Tac1 Signaling Is Required for Sexual Maturation and Responsiveness of GnRH Neurons to Kisspeptin in the Male Mouse.

The tachykinins substance P (SP) and neurokinin A (Tac1) have emerged as novel regulators of kisspeptin/GnRH release. Recently, we documented that SP modulates reproductive function in the female mouse. Here, we extended this characterization to the male mouse. Tac1-/- male mice showed delayed puberty onset. They also presented significantly decreased expression levels of Pdyn (dynorphin) and Nos1 (nitric oxide synthase) in the mediobasal hypothalamus and elevated Gnrh1 levels. Unexpectedly, the response of Tac1-/- mice to central kisspeptin or senktide (neurokinin B receptor-agonist) administration was significantly decreased compared with controls, despite the preserved ability of GnRH neurons to stimulate luteinizing hormone release as demonstrated by central N-methyl-D-aspartate receptor administration, suggesting a deficit at the GnRH neuron level. Importantly, we demonstrated that kisspeptin receptor and SP receptor (NK1R) heterodimerize, indicating that changes in the SP tone could alter the responsiveness of GnRH neurons to kisspeptin. Finally, electrophysiological recordings from arcuate Kiss1 neurons showed that, although virtually all Kiss1 neurons responded to NKB and senktide, only half responded to an NK1R agonist and none to the neurokinin A receptor agonist at a 1-µM dose. In summary, we provide compelling evidence for a role of Tac1 in the control of reproductive function in the male mouse, suggesting a predominant central action that may involve a change in the balance of neural factors that control GnRH expression.

GABA interneurons mediate the rapid antidepressant-like effects of scopolamine.

Major depressive disorder (MDD) is a recurring psychiatric illness that causes substantial health and socioeconomic burdens. Clinical reports have revealed that scopolamine, a nonselective muscarinic acetylcholine receptor antagonist, produces rapid antidepressant effects in individuals with MDD. Preclinical models suggest that these rapid antidepressant effects can be recapitulated with blockade of M1-type muscarinic acetylcholine receptors (M1-AChR); however, the cellular mechanisms underlying activity-dependent synaptic and behavioral responses to scopolamine have not been determined. Here, we demonstrate that the antidepressant-like effects of scopolamine are mediated by GABA interneurons in the medial prefrontal cortex (mPFC). Both GABAergic (GAD67+) interneurons and glutamatergic (CaMKII+) interneurons in the mPFC expressed M1-AChR. In mice, viral-mediated knockdown of M1-AChR specifically in GABAergic neurons, but not glutamatergic neurons, in the mPFC attenuated the antidepressant-like effects of scopolamine. Immunohistology and electrophysiology showed that somatostatin (SST) interneurons in the mPFC express M1-AChR at higher levels than parvalbumin interneurons. Moreover, knockdown of M1-AChR in SST interneurons in the mPFC demonstrated that M1-AChR expression in these neurons is required for the rapid antidepressant-like effects of scopolamine. These data indicate that SST interneurons in the mPFC are a promising pharmacological target for developing rapid-acting antidepressant therapies.

Electrophysiology of kisspeptin neurons.

Kisspeptin is an important regulator of reproduction. Electrophysiological studies show that kisspeptin neurons of the arcuate nucleus that co-localize neurokinin B and dynorphin (aka KNDy neurons) fire action potentials in a tonic, irregular, or burst firing manner. Gonadectomy dramatically alters the membrane properties of KNDY neurons from male mice and induces somatic hypertrophy. NMDA, leptin, and neurokinin B are potent activators of KNDY neuron electrical activity and GABA inhibits KNDY neurons. The firing pattern of kisspeptin neurons located in the RP3V fluctuates with the estrus cycle and is strongly modulated by glutamate and GABA. Thus, kisspeptin neurons are capable of burst firing, and their activity is modulated by sex steroids and other regulatory factors.

Melanin-concentrating hormone directly inhibits GnRH neurons and blocks kisspeptin activation, linking energy balance to reproduction.

A link between energy balance and reproduction is critical for the survival of all species. Energy-consuming reproductive processes need to be aborted in the face of a negative energy balance, yet knowledge of the pathways mediating this link remains limited. Fasting and food restriction that inhibit fertility also upregulate the hypothalamic melanin-concentrating hormone (MCH) system that promotes feeding and decreases energy expenditure; MCH knockout mice are lean and have a higher metabolism but remain fertile. MCH also modulates sleep, drug abuse behavior, and mood, and MCH receptor antagonists are currently being developed as antiobesity and antidepressant drugs. Despite the clinical implications of MCH, the direct postsynaptic effects of MCH have never been reported in CNS neurons. Using patch-clamp recordings in brain slices from multiple lines of transgenic GFP mice, we demonstrate a strong inhibitory effect of MCH on an exclusive population of septal vGluT2-GnRH neurons that is activated by the puberty-triggering and preovulatory luteinizing hormone surge-mediating peptide, kisspeptin. MCH has no effect on kisspeptin-insensitive GnRH, vGluT2, cholinergic, or GABAergic neurons located within the same nucleus. The inhibitory effects of MCH are reproducible and nondesensitizing and are mediated via a direct postsynaptic Ba(2+)-sensitive K(+) channel mechanism involving the MCHR1 receptor. MCH immunoreactive fibers are in close proximity to vGluT2-GFP and GnRH-GFP neurons. Importantly, MCH blocks the excitatory effect of kisspeptin on vGluT2-GnRH neurons. Considering the role of MCH in regulating energy balance and of GnRH and kisspeptin in triggering puberty and maintaining fertility, MCH may provide a critical link between energy balance and reproduction directly at the level of the kisspeptin-activated vGluT2-GnRH neuron.

Gonadotropin inhibitory hormone inhibits basal forebrain vGluT2-gonadotropin-releasing hormone neurons via a direct postsynaptic mechanism.

The novel hypothalamic peptides avian gonadotropin inhibitory hormone (GnIH) and its mammalian analogue RFRP-3, are emerging as key negative regulators of reproductive functions across species. GnIH/RFRP-3 reduces gonadotropin release and may play an inhibitory role in ovulation and seasonal reproduction, actions opposite to that of the puberty-promoting kisspeptins. GnIH directly inhibits gonadotropin release from the anterior pituitary in birds. GnIH/RFRP-3-immunoreactive fibres also abut the preoptic-septal gonadotropin-releasing hormone (GnRH) neurons, suggesting an additional site of action that has not been studied at the cellular level. Using anatomical labelling and electrophysiological recordings in septal brain slices from GnRH-GFP, vGluT2-GFP and GAD67-GFP mice, we report inhibitory actions of GnIH/RFRP-3 on kisspeptin-activated vGluT2 (vesicular glutamate transporter 2)-GnRH neurons as well as on kisspeptin-insensitive GnRH neurons, but not on cholinergic or GABAergic neurons (n = 531). GnIH and RFRP-3 produced a strikingly similar non-desensitizing hyperpolarization following brief 15 s applications (GnIH: 9.3 +/- 1.9 mV; RFRP-3: 9.0 +/- 0.9 mV) with IC(50) values of 34 and 37 nm, respectively. The inhibitory effect was mediated via a direct postsynaptic Ba(2+)-sensitive K(+) current mechanism and could prevent or interrupt kisspeptin-induced activation of vGluT2-GnRH neurons. GnIH-immunoreactive fibres were in apparent contact with vGluT2-GFP neurons. Thus, GnIH/RFRP-3 could reduce GnRH and glutamate release in target brain regions and in the median eminence via a direct inhibition of vGluT2-GnRH neurons. This in turn could suppress gonadotropin release, influence reproductive development and alter sex behaviour.

Excitatory effects of the puberty-initiating peptide kisspeptin and group I metabotropic glutamate receptor agonists differentiate two distinct subpopulations of gonadotropin-releasing hormone neurons.

Activation of the G-protein-coupled receptor GPR54 by kisspeptins during normal puberty promotes the central release of gonadotropin-releasing hormone (GnRH) that, in turn, leads to reproductive maturation. In humans and mice, a loss of function mutations of GPR54 prevents the onset of puberty and leads to hypogonadotropic hypogonadism and infertility. Using electrophysiological, morphological, molecular, and retrograde-labeling techniques in brain slices prepared from vGluT2-GFP and GnRH-GFP mice, we demonstrate the existence of two physiologically distinct subpopulations of GnRH neurons. The first subpopulation is comprised of septal GnRH neurons that colocalize vesicular glutamate transporter 2 and green fluorescent protein and is insensitive to metabotropic glutamate receptor agonists, but is exquisitely sensitive to kisspeptin which closes potassium channels to dramatically initiate a long-lasting activation in neurons from prepubertal and postpubertal mice of both sexes. A second subpopulation is insensitive to kisspeptin but is uniquely activated by group I metabotropic glutamate receptor agonists. These two physiologically distinct classes of GnRH cells may subserve different functions in the central control of reproduction and fertility.

Neurokinins robustly activate the majority of septohippocampal cholinergic neurons.

In the brain, tachykinins acting via the three cloned neurokinin (NK) receptors are implicated in stress-related affective disorders. Hemokinin-1 is a novel tachykinin that reportedly prefers NK1 to NK2 or NK3 receptors. Although NK1 and NK3 receptors are abundantly expressed in the brain, NK2-receptor-mediated electrophysiological effects have rarely been described as NK2 receptors are expressed only in a few brain regions such as the nucleus of the medial septum/diagonal band. Medial septal/diagonal band neurons that control hippocampal mnemonic functions also colocalize NK1 and NK3 receptors. Functionally, intraseptal activation of all three NK receptors increases hippocampal acetylcholine release and NK2 receptors have specifically been implicated in stress-induced hippocampal acetylcholine release. Electrophysiological studies on the effects of NKs on septohippocampal cholinergic neurons are lacking and electrophysiological effects of hemokinin-1 have thus far not been reported in brain neurons. In the present study we examined the electrophysiological and pharmacological effects of multiple NKs on fluorescently tagged septohippocampal cholinergic neurons using whole-cell patch-clamp recordings in a rat brain slice preparation. We demonstrate that a vast majority of septohippocampal cholinergic cells are activated by NK1, NK2 and NK3 receptor agonists as well as by hemokinin-1 via direct post-synaptic mechanisms. Pharmacologically, hemokinin-1 recruits not only NK1 but also NK2 and NK3 receptors to activate septohippocampal cholinergic neurons that are the primary source of acetylcholine for the hippocampus.

Muscarine activates the sodium-calcium exchanger via M receptors in basal forebrain neurons.

Neurons of the medial septum/diagonal band of Broca (MSDB) project to the hippocampus. Muscarinic cholinergic mechanisms within the MSDB are potent modulators of hippocampal functions; intraseptal scopolamine disrupts and intraseptal carbachol facilitates hippocampus-dependent learning and memory tasks, and the associated hippocampal theta rhythm. In earlier work, we demonstrated that, within the MSDB, the septohippocampal GABAergic but not cholinergic neurons are the primary target of muscarinic manipulations and that muscarinic activation of septohippocampal GABAergic neurons is mediated directly via M(3) receptors. In the present study, we examined the ionic mechanism(s) underlying the excitatory actions of muscarine in these neurons. Using whole-cell patch-clamp recording techniques in rat brain slices, we demonstrated that M(3) receptor-mediated muscarinic activation of MSDB neurons is dependent on external Na(+) and is also reduced by bath-applied Ni(2+) and KB-R7943 as well as by replacing external Na(+) with Li(+), suggesting a primary involvement of the Na(+)-Ca(2+) exchanger. We conclude that the M(3) receptor-mediated muscarinic activation of MSDB septohippocampal GABA-type neurons, that is important for cognitive functioning, is mediated via activation of the Na(+)-Ca(2+) exchanger.

Histamine innervation and activation of septohippocampal GABAergic neurones: involvement of local ACh release.

Recent studies indicate that the histaminergic system, which is critical for wakefulness, also influences learning and memory by interacting with cholinergic systems in the brain. Histamine-containing neurones of the tuberomammillary nucleus densely innervate the cholinergic and GABAergic nucleus of the medial septum/diagonal band of Broca (MSDB) which projects to the hippocampus and sustains hippocampal theta rhythm and associated learning and memory functions. Here we demonstrate that histamine, acting via H(1) and/or H(2) receptor subtypes, utilizes direct and indirect mechanisms to excite septohippocampal GABA-type neurones in a reversible, reproducible and concentration-dependent manner. The indirect mechanism involves local ACh release, is potentiated by acetylcholinesterase inhibitors and blocked by atropine methylbromide and 4-DAMP mustard, an M(3) muscarinic receptor selective antagonist. This indirect effect, presumably, results from a direct histamine-induced activation of septohippocampal cholinergic neurones and a subsequent indirect activation of the septohippocampal GABAergic neurones. In double-immunolabelling studies, histamine fibres were found in the vicinity of both septohippocampal cholinergic and GABAergic cell types. These findings have significance for Alzheimer's disease and other neurodegenerative disorders involving a loss of septohippocampal cholinergic neurones as such a loss would also obtund histamine effects on septohippocampal cholinergic and GABAergic functions and further compromise hippocampal arousal and associated cognitive functions.

Intrinsic vesicular glutamate transporter 2-immunoreactive input to septohippocampal parvalbumin-containing neurons: novel glutamatergic local circuit cells.

Glutamatergic influence on the medial septum diagonal band of Broca complex (MSDB) is a crucial and powerful driver of hippocampal theta rhythm and associated memory processes, in the rat. The recent discovery of vesicular glutamate transporters (VGLUT) provided a specific marker for glutamatergic neuronal elements. Therefore, this study aimed to address two specific questions: (1) do glutamatergic axons innervate MSDB gamma-aminobutyric acid (GABA)ergic, parvalbumin (PV)-containing septohippocampal neurons that are known to have a great influence on the electric activity of the hippocampus; and (2) is the origin of these glutamatergic axons extrinsic and/or intrinsic to the septum. The results of the correlated light and electron microscopic double-labeling immunohistochemistry for VGLUT2 and PV, and single immunostaining for VGLUT2 in colchicine-treated animals, showed that (1) VGLUT2-containing boutons establish asymmetric synaptic contacts with PV-positive perikarya and dendrites; (2) a large population of VGLUT2-immunoreactive neurons is located primarily in the posterior division of the septum; and (3) following surgical fimbria/fornix transection and septal undercut, most VGLUT2-containing axons, including those terminating on MSDB PV cells, remains intact. The latter two observations suggest that the major portion of MSDB glutamate axons have an intraseptal origin and raise a novel functional aspect of glutamatergic cells as local circuit neurons. A constant impulse flow in the septohippocampal GABA pathway is essential for the generation of theta rhythm. Thus, the heavy glutamatergic innervation of these septohippocampal GABA cells establishes the morphological basis for the powerful glutamatergic influence upon theta rhythm and hippocampus-associated memory processes.

Hippocampal theta rhythm is reduced by suppression of the H-current in septohippocampal GABAergic neurons.

Hippocampal learning and memory tasks are tightly coupled to the hippocampal theta rhythm, which is critically dependent on the medial septum/diagonal band of Broca (MSDB) although the underlying mechanisms remain unclear. The MSDB sends both cholinergic and GABAergic projections to the hippocampus. Here we show that: (i) septo-hippocampal GABAergic but not cholinergic neurons have a pacemaking current, the H-current, and that its selective blockade by ZD7288 reduces their spontaneous firing in rat brain slices; and (ii), local infusions of ZD7288 into the MSDB reduce exploration and sensory evoked hippocampal theta bursts in behaving rats. Thus, the H-current in septohippocampal GABAergic neurons modulates the hippocampal theta rhythm.

Hypocretin/orexin innervation and excitation of identified septohippocampal cholinergic neurons.

Hypothalamic fibers containing the wake-promoting peptides, hypocretins (Hcrts) or orexins, provide a dense innervation to the medial septum-diagonal band of Broca (MSDB), a sleep-associated brain region that has been suggested to show intense axonal degeneration in canine narcoleptics. The MSDB, via its cholinergic and GABAergic projections to the hippocampus, controls the hippocampal theta rhythm and associated learning and memory functions. Neurons of the MSDB express very high levels of the Hcrt receptor 2, which is mutated in canine narcoleptics. In the present study, we investigated the electrophysiological effects of Hcrt peptides on septohippocampal cholinergic neurons that were identified in living brain slices of the MSDB using a selective fluorescent marker. Hcrt activation of septohippocampal cholinergic neurons was reversible, reproducible, and concentration dependent and mediated via a direct postsynaptic mechanism. Both Hcrt1 and Hcrt2 activated septohippocampal cholinergic neurons with similar EC(50) values. The Hcrt effect was dependent on external Na(+), reduced by external Ba(2+), and also reduced in recordings with CsCl-containing electrodes, suggesting a dual underlying ionic mechanism that involved inhibition of a K(+) current, presumably an inward rectifier, and a Na(+)-dependent component. The Na(+) component was dependent on internal Ca(2+), blocked by replacing external Na(+) with Li(+), and also blocked by bath-applied Ni(2+) and KB-R7943, suggesting involvement of the Na(+)-Ca(2+) exchanger. Using double-immunolabeling studies at light and ultrastructural levels, we also provide definitive evidence for a hypocretin innervation of cholinergic neurons. Thus Hcrt effects within the septum should increase hippocampal acetylcholine release and thereby promote hippocampal arousal.

Group I metabotropic glutamate receptor activation produces a direct excitation of identified septohippocampal cholinergic neurons.

Septohippocampal cholinergic neurons innervate the hippocampus and provide it with almost its entire acetylcholine. Axon collaterals of these neurons also release acetylcholine within the septum and thereby maintain the firing activity of septohippocampal GABAergic neurons. A loss of septohippocampal cholinergic neurons occurs in various neurodegenerative disorders associated with cognitive dysfunctions. group I metabotropic glutamate receptors have been implicated in septohippocampal-dependent learning and memory tasks. In the present study, we examined the physiological and pharmacological effects of a potent and selective group I metabotropic glutamate receptor (mGluR) agonist S-3,5-dihydroxyphenylglycine (DHPG) on rat septohippocampal cholinergic neurons that were identified in brain slices using a selective fluorescent marker. In whole cell recordings, DHPG produced a reversible, reproducible and a direct postsynaptic and concentration-dependent excitation in 100% of septohippocampal cholinergic neurons tested with an EC(50) of 2.1 microM. Pharmacologically, the effects of DHPG were partially/completely reduced by the mGluR1 antagonists, 7-hydrox-iminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester and (+)-2-methyl-4-carboxyphenylglycine. Addition of the mGluR5 antagonist, 2-methyl-6-(phenylethnyl)pyridine hydrochloride, reduced the remaining response to DHPG, suggesting involvement of both receptor subtypes in a subpopulation of septohippocampal cholinergic neurons. In double-immunolabeling studies, 74% of septohippocampal cholinergic neurons co-localized mGluR1alpha-immunoreactivity and 35% co-localized mGluR5-immunoreactivity. Double-immunolabeling studies at the light and electron-microscopic levels showed that vesicular glutamate transporter 2 terminals make asymmetric synaptic contacts with septohippocampal cholinergic neurons. These findings may be of significance in treatment of cognitive deficits associated with neurodegenerative disorders as a group I mGluR-mediated activation of septohippocampal cholinergic neurons would enhance the release of acetylcholine both in the hippocampus and in the septum.

Acetylcholinesterase inhibitors activate septohippocampal GABAergic neurons via muscarinic but not nicotinic receptors.

Acetylcholinesterase (AChE) inhibitors, which increase synaptic levels of available acetylcholine (ACh) by preventing its degradation, are the most extensively prescribed drugs for the treatment of Alzheimer's disease. In animals, AChE inhibitors improve learning and memory, reverse scopolamine-induced amnesia, and produce hippocampal theta rhythm. The medial septum/diagonal band of Broca (MSDB), which maintains hippocampal theta rhythm and associated mnemonic functions via the septohippocampal pathway, is considered a critical locus for mediating the effects of AChE inhibitors. Using electrophysiological recordings and fluorescent labeling techniques to identify living septohippocampal neurons in rat brain slices, we report that AChE inhibitors, in the absence of exogenous ACh, produce a profound excitation in 94% of septohippocampal GABAergic neurons and an inhibition in 24% of septohippocampal cholinergic neurons. The inhibitory and excitatory effects of AChE inhibitors, presumably, occur due to accumulation of ACh that is released locally within the MSDB via axon collaterals of septohippocampal cholinergic neurons. The excitatory effects of AChE inhibitors on septohippocampal GABAergic neurons were blocked by muscarinic but not nicotinic receptor antagonists, especially by the M3 receptor antagonist, 4-diphenylacetoxy-N-methylpiperidine mustard, and not by M1 or M2/M4 muscarinic receptor antagonists. M3 muscarinic receptor mRNA colocalized with the calcium-binding protein, parvalbumin, a marker of septohippocampal GABAergic neurons. These findings may be useful in designing therapeutic strategies that do not depend on endogenous ACh and may therefore be effective in situations where AChE inhibitors cease to be effective, such as in progressive neurodegeneration.

Nicotine recruits a local glutamatergic circuit to excite septohippocampal GABAergic neurons.

Tonic impulse flow in the septohippocampal GABAergic pathway is essential for normal cognitive functioning and is sustained, in part, by acetylcholine (ACh) that is released locally via axon collaterals of septohippocampal cholinergic neurons. Septohippocampal cholinergic neurons degenerate in Alzheimer's disease and other neurodegenerative disorders. While the importance of the muscarinic effects of ACh on septohippocampal GABAergic neurons is well recognized, the nicotinic effects of ACh remain unstudied despite the reported benefits of nicotine on cognitive functioning. In the present study, using electrophysiological recordings in a rat brain slice preparation, rapid applications of nicotine excited 90% of retrogradely labelled septohippocampal GABA-type neurons with an EC50 of 17 microm and increased the frequency of spontaneously occurring, impulse-dependent fast GABAergic and glutamatergic synaptic currents via the alpha4beta2-nicotinic receptor. Interestingly, tetrodotoxin blocked all effects of nicotine on septohippocampal GABAergic type neurons, suggesting involvement of indirect mechanisms. We demonstrate that the effects of nicotine on septohippocampal GABA-type neurons involve recruitment of a novel, local glutamatergic circuitry as (i). Group I metabotropic glutamatergic receptor antagonists reduced the effects of nicotine; (ii). the number of nicotine responsive neurons was significantly reduced in recordings from slices that had been trimmed so as to reduce the number of glutamate-containing neurons within the slice preparation; (iii). in light and ultrastructural double immunocytochemical labelling studies vesicular glutamate 2 transporter immunoreactive terminals made synaptic contacts with parvalbumin-immunoreactive septohippocampal GABAergic neurons. The discovery of a local glutamatergic circuit within the septum may provide another avenue for restoring septohippocampal GABAergic functions in neurodegenerative disorders associated with a loss of septohippocampal cholinergic neurons.

Hypocretin increases impulse flow in the septohippocampal GABAergic pathway: implications for arousal via a mechanism of hippocampal disinhibition.

Hypocretins (Hcrts), or orexins, are a recently described set of hypothalamic peptides that have been implicated in feeding, neuroendocrine regulation, sleep-wakefulness, and disorders of sleep, such as narcolepsy. Hcrt-containing neurons, which are located exclusively in the lateral hypothalamic area, provide a dense innervation to the medial septum/diagonal band of Broca (MSDB), a sleep-associated brain region that has been suggested to show intense axonal degeneration in canine narcoleptics. The MSDB, via its cholinergic and GABAergic projections to the hippocampus, controls the hippocampal theta rhythm and associated learning and memory functions that occur during exploratory behavior and rapid eye movement sleep. Neurons of the MSDB express the Hcrt receptor 2, which is mutated in canine narcoleptics, but lack the Hcrt receptor 1 mRNA. In the present study, we investigated the electrophysiological effects of Hcrt2 on MSDB neurons from rat brain slices. We report that Hcrt2 produces a reversible, reproducible, concentration-dependent and direct postsynaptic excitation of GABA-type neurons of the MSDB with an EC50 of 207 nm. This effect is sodium dependent but not potassium or chloride dependent and is attenuated by blockers of the Na+-Ca+ exchanger. Hcrt2 also increases impulse-dependent release of GABA within the MSDB. Using recordings from retrogradely labeled septohippocampal neurons, we found that Hcrt2-excited MSDB neurons project to the hippocampus and have a GABAergic physiological signature. Double-immunolabeling studies confirmed the presence of Hcrt receptor-2 immunoreactivity in septohippocampal GABAergic neurons, as well as the presence of Hcrt fibers adjacent to these neurons. Based on these results, we speculate that Hcrt2-induced activation of septohippocampal GABAergic neurons will, by engaging disinhibitory mechanisms in the hippocampus, promote generation of the hippocampal theta rhythm and associated behaviors.

The formalin test: a tonic pain model in the primate.

The formalin test has been used in monkeys for assessing pain. After formalin injection in the palmar surface of the hand just proximal to the base of the fingers, the monkey's responses are rated for 1 h according to objective behavioral criteria. The present 'tonic' pain model has a fair degree of objectivity, validity, reproducibility and quantifiability. The analgesic effects of morphine and pethidine have been evaluated.