Motoneurone excitability during antidromically evoked inhibition after administration of a Thyrotrophin Releasing Hormone (TRH) analogue

Acute experiments have been performed in urethane anaesthetised rats to determine the effect of a TRH analogue (RX77368) on the excitability of motoneurone pools in the lumbar region of the spinal cord to paired antidromic stimuli (interval 10ms) delivered to the ventral root, by measuring the field potentials produced. Following the response (CR) to the first stimulus (conditioning stimulus), the response (TR) to the second stimulus (test stimulus) is reduced in size. In control experiments, the greater the amplitude of CR, the smaller TR becomes. After RX77368 injection, CR progressively increases over the initial post injection period. It would be expected therefore that during this time there would be a progressive decline in TR relative to CR. It has been shown however, that over this period there is no decline of TR relative to CR. There is speculation on a possible role of putative neuromodulators such as TRH in the maintenance of control parameters during motoneurone recruitment and/or increases in discharge frequency.     

Potentiation of motoneurone excitability by combined administration of 5-HT agonist and TRH analogue

Motoneurone field potentials have been recorded from the lumbar region of the spinal cord, to antidromic stimulation of a ventral root, in rats anaesthetised with urethane. Injection of the thyrotropin releasing hormone (TRH) analogue RX77368 (1mg/kg) plus the 5-hydroxytryptamine (5-HT) receptor agonist 5-methoxy-N, N-dimethyl-tryptamine (5MeODMT 0.4mg/kg) resulted in a potentiation of the increase in amplitude and duration of response, compared to when the drugs were given singly. These results are discussed in the context of possible interactions between 5-HT and TRH systems.     

Further studies on the effects of a thyrotropin releasing hormone analogue on locomotor activity in the rat

In these experiments, the relationship between two of the variables of locomotion, stepping frequency and velocity, after injection of the TRH analogue RX77368 (10 mg/Kg i.p.), has been studied. A shift towards higher stepping frequencies was observed, confirming previous observations. However, there was no difference between the velocities of locomotion produced by treated and control rats. In the treated rats the relationship between stepping frequency and velocity was disturbed, such that a higher stepping frequency was employed to attain any particular velocity. This was accompanied by a reduction in stride length. Possible reasons for this disturbance are discussed in terms of changes in muscle stiffness and proprioceptive reflexes.     

Presence of a membrane bound pyroglutamyl amino peptidase degrading thyrotropin releasing hormone in rat brain

In the present work we studied the pattern of degradation of [3H-Pro]-TRH by soluble and membrane fractions from rat brain. Demonstration of the membrane bound or soluble nature of the activities was obtained by comparing their distribution to that of lactate dehydrogenase and by looking at the effect of NaCl washes on the membrane fractions. We observed that the pyroglutamyl amino peptidase activity detected in brain homogenates is a result of two different enzymes. One of them is a soluble enzyme previously characterized, that needs DTT and EDTA for its expression, is inhibited by SH-blocking agents such as iodoacetamide and utilizes p-glu-beta-naphtylamide as a substrate. The other one, a membrane enzyme, is inhibited by chelating agents such as EDTA and DTT, is not affected by iodoacetamide and does not degrade p-glu-beta-naphtylamide. The later presents some specificity towards TRH as shown by competition experiments with TRH analogs. We were able to corroborate that the post proline cleaving enzyme acting on TRH is a soluble enzyme. In membranes we demonstrated also the presence of a post-proline dipeptidyl aminopeptidase. The membrane bound pyroglutamidase activity is a potential new source of L-his-L-pro-diketopiperazine in brain. The presence of a TRH degrading enzyme in membrane fractions is of particular importance in searching an inactivation mechanism of this peptide once it is released into the synaptic cleft.     

Growth hormone releasing factor immunoreactivity in rat hypothalamus

Neurones immunoreactive to antibodies against human pancreatic growth hormone releasing factor1-40 (hpGRF) were identified in the hypothalamus of the rat after pretreatment with colchicine. Reactive perikarya were concentrated in the arcuate nucleus and were also present around the anterior commissure. hpGRF immunoreactive fibres were observed in the median eminence and preoptic area where they tended to complement the distribution of somatostatin immunoreactive fibres. The distribution of GRF-immunoreactive perikarya in the rat hypothalamus is similar to that reported in monkey, and is consistent with other studies which suggest that neural mechanisms stimulatory for growth hormone secretion in the rat are situated in the medial basal hypothalamus.     

The effects of vasopressin on positively rewarded responding and on locomotor activity in rats

It has been suggested that arginine-vasopressin (AVP) enhances cognitive, and especially mnemonic, ability. Most studies have employed shock avoidance paradigms; we report the results of a study in which saline or vasopressin (0, 0.5 or 1 microgram, mcg, per rat, subcutaneous) pre-treated rats learned to press a lever for food reward. AVP was found to have a disruptive effect on aquisition, particularly when the tendency for these rats to produce extreme learning scores was taken into account. Locomotor activity, with and without vasopressin pre-treatment (0, 0.5, 1 or 2 mcg/rat), was also studied. Only the highest dose significantly reduced activity; therefore, the effects of AVP on acquisition are unlikely to have been caused by motor disruption. The results are discussed in terms of an hypothesis which suggests that AVP enhances arousal, hence influencing performance.     

Effects of thyrotropin releasing hormone on hypothalamic thermosensitive neurons of the rat

The actions of thyrotropin releasing hormone (TRH) were tested on the firing rat of temperature-sensitive and temperature-insensitive neurons in the rat preoptic/anterior area of the hypothalamus (POA). Iontophoretic application of TRH resulted in inhibition of the firing rate of temperature-insensitive, warm-sensitive and cold-sensitive neurons. The inhibitory response of cold sensitive neurons in TRH was relatively mild, however, and 2 of 6 of these neurons inhibited by TRH also displayed rebound increases in discharge rate following cessation of TRH application. These results parallel the hyperthermic effects of microinjected TRH and lend further support of the hypothesis that TRH acts as a neurotransmitter or neuromodulator in the POA portion of the CNS thermoregulatory network.     

Electrophysiological observations in patients with motor neuron disease receiving a thyrotropin releasing hormone analogue (RX77368).

Twenty nine patients with motor neuron disease receiving a thyrotropin releasing hormone analogue showed acute 25-30% increase in mean corrected fibre density and mean macro EMG median amplitude and area in brachial biceps muscle. The data are consistent with a direct or indirect action of the drug on anterior horn cells.     

Demonstration of a putative receptor site for cholecystokinin in rat brain

High affinity (apparent K_d ? 0.65 nM) and saturable (B_max ? 6.6 fmoles/mg protein) binding sites for cholecystokinin (CCK) have been demonstrated in crude synaptosomal membranes from rat cerebral cortex. Scatchard analysis of the equilibrium binding data indicates a single population of non-interacting sites. The specific binding of ¹²⁵I-CCK₃₃ to brain membranes is reversible ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 6 minutes}$), and can be inhibited by structurally related CCK analogues but not by unrelated neuropeptides or drugs. Specific CCK binding reaches equilibrium at a temperature-dependent rate, and is abolished when membranes are pre-treated with heat. Kinetic and competition studies suggest that these high affinity binding sites represent physiologically-relevant receptors for CCK in brain.     

Segmental distribution of thyrotropin releasing hormone in rat spinal cord

The segmental and laminar distribution of thyrotropin releasing hormone (TRH) was determined in the rat spinal cord using radioimmunoassay (RIA) and immunocytochemistry (ICC). Immunoreactive TRH was found in sensory, autonomic, and motor spinal columns. Dorsal horn TRH-containing fibers and cell bodies in lamina II and along the lamina II/III border were seen by ICC in all spinal cord segments. ICC showed dense TRH immunoreactivity in the sympathetic areas of the thoracic cord. Densely staining TRH-containing fibers were seen in the ventral horn of all spinal segments. RIA of whole segment extracts showed high concentrations in C6-T1 and T12-L6. Low levels were seen in C2-C4 and T5-T6. Other segments were intermediate in concentration. RIA and ICC results were comparatively evaluated.     

Properties of opiate binding in the rat spinal cord

Receptor sites for etorphine in spinal cord homogenates are sensitive to treatment with trypsin, phospholipase A₂, and -hydroxy-mercuribenzoate. The binding of enkephalin analogues selective for μ- and δ-opiate sites, and etorphine is decreased in the presence of NaCl (100 mM) and EDTA (5 mM). The effect of NaCl is reversed by MgCl₂. On the other hand ethylketocyclazocine (putative κ-ligand) binding is enhanced by NaCl. All ligands show decreased binding in the presence of GTP, however in the presence of NaCl the binding of ethylketocyclazocine becomes GTP-insensitive.     

Immunoreactive dynorphin in rat tissues and plasma

Distribution of immunoreactive dynorphin (ir-dyn) has been determined in rat tissues using a highly specific antiserum. Concentrations are highest in the pituitary, brain and spinal cord, as previously reported. In peripheral tissues highest levels occur in the stomach and upper intestine, but ir-dyn has also been detected in several other tissues, including heart and skeletal muscle. Unilateral vagotomy did not affect the amount of ir-dyn in heart or stomach, and denervation did not affect the amount in skeletal muscle. Gel permeation chromatography of the ir-dyn in gastrointestinal tract, heart, and skeletal muscle revealed a larger apparent molecular weight than that of the 17-residue dynorphin. Using octadecylsilyl-silica cartridges, it was possible to extract and concentrate ir-dyn from rat plasma. By gel permeation chromatography plasma immunoreactivity was found in two peaks, both of higher apparent molecular weight than dynorphin. This immunoreactivity was unaltered by hypophysectomy or adrenalectomy.     

Neurohypophysial peptides and the hippocampus. II. Excitation of rat hippocampal neurones by oxytocin and vasopressin applied in vitro

Neurohypophysial peptides were applied by superfusion to rat hippocampal slices. The peptides, arginine vasopressin, lysine vasopressin, arginine vasotocin and oxytocin, increased the activity of 88% of spontaneously active cells in the CA1 region and induced firing in many neurones that were not spontaneously active. The peptide sensitive cells appeared to be pyramidal cells rather than interneurones. The four peptides were found to be of roughly equivalent potency, producing a reversible, dose-dependent response in the range 10(-9) to 10(-6)M. Most of the cells were tested with more than one peptide and were always found to respond either to all or to none of them. The analogue [7-glycine]oxytocin and the deamino, dicarba derivatives of oxytocin and vasopressin were about as active as the parent compounds, but the oxytocin fragment prolyl-leucyl-glycinamide had no effect, and desglycinamide vasopressin was extremely weak. Responses to the peptides could be blocked by "specific" antagonists. The results suggest that all of the peptides are acting upon a single class of receptor.     

Neurohypophysial peptides and the hippocampus. I. Vasopressin immunoreactivity in the rat hippocampus

Immunocytochemical studies have demonstrated that nerve fibres containing immunoreactive vasopressin project to many areas of the central nervous system. In the present investigation, the presence of immunoreactive arginine vasopressin (IR-AVP) in the hippocampus of Wistar rats was confirmed by radioimmunoassay. The vasopressin content of the dorsal hippocampus was 30.3 ± 7.3 pg IR-AVP/mg soluble protein (mean ± SEM, n=9) and that of the ventral hippocampus was 81.4 ± 8.3 pg IR-AVP/mg soluble protein (n=9), while tissue from the cerebral cortex contained no detectable vasopressin. That the immunoreactivity was due to vasopressin was confirmed by its absence in hippocampal or cortical tissue from homozygous Brattleboro rats, which are genetically unable to synthesize vasopressin.     

A direct comparison of the rat and bovine arginine vasopressin/neurophysin II common precursor

Translation of polyA+ RNA from rat hypothalami in a reticulocyte lysate system yields a 19 000 dalton (19k) preproform which reacts with antibodies against bovine neurophysin II (Np II) and arginine vasopressin (AVP). Co-translational addition of canine microsomal membranes gives rise to a glycosylated 22 000 (22k) proform which co-migrates with an $\text{AVP}\text{Np II}$ common precursor extracted from rat hypothalamus. These rat precursors correspond to those obtained by translation of bovine mRNA except that the latter have molecular weights of 21 000 (21k) and 23 000 (23k), respectively (Schmale and Richter, 1981). Also as in the bovine system a second precursor with a molecular weight of 16 000 (16k) is synthesized from rat mRNA which reacts with anti Np II but not with anti AVP.     

Cataleptogenic and anticataleptic activity produced by cholecystokinin octapeptides in mice

The catalepsy induced by subcutaneously (sc.) and intracerebroventricularly (icv.) administered cholecystokinin octapeptide sulfate ester (CCK-8-SE) and desulfated cholecystokinin octapeptide (CCK-8-NS), and the effects of CCK-8-SE and CCK-8-NS on haloperidol-induced catalepsy, were investigated in mice. The results demonstrate the bimodal effect of CCK octapeptides in a catalepsy test. With sc. administration CCK-8-SE in the doses of 0.4 or 0.8 mumole/kg, but not CCK-8-NS at any dose, induced catalepsy. Furthermore, the catalepsy induced by CCK-8-SE was of short duration. With icv. administration only 40 pmole CCK-8-NS induced significant catalepsy. When 0.2, 0.4 and 0.8 mumole/kg sc. doses of CCK-8-NS or 0.4 pmole icv. dose of CCK-8-SE or CCK-8-NS was given in combination with intraperitoneal (ip.) administration of 1.0 mg/kg haloperidol, the total duration of catalepsy was suppressed. Finally, CCK-8-SE sc. when given in combination with haloperidol ip., exerted a biphasic, synergistic-antagonistic effect on the haloperidol-induced catalepsy.     

The distribution and characterisation of thyrotrophin releasing hormone (TRH) in the human brain

Thyrotrophin releasing hormone-like immunoreactivity (TRHLI) extracted from human hypothalamus was found to be homogeneous and identical to authentic TRH in its chromatographic behaviour on reverse-phase high performance liquid chromatography. The majority of extrahypothalamic TRH also behaved as authentic TRH on HPLC. Study of the regional distribution of TRHLI showed a high concentration in the hypothalamus, periaqueductal grey and amygdala, with lower amounts in the basal ganglia and only trace amounts of TRHLI in the cerebral cortex and cerebellum.     

Localization of immunoreactive thyrotropin releasing hormone in the lower brainstem of the rat

The distribution of thyrotropin releasing hormone (TRH) in individual nuclei of the rat brainstem was examined by radioimmunoassay. TRH was detectable in 36 of 40 brainstem nuclei investigated and the localization of TRH in the brainstem was unlike other known brainstem neuropeptides. By far the highest concentration of TRH in the brainstem (1.2 ng/mg protein) is present in the nucleus of the solitary tract. The concentration of TRH was relatively high in the motor nuclei of the IIIrd, Vth, VIIth, Xth and XIIth cranial nerves, and less high in the area postrema, nucleus gracilis, locus coeruleus, lateral reticular nucleus (A1-catecholamine cell group), dorsal raphe and central gray matter. Cerebellum and pontine nuclei contained very low levels (<0.03ng/mg protein) of TRH.     

Analysis of acetylcholinesterase synthesis and transport in the rat hippocampus: recovery of acetylcholinesterase activity in the septum and hippocampus after administration of diisopropylfluorophosphate

The site and rate of synthesis, as well as the transport dynamics, of newly synthesized acetylcholinesterase (AChE) has been studied in the septum and hippocampus of the rat. Histochemical and biochemical techniques were employed to study time-dependent changes in AChE activity of the medial septal nucleus and hippocampus following systemic administration of the anticholinesterase diisopropyl-fluorophosphate. In both septum and hippocampus, an initially rapid phase of recovery was followed by a slower recovery with similar rate constants for the two regions. Analysis of AChE synthesis in the septo-hippocampal system revealed that recovery of activity in the septum(t_1/2for recovery= 140h) preceded that in the hippocampus(t_1/2for recovery= 400h). The data suggested that the bulk of septal AChE in the hippocampus is transported via the slow component of axoplasmic flow (about 2 mm/day). A fast component may exist, but the experimental arrangement did not permit the clear demonstration of any rapid axoplasmic flow.