Kevin C. F. Fone

Involvement of catecholaminergic neurones and α-adrenoceptors in the Wet-dog shake and forepaw licking behaviour produced by the intrathecal injection of an analogue of thyrotrophin-releasing hormone (CG 3509)

Intrathecal injection of the analogue of TRH, CG 3509, into conscious rats produced dose-related wet-dog shakes and forepaw licking, which showed a bell-shaped relationship of intensity to dose. Pretreatment with alpha-MPT intraperitoneally, markedly reduced levels of noradrenaline and dopamine in the spinal cord and brainstem and attenuated both CG 3509-induced responses, while intrathecal treatment with DSP4 selectively reduced noradrenaline in the spinal cord without affecting either behaviour. Since denervation supersensitivity may develop following treatment with DSP4, these results are not inconsistent with a proposal that bulbospinal noradrenergic neurones modulate the behaviour induced by CG 3509. Wet-dog shakes and forepaw licking induced by CG 3509 were reduced by pretreatment with phenoxybenzamine or prazosin, suggesting that a tonic noradrenergic pathway may facilitate both behavioural responses through alpha 1-adrenoceptors. Methoxamine, combined with CG 3509 partially attenuated the wet dog shake behaviour, but methoxamine produced marked hindlimb jerking which might physiologically antagonise wet-dog shakes. Concomitant administration of clonidine and CG 3509 potently reduced wet-dog shakes in a dose-related manner but did not significantly affect forepaw licking, while idazoxan did not significantly affect either response. The latter findings imply that alpha 2-adrenoceptors play different roles in modulating the two behavioural responses and the possible synaptic location of the receptors is discussed. Taken together these results suggest that CG 3509 may release noradrenaline from bulbospinal neurones regulating motor function.

TRH-catecholamine interactions in brain and spinal cord.

It is well-established that the central actions of thyrotropin-releasing hormone (TRH) are not confined to its role as a hormone in the hypothalamus-pituitarythyroid axis. In rodents, this tripeptide has been shown to produce behavioral excitation and increased locomotor activity when injected either peripherally- or into specific brain regions such as the nucleus a c c u m b e n ~ ~ ~ ~ . ~ and hypothalamus. lo Various pharmacological and biochemical investigations have indicated that TRH probably produces some of these behavioral effects by increasing dopamine turnover and release in the n. a c c ~ m b e n s . ~ ~ ~ ~ ~ l ~ This interaction between TRH and the mesolimbic dopamine system is likely to be of physiological importance because (a) this brain region is densely innervated with TRH-containing nerve terminals and fibers and contains a large number of TRH receptors,15-17 and (b) changes in response to a TRH analogue injected into the n. accumbens is sensitized following central infusion of TRH antibodies. I* The evidence that TKHinduced hyperactivity may, in part, involve the interaction of this peptide with mesolimbic (and possibly striatal) dopamine has been reviewed recently elsewhere. l9 There is evidence to suggest that TRH may also influence the function of another catecholamine neurotransmitter, noradrenaline. For example, rearing behavior in rats appears to be mediated by a noradrenergic mechanism.2o Various groups have shown previously that peripheral administration of TRH increases noradrenaline turnover in rodent b r a i r ~ . ~ ~ ~ Furthermore, various behavioral effects of TRH or a TRH analogue (MK 771) were inhibited using adrenoceptor antagonists, and the depletion of brain noradrenaline by a-methyl-p-tyrosine was enhanced by TRH.25 Ascending noradrenergic pathways in brain are well-documented and noradrenaline-containing neurones richly innervate cortical and limbic regions, including the hypothalamus.26 In addition to ascending pathways,

A comparison of the motor behaviours produced by the intrathecal administration of thyrotrophin-releasing hormone and thyrotrophin-releasing hormone analogues in the conscious rat

The behaviours evoked by the intrathecal injection of thyrotrophin-releasing hormone and a variety of analogues (RX77368, CG3509 and CG3703) were examined in conscious rats and the spread of injectate at the peak of the behavioural response was determined using 14C-labelled RX77368. The number of wet-dog shakes observed following intrathecal injection of thyrotrophin-releasing hormone, RX77368, CG3509 and CG3703 was linearly related to log10 dose (0.01-200 micrograms) in the first 6 min with the relative potencies being 1:7:10:60 respectively. The thyrotrophin-releasing hormone analogues also produced a marked forepaw-licking behaviour, but this did not increase with dose. Intrathecal or intraperitoneal pretreatment with prazosin (0.5 microgram and 1 or 2 mg/kg, respectively) attenuated both the wet-dog shake and forepaw-licking behaviours normally produced by the thyrotrophin-releasing hormone peptides. Following intrathecal [14C]RX77368 the radioactivity was principally restricted to the spinal cord with only limited amounts rostral to the rhombencephalon. These results imply that a tonically active bulbospinal noradrenergic pathway facilitates both thyrotrophin-releasing hormone-induced behaviours via alpha 1-adrenoceptors.

Comparative behavioural and biochemical effects of repeated intrathecal administration of thyrotrophin-releasing hormone (TRH) or two analogues of TRH in adult rats.

The effect of repeated intrathecal injection of thyrotrophin-releasing hormone (TRH) and two analogues of TRH, C-terminally modified RX 77638 and N-terminally modified CG 3509, were examined on behavioural (wet-dog shakes and forepaw licking) and biochemical markers for spinal motoneurones, bulbospinal raphe nerve terminals and the pituitary-thyroid axis in rats. Saline (10 microliters washed in with 15 microliters), TRH (20 micrograms), RX 77368 (2 micrograms) or CG 3509 (2 micrograms) were administered intrathecally (twice daily for 3 or 5 days), after which levels of plasma-free thyroxine and thyroid-stimulating hormone (TSH) were measured and the dorsal and ventral portions of the thoracolumbar spinal cord, brainstem and hypothalamus were assayed for TRH- and calcitonin gene-related peptide (CGRP)-like immunoreactivity, levels of indoleamines and the activity of choline acetyltransferase (ChAT). Behavioural tolerance developed rapidly with consecutive injections of RX 77368, such that wet-dog shakes were significantly reduced and forepaw-licking tended to be decreased by the third intrathecal injection. Five, but not 3, days of administration of RX 77368 selectively elevated levels of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid in the ventral spinal cord, where these substances are principally located in bulbospinal raphe nerve terminals. The time course of the change in indoleamines suggests that administration of TRH peptides elevated the synthesis, rather than the release, of 5-HT from these nerve terminals.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional Interactions between TRH and 5‐Hydroxytryptamine (5‐HT) and Proctolin in Rat Brain and Spinal Cord

There are now several studies indicating a functional interaction between particular neuropeptides and amine neurons either in the short-term, such as alterations in release (e.g. TRH increases dopamine and noradrenaline release), or in the long-term by acting as trophic factors..* Such studies have lead to the concept of neuropeptides acting as neuromodulators in the CNS, and particular attention has been given to those peptides that coexist with conventional amine neurotransmitters. The present paper will discuss the evidence that thyrotropinreleasing hormone (TRH) is involved in the regulation and modification of certain 5-HT responses in the CNS. TRH is present in the brain in several regions other than the hypothalamus, the site of its neuroendocrine role; these include limbic areas, such as the nucleus accumbens and the septum as well as the ventral horn of the spinal cord. In these regions, high levels of TRH immun~reactivity,~,~ and saturable TRH binding are found, and Ca++-dependent release of TRH can be demonstrated in ~ i t r o . ~ * * In the spinal cord, TRH immunoreactivity is found in the same descending medullary raphe neurons as 5-HT and substance P. Evidence for this comes from the observation that administration of the serotonergic neurotoxins 5,7and 5,6dihydroxytryptamine (5,7and 5,6-DHT) result in the loss of TRH and substance P together with 5-HT.9.0 5-HT and the two peptides also have similar electrophysiological effects on motor neurons within the spinal cord,11 and some data are consistent with the view that TRH and 5-HT interact at the receptor level to facilitate spinal motor neuron function.* More recently, Holets et a1.I3 have shown that there is proctolin-like immunoreactivity within the same rat bulbospinal neurons as TRH and 5-HT, though there is no information available about the effects of this invertebrate neuropeptide on TRH-induced behavioral or biochemical responses. There is also evidence that TRH and 5-HT interact, though not necessarily coexist, within certain brain areas. Thus 5,7-DHT lesions reduce TRH levels in the nucleus accumbens but not in other brain regions, and a similar selective loss of TRH is observed after inhibition of tryptophan hydroxylase to

Spinal effects of chronic intrathecal administration of the thyrotrophin-releasing hormone analogue (CG 3509) in rats

The effect of repeated intrathecal administration of a thyrotrophin-releasing hormone (TRH) analogue (CG 3509; 2 micrograms twice daily for 5 days) on wet-dog shake (WDS) and forepaw-licking (FPL) behaviours and spinal cord TRH and indoleamine levels and choline acetyltransferase (ChAT) activity was examined in adult rats. A rapid behavioural tolerance developed to repeated intrathecal injections of CG 3509; WDS and FPL behaviours were reduced by 57% and 34%, respectively, following the fifth injection and remained reduced at the ninth injection. Repeated CG 3509 administration selectively elevated ChAT activity and the level of 5-hydroxytryptamine (5-HT) in the ventral but not in the dorsal horn of the spinal cord, while 5-hydroxyindoleacetic acid (5-HIAA) and TRH levels were unaltered in either region. As ventral horn ChAT activity is principally located within motoneurones this data implies that TRH exerts a trophic-like influence on mature rat motoneurones in vivo. The results also suggest that long-term intrathecal TRH administration may decrease the release of 5-HT from bulbospinal raphe neurones.

Ventral Horn Neuropeptides Modulate the Release of Noradrenaline from Tissue Slices of Rat Brainstem and Ventral Thoracic Spinal Cord

The release of endogenous noradrenaline (NA) from slices of adult rat brainstem and ventral thoracic spinal cord was investigated using a fixed‐volume incubation technique and HPLC with electrochemical detection. Incubation with potassium (15–50 mM) produced a dose‐related increase in basal NA release that was calcium dependent The potassium‐evoked release of NA from spinal cord or brainstem slices was potentiated according to dose by preincubation with either (a) the selective α2‐adrenoceptor antagonist idazoxan (10−6‐10−4M) or (b) the thyrotrophin‐releasing hormone (TRH) analogue RX 77368 (pGlu‐His‐3,3′‐dimethyl ProNH2; 10−5 and 10−4M). Incubation of spinal cord slices with the NA uptake inhibitor maprotiline (1 μM) enhanced the effect of idazoxan but inhibited that of RX 77368. The effects of RX 77368 and potassium alone (15 mM) on NA release from both spinal cord and brainstem slices were reduced to basal levels with tetrodotoxin (10−7M). Similarly, preincubation of spinal cord, but not brainstem, slices with the insect neuropeptide proctolin (10−4M) significantly attenuated the potassium‐or RX 77368‐induced release of NA, whereas substance P (3 × 10−5 and 1 × 10−4M) had no effect on either tissue. These results suggest that changes in NA release in the spinal cord and brainstem may mediate some of the actions of neuropeptides in ventral spinal cord, although the peptides may not be acting directly on the noradrenergic nerve terminals in these tissues.

Repeated Intrathecal TRH Analogue Injection, but Not 57DHT Treatment, Induces Tolerance to Wet‐Dog Shake Behavior and Increases Ventral Horn ChAT Activitya

Thyrotropin-releasing hormone (TRH) coexists with 5-hydroxytryptamine (5HT) in bulbospinal raphe neurones that innervate the ventral horn of the spinal cord and regulate somatic motoneurone activity. Intrathecal injection of TRH or TRH analogues produces wet-dog shakes (WDS) and forepaw-licking (FPL) behavior’, but it is unknown if this behavior is mediated by bulbospinal serotonergic neurones. Both SHT and TRH, however, produce subthreshold depolarizations of motoneurones* and augment the spinal reflex activation of motoneurone~.~ In addition, TRH also exerts a long-term trophic effect on cultured motoneurones, elevating their choline acetyltransferase (ChAT) ~ o n t e n t . ~ The present study examined the effect of a TRH analogue (CG 3509) on WDS and FPL behavior in the presence of an intact bulbospinal 5HT pathway and tested whether this analogue had similar trophic effect on mature motoneurones in vivo. An intrathecal cannula was implanted in male Wistar rats (260 and 320 g) under sodium methohexitone (60 mg/kg i.p.) anesthesia so that the cannula tip was at the thoracolumbar junction of the spinal cord. Following seven days recovery, rats received intrathecal injections of either (a) saline (n = 8) or CG 3509 (2 pg twice daily for five days: n = 8) or (b) a single injection of CG 3509 (0.5 yg) two days before and twelve days after vehicle (n = 5 ) or 5,7 dihydroxytryptamine (57DHT, 2 x 150 pg intrathecally 1 hr after desipramine, 25 mglkg Lp.; n =6). The number of WDS and the time spent FPL in 30 min after saline or CG 3509 were recorded. After the behavioral study, the thoracolumbar spinal cord and brainstem levels of 5HT and 5-hydroxyindoleacetic acid (5HIAA) were assayed using HPLC with electrochemical detection, TRH, and substance P by RIA, and the thoracolumbar spinal cord ChAT activity was determined using a radioenzymatic ~ n e t h o d . ~ Values given are mean * SEM, and Student’s t test was used for statistical analysis. Intrathecal CG 3509 injection produced significantly more (p < 0.05) WDS and FPL than saline. When compared with the response to the first injection, WDS