-324 [6]; H. Onimaru, I. Homma, Whole cell recordings from respiratory neurons in the medulla of brainstem-spinal cord preparations isolated from newborn rats, Pflügers Arch. 420 (1992) 399-406 [7]; J.C. Smith, K. Ballanyi, D.W. Richter, Whole-cell patch clamp recordings from respiratory neurons in neonatal rat brainstem in vitro, Neurosci. Lett. 314 (1992) 153-156 [10]; T. Suzue, Respiratory rhythm generation in the in vitro brainstem-spinal cord preparation of the neonatal rat, J. Physiol. (London) 354 (1984) 173-183 [11], reviewed in H. Onimaru, A. Arata, I. Homma, Neuronal mechanisms of respiratory rhythm generation: An approach using in vitro preparation, Jpn. J. Physiol. 47 (1997) 385-403 [8]]. Recently, the dissecting method of the preparation was modified to introduce a brainstem preparation with the trigeminal primary afferents attached for pain studies [M. Hamba, Repetitive stimulation potentiated the stimulus-evoked firing in the trigeminal caudalis-in vitro study. Neurosci. Res. 20 (1996) s163 [2]; M. Hamba, Stimulation-induced responses of the trigeminal caudal neurons in the brainstem preparation isolated from newborn rats, Brain Res. 785 (1998) 66-74 [3]]. As reported previously [3], the activity-dependent change in the excitability of pain-processing neurons, wind-up phenomenon, was studied in the trigeminal caudalis by stimulating the mandibular nerve, using a modified brainstem preparation isolated from newborn rats. The caudalis, the medulla dorsal horn, is known as the center for processing pain and sensory information from the cranio-facial area. The results indicated that the brainstem preparation is applicable for studies on the neuroplasticity at the pain-processing synapses. Here, we describe the method for isolation of a brainstem preparation with the trigeminal mandibular nerve attached and for recording the synaptic response evoked in the caudal neurons, using a whole-cell patch clamp technique. In the present study, we demonstrated repetitive stimulation-induced responses of caudal neurons at postnatal day 1 as an example showing the feasibility of the preparation for pain studies.