Michael E. Hall

Evidence in postmortem brain tissue for decreased numbers of hippocampal nicotinic receptors in schizophrenia

This study tests the hypothesis that nicotinic cholinergic receptors, including those sensitive to the antagonist alpha-bungarotoxin, are decreased in the hippocampus of schizophrenics. The hypothesis is derived from the finding that alpha-bungarotoxin causes a defect in the inhibitory gating of auditory-evoked potentials in laboratory animals that resembles a defect in auditory sensory gating observed in schizophrenics. Nicotine transiently normalizes this psychophysiological deficit in schizophrenic patients. Postmortem brain tissue was obtained from eight schizophrenic and eight age-matched nonschizophrenic subjects. Sections of the hippocampus were labeled with [125I alpha-bungarotoxin and imagined by autoradiography. Binding of the nicotinic agonist [3H]-cytisine was determined in tissue homogenates. alpha-Bungarotoxin labeled a population of putative interneurons in the hippocampus, primarily in the dentate gyrus and the CA3 region of Ammons horn. This labeling was significantly decreased in the tissue from the schizophrenic patients, with seven or eight patients below the range of the nonschizophrenic subjects. There was also a significant decrease in the binding of cytisine. The results were not related to generalized hippocampal cell loss, drug exposure at time of death, or smoking history. This initial study suggests that schizophrenic patients have fewer nicotinic receptors in the hippocampus, a condition which may lead to failure of cholinergic activation of inhibitory interneurons, manifest clinically as decreased gating of response to sensory stimulation.

Genetic correlation of inhibitory gating of hippocampal auditory evoked response and α-bungarotoxin-binding nicotinic cholinergic receptors in inbred mouse strains

One function of the hippocampus is to ascertain the novelty of incoming sensations and encode significant new information into memory. The regulation of response to repeated stimuli may prevent overloading of this function by redundant sensory input. Recent pharmacological studies implicate the role of α-bungarotoxin-sensitive nicotinic cholinergic receptors in the inhibition of hippocampal response to repeated auditory stimuli. The number of hippocampal α-bungarotoxin-sensitive receptors has a major genetic determinant, as demonstrated by a significant variance between different inbred mouse strains. The purpose of the present study was to determine whether there was a related genetic correlation for the gating of auditory response. Nine inbred mouse strains, representing a continuum of hippocampal α-bungarotoxin binding, were tested for the electrophysiological response to repeated auditory stimulation, followed by whole hippocampus membrane α-bungarotoxin binding studies. Several parameters of the auditory evoked response showed significant genetic variance over the nine strains, and a significant correlation was found between hippocampal α-bungarotoxin binding and both the amplitude of the initial evoked response and its inhibition to repeated auditory stimuli. There was no correlation of the auditory evoked response with high-affinity nicotine binding. These data further support the hypothesis that α-bungarotoxin-sensitive nicotinic receptors are involved in the regulation of hippocampal response to repeated auditory stimuli and suggest that this function is genetically controlled.

Rapid extraction of leukotrienes from biologic fluids and quantitation by high-performance liquid chromatography.

Previous methods for the recovery and quantitation of leukotrienes have involved tedious extraction procedures, and high-performance liquid chromatographic (HPLC) techniques with significant limitations. We have designed a method to extract leukotrienes from biologic fluids using commercially available silica mini-columns requiring minimal preparation. Sample clarification is followed by a sensitive and reproducible HPLC technique which separates and quantifies the leukotrienes LTC4, LTD4, LTB4 (and at least three of their isomers). The entire procedure requires less than one hour per sample.

Characterization of [3H]cytisine binding to human brain membrane preparations

The binding characteristics of [3H]cytisine, a putative CNS nicotinic receptor ligand, were examined in 4 regions of the human brain. [3H]Cytisine was found to bind non-cooperatively with high affinity to a single site in tissue homogenates and to exhibit low non-specific association. The binding characteristics of this ligand were evaluated in thalamus at 4 degrees C and 24 degrees C. The association constants were found to be 0.234 and 0.308 min-1 nM-1, while the dissociation constants were 0.007 and 0.098 min-1, respectively. Saturation analysis of thalamus revealed the equilibrium Kd to be 147 pM (4 degrees C) and 245 pM (24 degrees C), values in good agreement with those determined kinetically. The Hill coefficient varied slightly between brain regions; however, the mean values in all regions examined were close to 1.0 at 0.95 +/- 0.03 (4 degrees C) and 0.91 +/- 0.04 (24 degrees C). [3H]Cytisine binding could be displaced using both nicotinic agonists and antagonists. Cytisine was the most potent displacer of [3H]cytisine binding with an Ki of 250 pM. Nicotine and acetylcholine were also potent displacers with Ki values of 1.8 and 8.1 nM, respectively. The nicotinic antagonists alpha-bungarotoxin and mecamylamine were ineffective competitors for the [3H]cytisine binding site while dihydro-beta-erythroidine had an Ki value of 109 nM. Thalamus showed the highest density of cytisine binding sites of all the regions examined (48 fmol/mg protein) while the hippocampus, cingulate gyrus and the cortex showed Bmax values of 18.9, 19.3 and 8.8 fmol/mg protein, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of enzymatic processing in the biological actions of substance P

There is considerable evidence that substance P (SP) is a neurotransmitter in the CNS. Current findings suggest that the effects of synaptically released SP are terminated by enzymatic breakdown, primarily by endopeptidase 3.4.24.11 (endo 24.11). The products of cleavage by endo 24.11 include the amino-terminal fragment SP(1-7). Evidence suggests that SP is involved in mediating baroreceptor reflex activity in the nucleus of the solitary tract (NTS). Microinjection of SP into the NTS lowered blood pressure and heart rate. Microinjection of SP(1-7) into the NTS reproduced the effects of SP on both heart rate and blood pressure. Intra-NTS injection of phosphoramidon, an inhibitor of endo 24.11 activity, completely blocked the effects of a subsequent injection of SP. This blocking effect of phosphoramidon was unaltered by pretreatment with the opiate inhibitor naloxone. In contrast, phosphoramidon failed to block the depressor and bradycardic effects of SP(1-7). The implications of these findings regarding the role of endo 24.11 in the metabolism of SP are discussed.

Effects of substance P and neurokinin A (substance K) on motor behavior: unique effect of substance P attributable to its amino-terminal sequence.

The effects of intraventricular injections of the neuropeptides substance P (SP) and neurokinin A (NK-A; also called substance K) on spontaneous motor behavior were examined in mice. SP and NK-A were essentially equipotent at enhancing grooming and scratching behavior, and at reducing sniffing behavior. However, SP significantly enhanced hindlimb rearing behavior, while NK-A reduced this behavior. The effects of 3 other tachykinins, physalaemin, eledoisin and kassinin, were comparable to those of NK-A, including the reduction in rearing. Thus, SP is unique among tachykinins in its potentiation of rearing behavior. It was further demonstrated that carboxy-terminal SP fragments with tachykinin activity on smooth muscle resemble NK-A, and not SP, in their effects on motor behavior. In contrast, amino-terminal SP fragments, devoid of tachykinin-like activity, reproduced the one motor effect unique to SP, enhanced rearing, while lacking those actions common to all tachykinins. The structural requirements for enhanced rearing behavior by amino-terminal fragments were quite specific, in terms of chain length and sensitivity to D-amino acid substitutions, with the natural amino-terminal hexa- and heptapeptides being most active. The implications of these findings are discussed in light of recent observations that these same amino-terminal SP fragments are produced in vivo as metabolites of SP.

The substance P fragment SP(1-7) stimulates motor behavior and nigral dopamine release.

Earlier studies have shown that the undecapeptide substance P (SP) alters motor behavior and dopamine metabolism following injection into the substantia nigra (SN) in rat, even though the SN appears largely devoid of SP-specific (NK-1) receptors. In this report, intra-nigral injections of the amino-terminal SP fragment SP(1-7) enhanced rearing, sniffing and locomotor activity, and increased the nigral DOPAC-to-DA ratio. In addition, SP(1-7) increased 3H-DA release from the SN in vitro. These findings suggest that some of the effects of nigral SP on motor behavior and dopamine release are mediated by amino-terminal fragments of SP.

Modulation of blood pressure by substance P: opposite effects of N- and C-terminal fragments on anesthetized rats

Considerable evidence suggests that substance P (SP) may be a transmitter mediating the depressor effects of baroreceptor reflex activation within the brainstem, yet intracerebroventricular administration of SP is reported to result in a pronounced pressor effect. In this study, SP injected into the 4th cerebral ventricle produced a biphasic effect; a brief decrease in blood pressure followed by a lengthy increase. Similar injections of a carboxy-terminal fragment of SP produced only a pressor effect of long duration. Injection of an amino-terminal SP fragment produced only a brief, rapid depressor effect. These results suggest that the amino-terminal sequence of SP may be involved in mediating the depressor effects of baroreceptor activation.

Comparison of fetal rabbit brain xenografts to three different strains of athymic nude rats: electrophysiological and immunohistochemical studies of intraocular grafts.

Interest in the use of neural tissue transplantation for the study of CNS development and maturation and the potential use of this technique for the treatment of certain degenerative CNS disorders has led to our use of transplantation of neural tissue across species lines. Prior to extensive transplantation studies using athymic rats as recipients, we wished to evaluate the currently available strains of athymic rat for their suitability as host animals for xenografts of neural tissue. Fetal cerebellar and cerebral cortex tissue from rabbit brain of gestational age 20-25 days was dissected and transplanted to the anterior chamber of the eye of Harlan Wisconsin, Fisher 344 Jnu, or NCI-Harlan athymic nude rat strains. The brain tissue grafts were allowed to mature for 3 mo during which time the size and vascularity of each graft was monitored through the cornea of anesthetized hosts. In each group all of the transplants survived and grew to varying extents in the anterior chamber of the eye. Following the growth study in vivo extracellular recording of single neuronal activity was performed. Spontaneous neural activity was found in most transplants in all three groups with no difference in the viability or discharge rates of neurons between the groups. Illumination of the ipsilateral eye increased the firing rate of neurons in all three groups, suggesting excitatory cholinergic innervation of the grafted neurons from the host parasympathetic iris ground plexus. Antibodies directed against neurofilament protein, glial fibrillary acidic protein, synapsin, and tyrosine hydroxylase were used to characterize the transplants immunocytochemically and revealed no differences between the grafts in the three groups of recipients. All transplants contained significant numbers of glial and neuronal elements with the distribution resembling that in adult brain tissue. Some of the transplants contained a sparse innervation of tyrosine hydroxylase–positive fibers from the sympathetic plexus of the host iris. Furthermore, synapsin-immunoreactivity suggested that synaptogenesis had taken place within the grafts. Histological examination of the grafts revealed that 67% of the grafts had been infiltrated, to varying extents, by lymphocytes which led to areas of cell lysis and necrosis. All host animals had populations of T-cell receptor positive cells, most of which also expressed the T-cell surface antigens CD4 and CD8. However, no transplants were overtly rejected over the 15 wk period of study. Our investigation demonstrates that all of the athymic strains used in this study are able to mount an immune response against grafted fetal tissue, despite the absence of rejection, and that none of these strains is superior to the others with respect to suitability as a host for the long-term study of fetal CNS xenografts in oculo.

Nigral 5-HT and substance P-induced enhancement of passive avoidance retention

Peripheral, posttraining injection of substance P (SP) has been shown to facilitate the retention of aversive and appetitive learning tasks, suggesting that SP may play a role in information processing. In addition, SP may modulate the release of nigrostriatal monoamines, which have also been linked with avoidance learning. This paper examines the interaction between SP and nigrostriatal monoamines by observing the behavioral effects of neurochemical lesions on SP-induced avoidance retention, and by measuring changes in nigrostriatal monoamine activity and receptor regulation following avoidance training and SP injection. In Expt. 1, 5,7-dihydroxytryptamine lesions of the substantia nigra, but not the caudate, attenuated the retention-enhancing effects of posttraining SP injection. Further, 6-hydroxydopamine lesions of the substantia nigra produced a deficit in avoidance conditioning that was reversed by posttraining SP injection. Expts. 2 and 3 demonstrated that although passive avoidance training and posttraining SP injections did not significantly alter nigral 5-hydroxytryptamine (5-HT) activity, SP increased 5-HT1 receptor density. It was concluded that SP may affect avoidance retention by modulating nigral 5-HT activity.