Life Sciences, Vol. 49, pp. 987-1002 Printed in the U.S.A.

Pergamon Press

MINIREVIEW T H E ELECTROPHYSIOLOGICAL ACTIONS OF NEUROTENSIN IN THE CENTRAL NERVOUS SYSTEM Zachary N. Stowe 1 and Charles B. Nemeroff*,2 Departments of Psychiatry1 and Pharmacology2, Duke University Medical Center Durham, North Carolina 27710 (Received in final form July 23, 1991)

Summary The endogenous neuropeptide, neurotensin (NT) alters the firing frequencies of certain neurons in the central nervous system (CNS). This is one of the findings that support the hypothesis that NT is a neurotransmitter substance. The direct application of NT on CNS neurons causes predominantly excitatory effects. These effects occur in a dose-related fashion via a calcium-dependent postsynaptic mechanism. The C-terminal hexapeptide fragment, NT 8-13 exerts similar electrophysiological effects to NT, while the N-terminal octapeptide fragment, NT 1-8 is devoid of such activity. NT produces a significant increase in the firing rates of individual neurons in the substantia nigra (SN), ventral tegmental area (VTA), medial prefrontal cortex (MPF), hypothalamus, and periacqueductal grey (PAG). This excitation occurs with a rapid onset and is readily reversible after cessation of NT application. In contrast, NT has no effect or weak inhibitory effects on the firing rates of neurons in the locus coeruleus (LC) and cerebellum. These electrophysiological actions of NT appear to be unique and not shared by other neurotransmitter and neuropeptide receptor anta-gonists and agonists that have been studied via direct co-application. NT attenuates dopamine (DA)-induced inhibition associated with direct application onto neurons in the SN and VTA both in vivo and in vitro. Intracellular recordings suggest that direct application of higher concentrations of NT appears to produce 'depolarization block' on individual neurons in the SN, VTA, MPF, and hypothalamus. The electrophysiological consequences of NT application not only show similarities to clinically efficacious antipsychotic medications, but also demonstrate the ability of NT to modulate the activity of dopamine (DA) neurons at the cellular level via specific NT binding sites. These findings further underscore the possibility that NT may play a pre-eminent role in the pathogenesis of, and psychopharmacological management of neurological and psychiatric disorders purportedly related to perturbation of CNS DA systems including schizophrenia.

It has been over fifteen years since neurotensin (NT) was originally isolated (1), sequenced (2), and characterized (3) from extracts of bovine hypothalami. Since then a multitude of studies have sought to elucidate the role(s) of this endogenous tridecapeptide (pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-OH) in the periphery (4, for 0024-3205/91 $3.00 + .00

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review), and within the central nervous system (CNS) (5,6 for review). NT has fulfilled most of the requisite criteria for classification as a neurotransmitter including: 1.) NT-containing neurons within the brain exhibit a heterogeneous distribution (7). 2.) NT is enriched in the synaptosomal fraction after density gradient centrifugation (7), and is contained in the synaptic vesicles as assessed by electron microscopic immunohistochemistry. 3.) Neuronal release of NT occurs after potassium-induced depolarization and this release is calciumdependent (8). 4.) Specific peptidases have been identified in the CNS that degrade NT (9). 5.) NT exhibits saturable and specific high affinity binding to receptors (10,11). 6.) Alterations in neuronal firing rates (12,13) and in the activity of second messenger systems following NT application have been reported. The direct injection of NT into the CNS produces hypothermia (14,15), potentiation of barbiturate-induced sedation (16,17), catalepsy (18), antinociception (19,20), blockade of psychostimulant-induced locomotor activity (21,22), and reduced food consumption (24). The effects observed after central administration of NT do not occur after systemic administration, suggesting that NT does not readily permeate the blood-brain barrier. The mechanism by which NT exerts CNS effects has not been conclusively delineated. Evidence derived from several biological systems including gastrointestinal tissue (25), anterior pituitary (26), and CNS (27), suggest that the initial NT-linked cellular events include an increase in membrane permeability to calcium. NT receptors may be linked with an inhibitory regulator of adenylate cyclase, and in HT 29 cells, NT receptors are coupled to inositol triphosphate-linked calcium mobilization (28). NT stimulates inositol phospholipid hydrolysis in both mouse neuroblastoma N1E 115 cell lines (29) and rat slices (30). The magnitude of this hydrolysis is significantly correlated with the number of NT binding sites. Our group has found that NT preincubation in the absence of calcium resulted in an increase in phosphorylation in vitro of CNS proteins with apparent molecular weights of 80,000, 73,500 and 50,000. Preliminary identification of these proteins based on molecular weight, sensitivity to cAMP and calcium gave initial identification of the 80kDa and 73.5kDa as Synapsin IA and IB, respectively. Both of these proteins are enriched in synaptic vesicles and are substrates for cAMP-dependent and calcium/calmodulin-dependent protein kinases, and the irreversible phosphorylation has been implicated as a consequential mechanism in the regulation of neurotransmitter release (31). Further exploration of NT-induced alterations of second messenger systems is warranted. NT exerts effects activity in a variety of CNS regions, and possesses a pharmacobehavioral profile remarkably similar to that of antipsychotic drugs. Yet, a unifying theory for the role(s) of NT in the CNS has not been established. The purpose of this review is to scrutinize the available electrophysiological data on the actions of NT in the CNS, and provide correlations with the anatomical, neurochemical, pharmacobehavioral, and clinical data concerning this peptide. Eleetropl~iologieal Aetiom of Neurotemia in the CNS Mesencephalon Neuroanatomically, neurotensin (NT) is closely associated with dopamine (DA) in the mesencephalon where it has the ability to selectively modulate the mesolimbic and mesocortical dopaminergic systems (6, for review). Briefly, high concentrations of NT are found in close proximity to D A systems (32) such as the substantia nigra (SN), the nucleus accumbens (NAS), and the caudate nucleus. Neurotensin-like immunoreactivity (NTLI) is co-localized within DA perikarya in the A8, A9, and A10 neurons in the mesencephalon (33,34). NT receptors are found on DA cell bodies and certain DA terminals in the

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nigroneostriatal system (35,36). The concentrations of NT and DA are positively correlated in the mesolimbic projections and negatively correlated in the mesostriatal projections (37). NT containing projections have been identified within the midbrain (6, for review), including a projection from the VTA to the NAS (38). Neurochemically, injection of NT into the ventral tegmental area (VTA) increases DA turnover in the nucleus accumbens, olfactory tubercles, and the diagonal band of Broca, as measured by the concentrations of the major dopamine metabolites, homovanillic acid (HVA) and dihydroxyphenylacetic acid (DOPAC), whereas in contrast DA applied to the VTA causes autoreceptor activation and decreases turnover in these terminal regions (39). Central application of NT also increases tyrosine hydroxylase activity (40) and stimulates DA release in the striatum (41), and the NAS (42). NT application produced a greater increase in the metabolism of DA in the NAS than in the striatum, and higher doses of NT partially reversed amphetamine-induced alterations in DA metabolism (43). NT does not alter basal DA-stimulated adenylate cyclase activity (44), nor does even high concentrations of NT decrease [3H] spiperone binding (44). These findings suggest that NT does not function simply as a D1 (adenylate cyclase-linked) or D2 (spiperone labelled) receptor antagonist. However, one group has reported that NT reduces the binding affinity of [3H]-npropylnorapomorphine ([3H]NPA), a DA agonist, in the striatum and nucleus accumbens (45,46). The same group has recently reported that the reduction in binding of [3H]NPA caused by NT is apparently independent of G-protein mechanisms (47). Similar reductions in [3H]NPA binding affinity associated with NT have been demonstrated in post-mortem human caudate-putamen (48). Acute and chronic treatment with clinically efficacious antipsychotic drugs (e.g., haloperidol, chlorpromazine) have consistently demonstrated an increase in NT concentrations in the nucleus accumbens and striatum, while phenothiazines that are not antipsychotics (e.g., promazine, promethazine) did not produce this increase (49). Chronic (8 months) treatment with haloperidol increases NT concentrations in these areas (50). A variety of studies using selective neurotransmitter receptor antagonists (51,52,53) indicate that these neurochemical alterations in NT concentrations are modulated predominantly by DA receptors. Still, an apparent confound is the finding that systemic administration of psychostimulants (e.g., cocaine, d-amphetamine) also increased NT concentrations in the mesolimbic areas (54), although relatively high doses of these are required. Behaviorally, central application of NT mimics the effects of systemically administered neuroleptics. It has been hypothesized that NT may represent an endogenous antipsychotic (55). Central application of NT produces inhibition of avoidance, but not escape responding, in a discrete trial, conditioned avoidance paradigm (i.e., "shuttle box" - a laboratory screen for antipsychotic activity). Injection of NT into the nucleus accumbens, like parenterally applied neuroleptics, blocks the increased locomotion and rearing produced by the indirect DA agonists methylphenidate, cocaine (21) and d-amphetamine (22). In contrast NT does not alter scopolamine and caffeine-induced hyperactivity (56). NT blocks the apparently rewarding electrical self-stimulation observed in rats with electrodes placed in the VTA (57). Rodents will preferentially lever press for intra-VTA injection of NT while ignoring the inactive lever (58). Thus, it has been proposed that NT may have a role in CNS reward mechanisms (58), by modulating DA neuronal activity. The effects of directly applied NT on the firing rates of individual neurons in the mesencephalon are summarized in Table I. NT consistently produced an increase in the firing rates of DA neurons in the zona compacta (ZC) of the SN and VTA both in vivo

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(12,59,60) a n d in vitro (13), while exerting no effect on n o n - d o p a m i n e r g i c n e u r o n s (59). A single in vivo investigation in the nucleus a c c u m b e n s d e m o n s t r a t e d p r e d o m i n a n t l y no r e s p o n s e o r inhibitory effects of NT, yet all cells tested w e r e inhibited by direct a p p l i c a t i o n of D A (61). TABLE I Effects of Neurotensin on Neurons in the Mesencephalon Region (Reference)

Number of Cells Tested

Effects on Neuronal Firing Rates Increased Decreased No change

A. Preparation - t~ v/vo/Iontophoretic Application Nucleus Accumbens (61)

31

2

Zona Compacta (12)

na

++

Zona Compacta (59) Zona Reticularis

na na

++

Zona Compacta (66)

12

12

Ventral Tegmental Area (67) Substantia Nigra

106

32

Substantia Nigra (63)

19

10

Substantia Nigra (64)

na

++

Ventral Tegmental Area (62) Substantia Nigra

na na

++ ++

Ventral Tegmental Area (13)

25

25

Ventral Tegmental Area (65) Substantia Nigra

na na

++ ++

13

15

nc

I0

64

B. Preparation -/n vitro~Bath Application

na nc ++ -- -

number of cells tested not available authors reported no significant change in firing rates authors reported generally increased firing rates in response to NT application authors reported generally decreased firing rates in response to NT application

T h e in vitro studies d e m o n s t r a t e d excitations that o c c u r r e d in a linear d o s e - r e s p o n s e fashion (13,63), a n d at higher c o n c e n t r a t i o n s o f N T ( > 300 n M ) the p e p t i d e p r o d u c e d a d e c r e a s e d a m p l i t u d e followed by cessation o f m e a s u r a b l e activity (13,64). T h e C - t e r m i n a l f r a g m e n t N T 8-13, n e u r o m e d i n N, a structurally r e l a t e d p e p t i d e , and xenopsin, a n o t h e r N T analog, possess similar activity (13,64,65). In contrast, the direct a p p l i c a t i o n of the N - t e r m i n a l fragments, N T 1-8 a n d N T 1-11 do not g e n e r a t e any m e a s u r a b l e a l t e r a t i o n in the firing rates o f d o p a m i n e r g i c n e u r o n s in vitro (13,65). It is n o t e w o r t h y that the NT-like o c t a p e p t i d e , xenopsin, also p r o d u c e d cessation o f D A n e u r o n a l activity at a c o n c e n t r a t i o n considerably less t h a n that o f N T (64). Investigations c o n d u c t e d in high m a g n e s i u m a n d low calcium m e d i a s u p p o r t the conclusion that the N T - i n d u c e d excitations occur via a c a l c i u m - d e p e n d e n t p o s t s y n a p t i c m e c h a n i s m (13). N T - i n d u c e d excitations were not affected by c o - a p p l i c a t i o n of y - a m i n o b u t y r i c acid ( G A B A ) (62) a n d g l u t a m a t e (Glu) (62). In addition, p r o g l u m i d e (a

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cholecystokinin (CCK) antagonist) (66), substance P (60), and N-methyl-D-aspartate (NMDA) (13) do not appreciably affect NT-induced increases in spontaneous firing rates. The application of NT to individual DA neurons after having attained stable responses to dopamine (DA) have shown that NT attenuates the DA-induced inhibition both in vivo (60,67) and in vitro (B.S. Bunney, personal communication). Intracerebroventricular (ICV) NT (50/~g) attenuated the inhibitory effects of systemically administered (iv) quinpirole, a specific D2 agonist, on midbrain DA neurons (68). The in vitro investigations have shown that the attenuation of DA-induced inhibition by NT occurs at concentrations lower (as low as 1 pM) than those required to produce excitation (B.S. Bunney, personal communication). A n in vitro study utilizing 8-Br-cAMP and forskolin found similar excitation of neurons and hypothesized that NT attenuation of DA-induced inhibition may occur via second messenger systems (62). The same group has also found that by increasing or decreasing the available intracellular cAMP concentrations with isobutylmethylxanthine (IBMX) or SQ22536 (9(tetrahydro-2-furyl)-adenine) respectively, significantly alters the modulatory actions of NT on DA-induced inhibitions (B.S. Bunney, personal communication). In another electrophysiological study, NT significantly increased the IC50 of the dopamine agonist, BHT 920 in the VTA (13).

Cerebral Cortex The effects of NT on cerebrocortical neurons have received considerably less attention than neurons in the midbrain. NT is extensively co-localized with DA in mesocortical neurons (69,70,71). The potential mechanisms for modulation of the mesocortical DA neurons have received considerable attention (72, for review). NT is also co-released with DA from nerve terminals after stimulation of D2 autoreceptors with apomorphine in vivo (69,70). While there are considerable similarities in the neurochemical and pharmacological effects of NT in the cortex and striatum, there are also several notable differences: 1.) Lower endogenous concentrations of NT are found in the cortex (73,74). 2.) In the cortex, the density of NT receptors is high whereas concentrations of NT are low (75). 3.) The distribution of NT binding sites in the cortex is similar to the distribution pattern of dopamine D1 receptors (76). 4.) The concentration of [lzsI]-NT binding sites in the frontal cortex increases with 6-OHDA induced destruction of DA neurons in the VTA. 5.) Acute or chronic treatment with phencyclidine (PCP), a psychotomimetic drug, produces a decrease in NT concentration in the frontal cortex; no change was observed in the mesencephalon (77). Our group has found that selective destruction of DA neurons with systemic 1-methyl-4phenyl-l,2,3,6 tetrahydropyridine (MPTP) in mice causes a reduction in NT concentration in the frontal cortex, preoptic area, and caudate nucleus (78). Finally, post-mortem studies in patients with schizophrenia demonstrate an increase in NT concentrations in Brodmann's Area (BA) 12 and 32, but not in BA 24, nucleus accumbens, hypothalamus, or striatum (79). There have only been three investigations that have scrutinized the electrophysiological actions of NT on cortical neurons. These results are summarized in Table II. Direct application of NT produced an increase in the firing rates of individual neurons in various cortical regions, both in vivo (80,81) and in vitro (82). The ability of NT to depolarize neurons in medial prefrontal cortex (layers II-V) slice preparations was not affected by pretreatment of the slice with phentolamine (n=4), propranolol (n=4), sulpiride (n=3), or fluphenazine (n=3). Similarly, the octapeptide NT 1-8 was without any effect on the spontaneous firing frequency o f individual cerebrocortical neurons (82); higher concentrations of NT itself produced 'depolarization block' (82). The persistence of NT-

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induced depolarization of cortical neurons in the presence of TI'X suggests that the effects of N T are not dependent on TTX Na-sensitive channels (82). It is noteworthy that the lowest concentration of NT used in the cortical slice preparation was 1/~M, and that one in v i v o investigations identified neurons based on corticospinal projections, and found a slow onset of action (81). TABLE II Effects on Neurotensin on CerebrocorticalNeurons Region (Reference)

Preparation

Number of Cells Tested

Effects on Neuronal Firing Rates Increased Decreased No change

Frontal Cortex (80)

in vivo/P

na

++

Corticospinal (81) Unidentified

in vivo/I in vivo/I

28 69

7 17

Medial Prefrontal (82)

in vitro/B

61"

49

21 52

numberof cells tested not available + + - authorsreported generallyincreased firingrates in response to NT application * authors did not report the effects on the remaining neurons tested I - iontophoresis P - pneumatic/pressure ejection B - bath application na

Hypothalamus As noted previously, NT was originally isolated from extracts of bovine hypothalami (1). High concentrations of NT are found in various hypothalamic nuclei, and NT is co-localized with D A in the perikarya of the arcuate and periventricular nucleus (34,35), the so-called A12 and A14 DA cells, respectively. To date, two NT- containing projections in the hypothalamus have been identified: 1.) From the arcuate and periventricular nucleus to the median eminence (83,84). 2.) From the medial nucleus of the amygdala to the ventromedial nucleus of the hypothalamus (85). The profound hypothermia induced by direct intracerebroventricular (ICV) injection of NT received considerable attention early in the investigations of a physiological role(s) for this tridecapeptide. The mechanism by which NT produces hypothermia is still unclear, but the hypothermic effect of NT probably involves an effect in the hypothalamus (86), and is independent of NT-induced dopamine release in this region (42). The hyperthermic effect of prostaglandin F--2(PGF~) is antagonized by CNS NT injection (87). A clinical study in children with febrile aseptic meningitis demonstrated that during the febrile period the concentration of CSF neurotensin-like immunoreactivity (NTLI) was significantly decreased with respect to controls, that normalized when patients were afebrile (88). This emphasizes the potential role for NT in modulating thermoregulation. It is of interest when discussing the hypothalamus to discuss the neuroendocrine effects of N T (32, for review). ICV NT decreases the secretion of thyroid-stimulating hormone (TSH), growth hormone (GH), prolactin (PRL), thyrotropin-releasing hormone (TRH), and inhibits PGE2-stimulated P R L release (89,90). In thyroidectomized rodents with elevated plasma TSH concentrations, intracisternal (IC) NT produced a significant reduction in plasma TSH concentrations (91). In contrast, IC NT produced an increase in somatostatin

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secretion (92). Effects of NT on the hypothalamic-pituitary adrenal axis (HPA) have not been carefully characterized. Unfortunately, there has been only one in vivo study of the electrophysiological actions of NT in the hypothalamus (80). No effect of pneumatically applied NT on hypothalamic neurons was observed. Yet, there have been several in vitro studies (93,94,95,96) that have found variable effects of NT on hypothalamic neurons. These findings are summarized in Table III. The direct application of NT onto neurons by iontophoresis or pressure ejection caused a significant increase in the firing rates of preoptic-anterior hypothalamic neurons (93,94,95) that persisted in a low calcium medium (93,95). Again, the C-terminal NT fragments, NT 8-13, and N-acetylated NT 8-13, produced similar effects, whereas the Nterminal NT 1-8 did not produce any alteration in firing frequency at the concentrations used (93,95). When NT concentrations exceeded 1 /~M measurable neuronal activity was abolished, but this effect was consistently reversed by direct application of GABA (93). This test has been utilized in studies with other neuropeptides (e.g., CCK) to confirm that the inactivation is not secondary to hyperpolarization. Arcuate nucleus neurons had little or no response to NT when applied via bath (96). TABLE III

Effects of Neurotensin on Neurons in the Hypothalamus Region (Reference)

N u m b e r of Preparation CellsTested

Effects on Neuronal Firing Rates Increased Decreased No change

Hypothalamus (80)

in v/vo/P

na

nc

Preoptie Anterior (93)

in vitro/P

100

71

16

13

Preoptic Anterior (94)

in vitro~liP

14

8

2

4

Hypothalamus (95)

in vitrolP

42

30

4

8

Arcuate Nucleus (96)

in vitro/B

20

4

5

11

n u m b e r o f cells tested not available. nc a u t h o r s r e p o r t e d n o significant c h a n g e in firing rates. P - pneumatic/pressure ejection B - bath application I - iontophoresis na -

Other Central Nervous System Regions The distribution of NT binding sites is considerably more diffuse than that of the peptide itself in the CNS. There have been several electrophysiological studies in a variety of CNS regions. Unfortunately, there is not the body of neurochemical, anatomical, and behavioral data available for correlation to the electrophysiological findings in these areas. It is noteworthy, that the modulatory effects of NT are not entirely unique to dopaminergic systems. NT also increases the turnover of serotonin (5HT) (97), acetylcholine (Ach) (21), and norepinephrine (97), though at greater doses than those required to alter D A neurotransmission. The ability of NT to alter the release of Ach from interneurons in striatal slices was found to be independent of DA neurotransmission (98). Several identified NT-containing projections in rodents not yet cited in this monograph include: 1.) A pathway from the central nucleus of the amygdala to (and through) the bed

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nucleus of the stria terminalis (99). 2.) From the endopiriform cortex to the anterior olfactory nucleus, the nucleus of the diagonal band and the medial-dorsal thalamic nuclei (100). 3.) From the periacqueductal grey, nucleus of the solitary tract and parabrachial nucleus to the nucleus raphe magnus (101). The electrophysiological effects of NT on neurons in several miscellaneous CNS regions are summarized in Table IV. The first study addressing the neurophysiological actions of NT was conducted on five neurons in the locus coeruleus (LC), NT did not alter the firing rates TABLE IV Effects of Neurotensin on Neurons in Other CNS Regions

Region (Reference)

Preparation

Number of Cells Tested

Effects on Neuronal Firing Rates Increased Decreased No change

Basal Complex (80) Thalamus

in vivo/P

27

Cerebellum (61)

in vivo/l

na

-- (weak)

Cerebellum (104)

in vivo/l in vivo/P

26 60*

2 48

24 2

Hippocampus (80)

in vivo/P

14

5

7

Locus Coeruleus (102)

in vivo/I

5

Locus Coeruleus (103)

in vivo/I

355"

Nucleus Solitarius (113) Tractus

in vivo/I/P

20

20

Periacqueductal Grey (111)

in vivo/P

50*

32

Bed Nucleus of the (105) Stria Terminalis

in vitro/B

11

11

Laminae I-III, cat (107)

in vivo/I

33

20

Spinal cord, rat (109)

in vivo/P

na

++

Laminae IV, cat (110)

in vivo/I na

na

+ + motor neurons -- sensory neurons

Motor neurons, frog (106)

in vitro/B

na

depolarized neurons tested

Motor neurons, frog (108)

in vitro/I

na

depolarized neurons tested

2

2

19

6

5 13

11

Spinal cord

na ++ -- -

13

number of ceils tested not available. authors reported generally increased firing rates in response to NT application. authors reported generally decreased firing rates in response to NT application. authors did not report the effects on remaining neurons tested. t the remaining neurons were localized outside the LC, and no significant change in f'tring rate reported. I - iontophoresis P - pneumatic/pressure ejection B - bath application *

-

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of the neurons tested (102). In contrast, a second study on LC neurons demonstrated an inhibitory effect in most cells tested that occurred with a rapid onset (103). Another area that received early attention was the cerebellum. Both studies reported that direct application of NT decreased the firing rates of Purkinje neurons (61,104). The second study (104) utilized pretreatment of the animals with haloperidol, or destruction of the dopaminergic neurons with 6-hydroxydopamine (6-OHDA), that resulted in not only abolishing the inhibitory effects of NT but demonstrating an excitatory effect. As part of an initial in vivo survey study (80), NT was found to produce predominantly inhibitory effects on neurons in the basal complex of the thalamus, and have a variety of effects on neurons in the hippocampus that were not affected by the GABA antagonist, bicuculline. A single in vitro study in the stria terminalis illustrated excitatory effects of NT (105). In the spinal cord of different species, NT exerted excitatory effects on motor neurons (106,107,108,109,110) that occurred with a relatively slow onset and persists as long as 2 hours after NT application. The depolarizing effect of NT was largely abolished with pretreatment of the tissue preparation with tetrodotoxin (106,107). There have also been differential effects of NT reported in the spinal cord, with excitatory and inhibitory effects being observed on motor and sensory neurons, respectively (110). Some interesting observations have been made in the areas involving the NT-containing projection noted previously involving the periacqueductal grey (PAG), nucleus raphe magnus (NRM), and the nucleus of the solitary tract. As noted (vide supra), the central administration of NT produces antinociception in a dose-dependent fashion. This effect is abolished after destruction of the nucleus raphe magnus (111) or lesioning of the stria terminalis (112). NT produces differential excitations of neurons in the PAG, eliciting both short acting and sustained increases in the firing frequencies (111). The injection of NT into the PAG caused predominantly excitation of neurons in the NRM (111). The electrophysiological actions of NT in the PAG and NRM were not affected by the opioid antagonist, naloxone (111). All neurons tested demonstrated an increase in firing rate associated with NT application (113). Discussion Overall, scrutiny of the extant literature reveals that NT exerts electrophysiological actions with a variety of direct application techniques. These actions appear to be independent of other neurotransmitter and neuropeptide receptors. There is good agreement between behavioral and electrophysiological investigations, concerning structureactivity relations in that the C-terminus is required for activity. Undoubtedly, the vast majority of the data for such potential correlation have been generated in the mesencephalon. The electrophysiological actions of NT in the mesencephalon continue to underscore the similarity between centrally administered NT and systemically administered antipsychotic drugs. The electrophysiological effects of acute and chronic neuroleptic treatment on midbrain dopaminergic neurons have been extensively studied (114, for review). Briefly, most clinically efficacious antipsychotics increase the firing rates of dopaminergic neurons in the VTA and zona compacta (115,116); repeated administration induces 'depolarization block'in dopaminergic neurons in the VTA and SN (117,118,119,120,121). Bath application of haloperidol, (-) sulpiride, and cis-flupenthixol have demonstrated an attenuation of DAinduced inhibition in the SN (122). Clozapine, an atypical antipsychotic, and the purported selective D1 antagonist SCH 23390 does not alter the DA-induced inhibitions in the SN and VTA (123). Therefore, while antipsychotic drugs apparently attenuate DA

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neurotransmission via interactions with the D2 receptor, NT produces similar effects without binding directly to the D2 receptor. Presently, it is unclear if the cessation of activity in the mesencephalon is related to 'depolarization block' or possibly toxic effects (67). However, if we extend the findings of GABA-reversal of NT-induced cessation of activity in the hypothalamus to the mesencephalon, and the depolarization described with intracellular recordings in the MPF, it is reasonable to hypothesize that direct application of higher concentrations of NT induces a 'depolarization block' in individual dopaminergic neurons in the midbrain. Clearly, NT produces electrophysiological alterations of dopaminergic neurons that is similar to the effects of both systemic and directly applied antipsychotic drugs. The mechanism by which NT attenuates DA-induced inhibitions has not been delineated. One group has proposed that this mechanism may occur via formation of 'NT-DA complex extracellularly (124)', however NT does not block DA-stimulated adenylate cyclase activity. Also complexation studies have shown that NT 1-11 binds dopamine, yet this N-terminal fragment does not possess measurable electrophysiological activity on DA neurons (65). Moreover the C-terminal fragment, NT 8-13, does not bind with DA and still possesses electrophysiological actions similar to NT. These findings support the hypothesis that NT modulates DA neurotransmission via specific NT-binding sites. The modulation of the electrophysiological actions of NT found with alterations of intracellular cAMP concentrations, taken together with the available intracellular data, provides preliminary evidence that NT modulation of DA neurotransmission may occur via second messenger systems. Further electrophysiological investigations using direct DA receptor agonists, and modification of intracellular events may clarify this mechanism. It is noteworthy that presently, it is not known if the various NT fragments attenuate DA-induced inhibition. Some of these questions are currently under investigation in our laboratory. The systemic administration of DA agonists and antagonists produces some conflicting data on the alterations of NT concentrations in the NAS. This provides a major confound in the neurochemical data on NT. The electrophysiological data does not provide a definitive explanation. Yet, the dose-dependent effects NT, and the differential electrophysiological effects of NT in the NAS may in part account for this contrasting data. It is noteworthy that antidromic stimulation of the NAS produces inhibition of V I A neurons and the feedback pathways and self-inhibitory somatodendritic mechanisms have been implicated in the effects of antipsychotic drugs on DA neurons (114, for review). The electrophysiological actions of NT in the VTA taken in conjunction with the selfadministration behavioral data and the increases in NT concentrations following systemic administration of amphetamine and cocaine underscores the potential role for NT in CNS reward mechanisms. Currently, any firm conclusions cannot be made until further investigations are completed. The limited studies of cortical and hypothalamic neurons precedes any firm conclusions. Despite the limitations, preliminary findings of the independence of the electrophysiological actions of NT from the a and fl noradrenergic, and dopaminergic Dz receptors in cerebrocortical neurons emphasizes the novel receptor via which NT apparently exerts its effects. Future investigations in the cortex using antidromic stimulation may provide correlation with the projections from the VTA. The data from the hypothalamus illustrates the physiological activity of NT in the various hypothalamic neurons. Another interesting finding is that many of the pharmacobehavioral effects of IC NT are antagonized by thyrotropin-releasing hormone (TRH). Briefly, the avoidance behavior, the reduced food

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consumption, antinociception, hypothermia, hypotension, and the ethanol-potentiating effects associated with central administration of NT are all antagonized by TRH (6, for review). In the cortex, TRH decreased the firing frequencies of neurons that were both excited or unaffected by NT, yet no electrophysiological interactions between the two neuropeptides has been investigated (81). It will be of interest to examine this interaction in subsequent studies, particularly in the preoptic-anterior hypothalamus, nucleus accumbens, and PAG, all areas where TRH and NT exert effects after direct microinjection. Also in future studies in the hypothalamus, assessment of the electrophysiological actions of NT on thermosensitive neurons may provide information about the thermoregulatory role of NT. With respect to the potential role for NT in modulation of CNS nociceptive pathways, the electrophysiological data provides some interesting information. Several groups have reported that the antinociception associated with central NT administration is not naloxonereversible (19,125,126), though others have reported opposite findings (127,128). Interestingly, mice rendered tolerant to morphine exhibit a decreased antinociceptive response to IC NT (129). The systemic administration of both DA antagonists and agonists fails to antagonize NT-induced antinociception (130). The electrophysiological actions of NT are not affected by naloxone in the PAG and NRM (111). This would imply that naloxone antagonism of NT-induced antinociception, if real, is related to an indirect mechanism rather than a direct effect on the cellular actions of NT. The spinal cord demonstrates some significant differences from the other CNS areas that have been studied. Most investigators report NT effects characterized by a slow onset and gradual return to baseline. The preliminary conclusions about the electrophysiological actions of NT in the spinal cord suggest that the excitatory actions of NT are dependent on spinal interneurons. The physiological significance of the actions of NT in the spinal cord remain obscure. Conclusion In summary, the available electrophysiological literature is in good agreement with the burgeoning data base of the significant modulatory actions of NT on brain dopaminergic systems. Neurotensin demonstrates a pharmacobehavioral and electrophysiological profile that is remarkably similar to clinically efficacious antipsychotic drugs. It is not uncommon, that once a compound has shown activity in the behavioral paradigms utilized to test for potential antipsychotic activity, that a series of electrophysiological studies are conducted. It has been hypothesized that antipsychotic medications may exert their efficacy via alteration in the synaptic availability of DA and neuropeptides (131), and that the delay in clinical response may be related to the temporal relationship of antipsychotic-induced 'depolarization block' (117,119,132). Clearly, NT produces similar electrophysiological effects to those produced by antipsychotics, and may contribute to the development of 'depolarization block'. The clinical data demonstrates a reduction in the mean CSF NT concentration in patients suffering from a schizophrenic psychosis (133,134,135). The magnitude of this reduction was positively correlated with a greater preponderance of hallucinations-delusions, thought disorder, and disorganized behavior (135). Following treatment with antipsychotic drugs the CSF NT concentrations tended towards normalization (134,135), and in one study this increase in CSF NT concentrations preceded clinical improvement (135). The electrophysiological data and the clinical findings underscore the potential importance of NT in the therapeutic actions of antipsychotic medications. Clarification of the role(s) of NT in the action of antipsychotic drugs and in the development of future treatment modalities may be forthcoming as NT receptor agonists and antagonists

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are developed. The recent cloning of the NT receptor (136) may help with the development of such agonists and antagonists.

Acknowledgments The authors gratefully acknowledge the generosity of Dr. B.S. Bunney (Yale University) foi his sharing of findings from his laboratory prior to their publication that greatly enhanced this review. The authors are supported by NIMH MH-39415.

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